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NF-??B Induces Expression of the Bcl-2 Homologue A1/Bfl-1 To Preferentially Suppress Chemotherapy-Induced Apoptosis
Abstract
Recent evidence indicates that the transcription factor NF-κB is a major effector of inducible antiapoptotic mechanisms. For example, it was shown that NF-κB activation suppresses the activation of caspase 8, the apical caspase in tumor necrosis factor (TNF) receptor family signaling cascades, through the transcriptional regulation of certain TRAF and IAP proteins. However, it was unknown whether NF-κB controls other key regulatory mechanisms in apoptosis. Here we show that NF-κB activation suppresses mitochondrial release of cytochrome
c
through the activation of the Bcl-2 family member A1/Bfl-1. The restoration of A1 in NF-κB null cells diminished TNF-induced apoptosis by reducing the release of proapoptotic cytochrome
c
from mitochondria. In addition, A1 potently inhibited etoposide-induced apoptosis by inhibiting the release of cytochrome
c
and by blocking caspase 3 activation. Our findings demonstrate that A1 is an important antiapoptotic gene controlled by NF-κB and establish that the prosurvival function of NF-κB can be manifested at multiple levels.
... Overexpression of CD147 leads to the dysregulation of the NF-κB pathway in cancer cells [26,30,38], primarily through the degradation of the inhibitor of κB (IκB), which allows for the activation and nuclear translation of p50/p65 dimers [39]. Once in the nucleus, active NF-κB dimers can promote the transcription of genes that contribute to the malignant phenotype [40][41][42][43][44]. Specifically, NF-κB can upregulate anti-apoptotic Bcl-2 proteins (e.g., Bcl-2 and Bcl-xL) [31,[45][46][47], which in turn suppress pro-apoptotic proteins (e.g., Bax and Bak), thereby promoting cell survival and proliferation [48][49][50]. Consequently, the inhibition of CD147 suppresses the NF-κB pathway and alters anti-and proapoptotic protein ratios to favor apoptosis induction. ...
... Overexpression of CD147 leads to the dysregulation of the NF-κB pathway in cancer cells [26,30,38], primarily through the degradation of the inhibitor of κB (IκB), which allows for the activation and nuclear translation of p50/p65 dimers [39]. Once in the nucleus, active NF-κB dimers can promote the transcription of genes that contribute to the malignant phenotype [40][41][42][43][44]. Specifically, NF-κB can upregulate anti-apoptotic Bcl-2 proteins (e.g., Bcl-2 and Bcl-xL) [31,[45][46][47], which in turn suppress pro-apoptotic proteins (e.g., Bax and Bak), thereby promoting cell survival and proliferation [48][49][50]. Consequently, the inhibition of CD147 suppresses the NF-κB pathway and alters anti-and pro-apoptotic protein ratios to favor apoptosis induction. ...
Acute myeloid leukemia (AML) is an aggressive blood cancer. With low survival rates, new drug targets are needed to improve treatment regimens and patient outcomes. Pseudolaric acid B (PAB) is a plant-derived bioactive compound predicted to interact with cluster of differentiation 147 (CD147/BSG). CD147 is a transmembrane glycoprotein overexpressed in various malignancies with suggested roles in regulating cancer cell survival, proliferation, invasion, and apoptosis. However, the detailed function of PAB in AML remains unknown. In this study, AML cell lines and patient-derived cells were used to show that PAB selectively targeted AML (IC50: 1.59 ± 0.47 µM). Moreover, proliferation assays, flow cytometry, and immunoblotting confirmed that PAB targeting of CD147 resulted in AML cell apoptosis. Indeed, the genetic silencing of CD147 significantly suppressed AML cell growth and attenuated PAB activity. Overall, PAB imparts anti-AML activity through transmembrane glycoprotein CD147.
... NF-κB target genes promote protective immunity against pathogens and/or anti-tumorigenic activity; for example, engaging in immune cell activation, antigen presentation and cytokine production. NF-κB transcribes genes encoding cytokines, chemokines, immunoreceptors, growth factors as well as both activators and inhibitors for various cell death types (Bauernfeind et al., 2009;Hinz et al., 1999;Kreuz et al., 2001;Liu et al., 2017;Micheau et al., 2001;Schwenzer et al., 1999;Stehlik et al., 1998;Wang et al., 1998Wang et al., , 1999Yang et al., 2015). The oscillation of NF-κB between nuclear and cytosol, is dependent on stimuli type, dosage and timing as well as cell type (Butera et al., 2022;Nelson et al., 2004); leading to different transcriptional outcomes (Colombo et al., 2018;Tian et al., 2005;Zambrano et al., 2016;Zhao et al., 2018). ...
... Under classical stimuli (e.g. TNFα), NF-κB promotes the rapid transcription of antiapoptotic genes including TRAF2, cIAPs, cFLIP and Bcl-2 family proteins (Kreuz et al., 2001;Schwenzer et al., 1999;Stehlik et al., 1998;Tian et al., 2005;Wang et al., 1998Wang et al., , 1999Zhao et al., 2018) but also apoptosis genes FAS and FASL and, under atypical stimuli (e.g. UV-C and anthracyclines), represses XIAP and Bcl-XL to promote apoptosis (Campbell et al., 2004;Ho et al., 2005;Liu et al., 2012; (which was not certified by peer review) is the author/funder. ...
Re-engineering NF-κB signalling towards enhancing beneficial outcomes such as tumour cell elimination, while minimising inflammatory damage, is a potential therapeutic avenue. In this study, we explored the ability of bacterial effectors injected into host cells by the type III secretion system to regulate NF-κB translocation dynamics. We used the enteropathogenic Escherichia coli effectors Tir (NF-κB activator), NleC (NF-κB protease) and NleE (TAB2/3 methyltransferase), to manipulate NF-κB translocation and cancer cell survival. We discovered that while these effectors have either limited or no cytotoxicity alone, they greatly enhanced caspase-8-dependent pancreatic cancer cell death in the presence of TNFα. Single cell analysis revealed that the sub-population of cells showing high NF-κB activation is less susceptible to cell death caused by NleC or NleE but instead is more susceptible to Tir. A combination of Tir, NleE and TNFα eliminated 95% cancer cells with limited NF-κB activation, potentially due to NleE-dependent blockage of the immediate pro-survival NF-κB activation without inhibiting Tir’s long-term NF-κB activation that promotes cell death. This work demonstrates that effector combinations could be used to re-engineer stress responses towards favourable outcomes.
... BCL2A1 protein contains 4 BH-domains and does not have any C-terminal transmembrane domain like other BCL2 family of protein but regulates anti-apoptotic function 21 . BCL2A1 mRNA expression has been shown to be generally overexpressed in many cancers including breast cancer 22,23 and is attributed to poor prognosis 24 and directly contributes to chemoresistance 25 . Studies have shown that CD40, PI3K and ERK signaling initiated by ICAM-1 binding has been found to induce NF-kB and subsequently BCL2A1 expression 26 . ...
... Several studies have shown that an increase in TNF-alpha expression induces cell survival and cancer progression 34,35 . In fibrosarcoma cells for example, BCL2A1 was rapidly induced after TNF-α stimulation 25 . Studies have also shown that TNF-alpha gene knockout in the TNBC cell line induces apoptosis 36 . ...
Breast cancer is the leading cause of cancer death among women. Triple-negative breast cancer (TNBC) has a poor prognosis and frequently relapses early compared with other subtypes. The Cold Atmospheric Plasma (CAP) is a promising therapy for prognostically poor breast cancer such as TNBC. The Canady Helios Cold Plasma (CHCP) induces cell death in the TNBC cell line without thermal damage, however, the mechanism of cell death by CAP treatment is ambiguous and the mechanism of resistance to cell death in some subset of cells has not been addressed. We investigate the expression profile of 48 apoptotic and 35 oxidative gene markers after CHCP treatment in six different types of breast cancer cell lines including luminal A (ER ⁺ PR +/− HER2 ⁻ ), luminal B (ER ⁺ PR +/− HER2 ⁺ ), (ER ⁻ PR ⁻ HER2 ⁺ ), basal-like: ER ⁻ PR ⁻ HER2 ⁻ cells were tested with CHCP at different power settings and at 4 different incubation time. The expression levels of the gene markers were determined at 4 different intervals after the treatment. The protein expression of BCL2A1 was only induced after CHCP treatment in TNBC cell lines (p < 0.01), whereas the HER2-positive and ER, PR positive cell lines showed little or no expression of BCL2A1. The BCL2A1 and TNF-alpha expression levels showed a significant correlation within TNBC cell lines (p < 0.01). Silencing BCL2A1 mRNA by siRNA increased the potency of the CHCP treatment. A Combination of CHCP and CPI203, a BET bromodomain inhibitor, and a BCL2A1 antagonist increased the CHCP-induced cell death (p < 0.05). Our results revealed that BCL2A1 is a key gene for resistance during CHCP induced cell death. This resistance in TNBCs could be reversed with a combination of siRNA or BCL2A1 antagonist-CHCP therapy.
... Increased Bfl-1 expression has been reported as a mechanism of resistance to cancer therapies, although its prevalence in mediating intrinsic resistance is lesser studied, particularly in the context of lymphoma. 6,7,22,23 Thus, we investigated the relationship between Bfl-1 expression and the sensitivity of BH3 mimetics across a lymphoma cell-line panel. Relative to lymphoma cell lines without detectable Bfl-1 protein ("Bfl-1 2 lymphomas"), Bfl-1 1 lymphomas exhibited reduced sensitivity, as measured by cleaved caspase-3/7 activation ("caspase activation"), to BH3 mimetics targeting Mcl-1 (AZD5991) and Bcl-2 (venetoclax) ( Figure 1E), which translated to significantly reduced cell death compared with that achieved in Bfl-1 2 lymphoma cell lines ( Figure 1F). ...
... 29 Across hematological cancer cell-line panels Bcl-2 and Mcl-1 inhibitors display enhanced antitumor potency in leukemia and myeloma-derived neoplasms compared with lymphomas. 9,11,23 Consistent with the preclinical data, venetoclax monotherapy treatment in 106 non-Hodgkin lymphoma patients produced an inferior overall response rate (44%) 30 compared with that observed for 116 CLL patients (79%). 2 To date, however, clinical response data for Mcl-1 inhibitors has not been reported. In the present study, we identified that Bfl-1 expression in preclinical lymphoma models reduces the apoptogenic potential of Bcl-2 and Mcl-1 inhibitors while Bfl-1 suppression sensitizes these lymphomas to BH3 mimetics in both monotherapy and combination settings. ...
BH3 mimetics like Venetoclax target pro-survival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2-inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional pro-survival proteins in this context. Importantly, we demonstrated that CDK9 inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3 mimetic resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in DLBCL PDX models expressing Bfl-1. This data underscores the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.
... Additionally, NF-κβ maintain the balance between tendon fibroblast proliferation and apoptosis and inhibit caspase 8 activation, thereby blocking apoptosis in different cell types in the tendon [99]. Moreover, NF-κβ suppresses apoptosis through activation of the Bcl-2 family member A1/Bfl-1 which reduces the release of proapoptotic cytochrome C from mitochondria [100]. Interestingly, this study aligned with our previously recorded findings of higher expression of Bcl-2 in cultured hypoxic tenocytes [101]. ...
... Apoptosis is widely acknowledged as one of the primary mechanisms of programmed cell death (35). The promoter of the protein that regulates apoptosis, the BCL2 family, is significantly regulated by NF-kB (36). A balance of anti-apoptotic and apoptotic molecules signifies cellular homeostasis. ...
Cancer treatment that involves chemotherapy often results in developed drug resistance which can be a major deterrent for the success of a treatment protocol. Increased inflammation, due to the drug's presence, contributes to disease progression and increased Cyclooxygenase-2 (COX2) expression (inflammatory marker). Integrative oncology is a field that focuses on combining natural herbal formulations with standard care to provide optimal clinical outcomes in a holistic manner, to minimize side effects, and to overcome drug resistance, including inflammation. 5-Flurouracil (5-FU) is a chemotherapeutic medication used to treat cancers. We studied drug resistance by deriving 5-FU-resistant HeLa cells (HeLa R) and evaluating the efficacy of AS20 in in vitro experiments. AS20 is an herbal formulation derived from the plant Amaranthus spinosus, an indigenous plant found in India. It contains different types of secondary metabolites such as saponins, polyphenols, alkaloids, terpenoids, and flavonoids. We hypothesized that AS20 has anti-inflammatory and anticancer properties on HeLa R. We investigated if AS20 could downregulate increased inflammatory markers such as COX2 in HeLa R. Also, we studied anti-apoptotic and pro-apoptotic genes in AS20 treated HeLa R cells. Our results demonstrated that the inhibitory concentration at 50% (IC50) values of HeLa wild type cells with AS20 treatment was significantly less than HeLa R cells. Also, AS20 brought down induced COX2 expression in HeLa R cells. AS20 treatment on HeLa R cells showed upregulation of BAX (pro-apoptotic) but no significant change in BAD (pro-apoptotic) and downregulation of BCL2 and BCL2L1 (anti-apoptotic genes) in HeLa R cells. Our results showed that AS20 has anti-inflammatory and pro-apoptotic properties when treated on HeLa R cells.
... NF-κB regulates the expression of many genes that have an important role in vital biologic processes, such as inflammation, apoptosis, cell growth, and immunity (Zinatizadeh et al. 2021). NF-κB becomes highly activated in many inflammatory disorders and cancers and triggers the transcription of chemokines, inducible nitric oxide, and pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), Interleukin 6 (IL-6), Interleukin 1 beta (IL-1β) and suppresses apoptosis activation (Serasanambati and Chilakapati 2016;Tak and Firestein 2001;Wang et al. 1999). Lipopolysaccharide (LPS) and Toll-like receptor-4 (TLR4) play critical roles in NF-κB activation in breast and ovarian cancers (Long et al. 2020;Wan et al. 2016;Woods et al. 2011). ...
Background
Studies have demonstrated that natural products, such as curcumin and artemisinin, possess anti-inflammatory effects, which can be beneficial for cancer treatment. Tehranolide, as a novel natural product, has a wide range of biological activities, including anti-cancer effects. However, many properties of Tehranolide, like its anti-inflammatory activity and its combination with curcumin, have not been investigated yet. This investigation examined the anti-inflammatory activity of Tehranolide, either alone or in combination with curcumin, via modulating the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and STAT3 (signal transducer and activator of transcription 3) signaling pathways in MDA-MB-231 and SKOV3, breast and ovarian cancer cell lines.
Methods
ELISA-based methods were employed to measure the pro-inflammatory cytokine levels and the NF-κB activity in lipopolysaccharide (LPS)-induced cells. The real-time PCR experiment and Griess test were performed to evaluate inducible nitric oxide synthase (iNOS) gene expression and nitrite levels, respectively. The STAT3 and NF-κB signaling pathways were investigated by Western blotting analysis. Tehranolide's anti-cancer activity was also assessed in a mouse model of breast cancer using the TUNEL (terminal deoxynucleotidyl transferase nick-end labeling) assay.
Results
Tehranolide diminished levels of pro-inflammatory cytokines in cancer cells. Additionally, it suppressed NF-κB DNA binding and STAT3 phosphorylation, reducing iNOS gene expression and nitrite production. Moreover, Western blotting showed that Tehranolide enhanced the inhibitory κB (IκBα) and Bcl-2 (B-cell lymphoma 2)-associated X (BAX) expression, and downregulated the expression of Bcl-2 proteins. Furthermore, the TUNEL assay demonstrated that Tehranolide induced apoptosis in a breast cancer mouse model. Curcumin potentiated all the anti-inflammatory effects of Tehranolide.
Conclusion
This investigation indicated for the first time that Tehranolide, either alone or in combination with curcumin, exerted its anti-inflammatory effects by suppressing NF-κB and STAT3 signaling pathways in SKOV3 and MDA-MB-231 cells.
... In response to DNA double-strand breaks (DSBs) generated by topoisomerase (topo) I and II poisons, IB-␣ is phosphorylated at Ser32 and Ser36 by the IB kinase (IKK) complex (6,10,15,37), which is formed by the IKK-1 and IKK-2 catalytic subunits and by a scaffold subunit termed IKK-␥/NEMO (32). As a consequence of phosphorylation, the IB-␣ protein is rapidly ubiquitinated on lysine residues 21 and 22 and degraded through the proteasome pathway, thereby allowing for migration of NF-B to the nucleus, where it regulates the expression of a variety of genes involved in cell survival (6,52). Consistent with this mechanism, inhibition of NF-B activity through ectopic expression of a degradationresistant mutant IB-␣ sensitized chemoresistant tumors to the topo I poison camptothecin (CPT) (51). ...
We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor B (NF-B) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladria-mycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90 rsk signaling cascade in a p53-independent fashion. In turn, p90 rsk interacts with the IB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-B activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-B pathway that opposes the apoptotic response following DNA damage.
... The transcription of multiple apoptosis-related genes has been found to be controlled by NF-κB [35][36][37][38] . NF-κB regulates the expression of two antiapoptotic members (BCL-XL and A1/Bfl-1) [39][40][41][42][43][44][45] , on the other hand, NF-κB2 suppresses the expression of the proapoptotic Bax protein 46 . The purpose of this research is to find the correlation between BCL2 and P52 in malignant cells of breast carcinoma. ...
... Nuclear factor kB (NF-kB) plays a major role in apoptosis by regulating the transcription of Bcl-2 [77]. Activation of the NF-kB pathway in PCa cells leads to Pca progression, metastasis, recurrence, and resistance [78,79]. ...
Simple Summary
The standard of care therapy for early prostate cancer (PCa) includes external beam radiation therapy (EBRT), brachytherapy, radical prostatectomy, active surveillance, or a combination approach. For advanced disease, androgen deprivation therapy (ADT) and other neoadjuvant therapies are considered. Nevertheless, castration-resistant prostate cancer (CRPC) develops in many patients. This instigated the development of novel therapeutic approaches using targeted therapies, including prostate cancer stem cell (PCSC)-targeted therapies. Here, we summarize the mechanisms of action of PCSC-targeted therapies and discuss avenues of future development.
Abstract
Prostate cancer (PCa) is the second-most commonly diagnosed cancer in men around the world. It is treated using a risk stratification approach in accordance with the National Comprehensive Cancer Network (NCCN) in the United States. The main treatment options for early PCa include external beam radiation therapy (EBRT), brachytherapy, radical prostatectomy, active surveillance, or a combination approach. In those with advanced disease, androgen deprivation therapy (ADT) is considered as a first-line therapy. However, the majority of cases eventually progress while receiving ADT, leading to castration-resistant prostate cancer (CRPC). The near inevitable progression to CRPC has spurred the recent development of many novel medical treatments using targeted therapies. In this review, we outline the current landscape of stem-cell-targeted therapies for PCa, summarize their mechanisms of action, and discuss avenues of future development.
... Several growth-factor and survival-factor receptors activate PI3K [94,95]. Stimulated by second messengers, phosphoinositide-dependent protein kinase-1 phosphorylates protein kinase B, resulting in activation of NF-κB and transcription of anti-apoptotic genes [96]. The PI3K inhibitor did not affect either the apoptosis-enhancing effect of GYY4137 or the apoptosisattenuating effect of cysteine (Figure 8), suggesting that both substances target signaling downstream of PI3K. ...
Impaired polymorphonuclear leukocyte (PMNL) functions contribute to increased infections and cardiovascular diseases in chronic kidney disease (CKD). Uremic toxins reduce hydrogen sulfide (H2S) levels and the anti-oxidant and anti-inflammatory effects of H2S. Its biosynthesis occurs as a side process of transsulfuration and in the disposal of adenosylhomocysteine, a transmethylation inhibitor and proposed uremic toxin. PMNL chemotaxis was measured by the under-agarose method, phagocytosis, and oxidative burst by flow cytometry in whole blood and apoptosis by determining DNA content by flow cytometry and morphological features by fluorescence microscopy. Sodium hydrogen sulfide (NaHS), diallyl trisulphide (DATS) and diallyl disulphide (DADS), cysteine, and GYY4137 were used as H2S-producing substances. Increased H2S concentrations did not affect chemotaxis and phagocytosis. NaHS primed PMNL oxidative burst activated by phorbol 12-myristate 13-acetate (PMA) or E. coli. Both DATS and cysteine significantly decreased E. coli-activated oxidative burst but had no effect on PMA stimulation. While NaHS, DADS, and cysteine attenuated PMNL apoptosis, GYY4137 decreased their viability. Experiments with signal transduction inhibitors suggest that the intrinsic apoptosis pathway is mainly involved in GYY4137-induced PMNL apoptosis and that GYY4137 and cysteine target signaling downstream of phosphoinositide 3-kinase.
... Several feedback mechanisms have been identified between ROS, caspases, JNK, and the NF-κB pathway [180,182]. The expression of anti-apoptotic genes such as Bcl-2 and caspase inhibitors are induced by NF-κB [189,190]. Moreover, NF-κB can transactivate multiple antioxidant genes, including manganese superoxide dismutase (MnSOD), Cu, Zn superoxide dismutase (SOD1), ferritin heavy chain (FHC), thioredoxin 1 (TRX1), TRX2, GSTP1, metallothionein-3 (MT3), NAD(P)H quinone dehydrogenase 1 (NQO1), heme oxygenase 1 (HO-1), glutathione peroxidase 1 (Gpx1), and dihydrodiol dehydrogenase (DDH1) [36]. ...
Glutathione-S-transferases (GSTs) are phase II detoxification isozymes that conjugate glutathione (GSH) to xenobiotics and also suppress redox stress. It was suggested that GSTs have evolved not to enhance their GSH affinity, but to better interact with and metabolize cytotoxic nitric oxide (NO). The interactions between NO and GSTs involve their ability to bind and store NO as dinitrosyl-dithiol iron complexes (DNICs) within cells. Additionally, the association of GSTP1 with inducible nitric oxide synthase (iNOS) results in its inhibition. The function of NO in vasodilation together with studies associating GSTM1 or GSTT1 null genotypes with preeclampsia, additionally suggests an intriguing connection between NO and GSTs. Furthermore, suppression of c-Jun N-terminal kinase (JNK) activity occurs upon increased levels of GSTP1 or NO that decreases transcription of JNK target genes such as c-Jun and c-Fos, which inhibit apoptosis. This latter effect is mediated by the direct association of GSTs with MAPK proteins. GSTP1 can also inhibit nuclear factor kappa B (NF-κB) signaling through its interactions with IKKβ and Iκα, resulting in decreased iNOS expression and the stimulation of apoptosis. It can be suggested that the inhibitory activity of GSTP1 within the JNK and NF-κB pathways may be involved in crosstalk between survival and apoptosis pathways and modulating NO-mediated ROS generation. These studies highlight an innovative role of GSTs in NO metabolism through their interaction with multiple effector proteins, with GSTP1 functioning as a “good Samaritan” within each pathway to promote favorable cellular conditions and NO levels.
... The gene encoding BFL-1/A1 is a direct target of NF-kB transcription factors [86]. The PI3K and JAK/STAT signalling pathways have also been reported to regulate the expression of BFL-1/A1. ...
Acquired resistance to cell death is a hallmark of cancer. The BCL-2 protein family members play important roles in controlling apoptotic cell death. Abnormal over-expression of pro-survival BCL-2 family members or abnormal reduction of pro-apoptotic BCL-2 family proteins, both resulting in the inhibition of apoptosis, are frequently detected in diverse malignancies. The critical role of the pro-survival and pro-apoptotic BCL-2 family proteins in the regulation of apoptosis makes them attractive targets for the development of agents for the treatment of cancer. This review describes the roles of the various pro-survival and pro-apoptotic members of the BCL-2 protein family in normal development and organismal function and how defects in the control of apoptosis promote the development and therapy resistance of cancer. Finally, we discuss the development of inhibitors of pro-survival BCL-2 proteins, termed BH3-mimetic drugs, as novel agents for cancer therapy.
... PPI networks of intrinsic apoptosis have been well studied [11] and specific interactions between pro-and anti-apoptotic proteins have been discovered [7]. More importantly, well-known cancer pathways, such as TP53, NF-κB, and TGF-β pathways, are closely linked to the BCL2 family [12][13][14][15][16][17][18][19][20], which implies that these pathways could be potential targets for combination treatment with apoptosis modulating agents. Given the BCL2 family PPIs and their association with cancer pathways, we speculated that the transcriptional signatures reflecting the complicated regulatory networks of the BCL2 family exist and may guide individualized treatment strategies targeting the BCL2 family. ...
Background
Although anti-apoptotic proteins of the B-cell lymphoma-2 (BCL2) family have been utilized as therapeutic targets in acute myeloid leukaemia (AML), their complicated regulatory networks make individualized therapy difficult. This study aimed to discover the transcriptional signatures of BCL2 family genes that reflect regulatory dynamics, which can guide individualized therapeutic strategies.
Methods
From three AML RNA-seq cohorts (BeatAML, LeuceGene, and TCGA; n = 451, 437, and 179, respectively), we constructed the BCL2 family signatures (BFSigs) by applying an innovative gene-set selection method reflecting biological knowledge followed by non-negative matrix factorization (NMF). To demonstrate the significance of the BFSigs, we conducted modelling to predict response to BCL2 family inhibitors, clustering, and functional enrichment analysis. Cross-platform validity of BFSigs was also confirmed using NanoString technology in a separate cohort of 47 patients.
Results
We established BFSigs labeled as the BCL2, MCL1/BCL2, and BFL1/MCL1 signatures that identify key anti-apoptotic proteins. Unsupervised clustering based on BFSig information consistently classified AML patients into three robust subtypes across different AML cohorts, implying the existence of biological entities revealed by the BFSig approach. Interestingly, each subtype has distinct enrichment patterns of major cancer pathways, including MAPK and mTORC1, which propose subtype-specific combination treatment with apoptosis modulating drugs. The BFSig-based classifier also predicted response to venetoclax with remarkable performance (area under the ROC curve, AUROC = 0.874), which was well-validated in an independent cohort (AUROC = 0.950). Lastly, we successfully confirmed the validity of BFSigs using NanoString technology.
Conclusions
This study proposes BFSigs as a biomarker for the effective selection of apoptosis targeting treatments and cancer pathways to co-target in AML.
... A growing body of evidence indicates abnormal activation of the NFκB pathway in multiple malignancies, suggesting a putative role for NFκB in tumorigenesis [134,[138][139][140][141] and chemotherapy resistance [142]. ...
The most common breast cancer (BC) subtypes are hormone-dependent, being either estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), or both, and altogether comprise the luminal subtype. The mainstay of treatment for luminal BC is endocrine therapy (ET), which includes several agents that act either directly targeting ER action or suppressing estrogen production. Over the years, ET has proven efficacy in reducing mortality and improving clinical outcomes in metastatic and nonmetastatic BC. However, the development of ET resistance promotes cancer survival and progression and hinders the use of endocrine agents. Several mechanisms implicated in endocrine resistance have now been extensively studied. Based on the current clinical and pre-clinical data, the present article briefly reviews the well-established pathways of ET resistance and continues by focusing on the three most recently uncovered pathways, which may mediate resistance to ET, namely receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK), nuclear factor kappa B (NFκB), and Notch. It additionally overviews the evidence underlying the approval of combined therapies to overcome ET resistance in BC, while highlighting the relevance of future studies focusing on putative mediators of ET resistance to uncover new therapeutic options for the disease.
... Additionally, the constitutive expression and activation of NF-κB has been observed in several types of human cancer, leading to increased cell proliferation, blocking apoptosis and promoting angiogenesis, invasiveness, and metastatic capacity ( [81]; for a review, see [82]). ...
Carbon monoxide (CO) is one of the most common causes of inhalation poisoning worldwide. However, it is also well known that CO is produced endogenously in the heme degradation reaction catalyzed by heme oxygenase (HO) enzymes. HO catalyzes the degradation of heme to equimolar quantities of CO, iron ions (Fe2+), and biliverdin. Three oxygen molecules (O2) and the electrons provided by NADPH-dependent cytochrome P450 reductase are used in the reaction. HO enzymes comprise three distinct isozymes: the inducible form, heme oxygenase-1 (HO-1); the constitutively expressed isozyme, heme oxygenase-2 (HO-2); and heme oxygenase-3 (HO-3), which is ubiquitously expressed but possesses low catalytic activity. According to some authors, HO-3 is rather a pseudogene originating from the HO-2 transcript, and it has only been identified in rats. Therefore, cellular HO activity is provided by two major isoforms—the inducible HO-1 and the constitutively expressed HO-2. For many years, endogenously generated CO was treated as a by-product of metabolism without any serious physiological or biochemical significance, while exogenous CO was considered only as an extremely toxic gas with lethal effects. Research in recent years has proven that endogenous and exogenous CO (which may be surprising, given public perceptions) acts not only as an agent that affects many intracellular pathways, but also as a therapeutic molecule. Hence, the modulation of the HO/CO system may be one option for a potential therapeutic strategy. Another option is the administration of CO by exogenous inhalation. As alternatives to gas administration, compounds known as CO-releasing molecules (CORMs) can be administered, since they can safely release CO in the body. The aim of this article is to provide a brief overview of the physiological and biochemical properties of CO and its therapeutic potential.
... it is well known that more than caspase cascade activation, Trail can also modulate nF-κB signaling (51,52), which is controversial as nF-κB can upregulate the expression of survival factors such as members of the apoptosis family inhibitor and Bcl-xL (53). Conversely, TRAIL-dependent nF-κB activation triggers apoptosis rather than survival in epithelial cell lines and T cells (54,55). ...
As the understanding of cancer grows, new therapies have been proposed to improve the well-known limitations of current therapies, whose efficiency relies mostly on early detection, surgery and chemotherapy. Mesenchymal stem cells (MSCs) have been introduced as a promissory and effective therapy. This fact is due to several useful features of MSCs, such as their accessibility and easy culture and expansion in vitro, and their remarkable ability for 'homing' towards tumors, allowing MSCs to exert their anticancer effects directly into tumors. Additionally, MSCs offer the practicability of being genetically engineered to carry anticancer genes, increasing their specificity and efficacy for fighting tumors. In the present study, the antitumoral efficacy and post-implant survival of mice bearing lymphomas implanted intratumorally were determined using mouse bone marrow-derived (BM)-MSCs transduced with soluble TRAIL (sTRAIL), full length TRAIL (flTRAIL), or interferon β (IFNβ), naïve BM-MSCs, or combinations of these. The percentage of surviving mice was determined once all not-implanted mice succumbed. It was found that the percentage of surviving mice implanted with the combination of MSCs-sTRAIL and MSCs-IFN-β was 62.5%. Lymphoma model achieved 100% fatality in the non-treated group by day 41. On the other hand, the percentage of surviving mice implanted with MSCs-sTRAIL was 50% and with MSCs-INFβ 25%. All the aforementioned differences were statistically significant (P<0.05). In conclusion, all implants exhibited tumor size reduction, growth delay, or apparent tumor clearance. MSCs proved to be effective anti-lymphoma agents; additionally, the combination of soluble TRAIL and IFN-β resulted in the most effective antitumor and life enlarging treatment, showing an additive antitumoral effect compared with individual treatments.
... The critical point of contention is whether NF-kB activities protect neurons after cerebral ischemia or whether its role as an inflammatory mediator worsens neuronal damage. NF-kB promotes cell survival by regulating caspase inhibitors, TNF-receptor associate factor (TRAF), and Bcl-2 family members (Wang et al. 1998(Wang et al. , 1999. However, these anti-apoptotic and subsistence genes can code for cell death resistance during pathological circumstances (Yang, Tao, and Chen 2007;Mettang et al. 2018). ...
Vitamin D deficiency has been linked to several major chronic diseases, such as cardiovascular and neurodegenerative diseases, diabetes, and cancer, linked to oxidative stress, inflammation, and aging. Vitamin D deficiency appears to be particularly harmful to the cardiovascular system, as it can cause endothelial dysfunctioning and vascular abnormalities through the modulation of various downstream mechanisms. As a result, new research indicates that therapeutic approaches targeting vitamin D inadequacies or its significant downstream effects, such as impaired autophagy, abnormal pro-inflammatory and pro-oxidant reactions, may delay the onset and severity of major cerebrovascular disorders such as stroke and neurologic malformations. Vitamin D modulates the various molecular pathways, i.e., Nitric Oxide, PI3K-Akt Pathway, cAMP pathway, NF-kB Pathway, Sirtuin 1, Nrf2, FOXO, in cerebrovascular disorder. The current review shows evidence for vitamin D's mitigating or slowing the progression of these cerebrovascular disorders, which are significant causes of disability and death worldwide.
... Fascinatingly, when we tested different anti-apoptotic genes, only Bcl-2A1 was induced by GM-CSF and not by M-CSF, confirming previous results [62]. Bcl2-A1 is a hematopoietic-specific protein and protects cells from apoptosis induced by a variety of apoptotic stimuli, such as DNAdamaging agents [63]. In response to GM-CSF, the BCL2-A1 gene is induced and is a direct transcriptional target of nuclear factor NF-κB [44]. ...
At inflammatory loci, pro-inflammatory activation of macrophages produces large amounts of reactive oxygen species (ROS) that induce DNA breaks and apoptosis. Given that M-CSF and GM-CSF induce two different pathways in macrophages, one for proliferation and the other for survival, in this study we wanted to determine if these growth factors are able to protect against the DNA damage produced during macrophage activation. In macrophages treated with DNA-damaging agents we found that GM-CSF protects better against DNA damage than M-CSF. Treatment with GM-CSF resulted in faster recovery of DNA damage than treatment with M-CSF. The number of apoptotic cells induced after DNA damage was higher in the presence of M-CSF. Protection against DNA damage by GM-CSF is not related to its higher capacity to induce proliferation. GM-CSF induces differentiation markers such as CD11c and MHCII, as well as the pro-survival Bcl-2A1 protein, which make macrophages more resistant to DNA damage.
... Levels of both BCL-2 and TRAF-6 proteins are elevated in inflammatory disorder conditions such as osteoarthritis and rheumatoid arthritis [18,19]. Also, BCL-2 and TRAF-6 proteins are capable of activating the NF-κB/MAPK pathway [20,21]. In addition to this AGEs have been reported to increase the levels of TRAF-6 [22] and BCL-2 [23] both of which are the important members of mitogenactivated protein kinase (MAPK) and Nuclear factor kappa-B (NF-κB) family proteins, hence MAPK and NF-κB cascades are regarded as potential targets for treating OA. ...
Background:
Osteoarthritis (OA) is degenerative joint disorder mainly characterized by long-term pain with limited activity of joints, the disease has no effective preventative therapy. Rutin (RUT) is a flavonoid compound, present naturally. The flavonoid shows range of biological activities such as anti-inflammatory and anti-cancer effect. We screened RUT for its activity against osteoarthritis with in vivo and in vitro models of osteoarthritis.
Methods:
Animal model of OA was developed using C57BL/6 mice by surgical destabilization of medial meniscus. For in vitro studies the human articular cartilage tissues were used which were collected from osteoarthritis patients and were processed to isolate chondrocytes. The chondrocytes were submitted to advanced glycation end products (AGEs) for inducing osteoarthritis in vitro. Cell viability was done by CCK-8 assay, ELISA analysis for MMP13, collage II, PGE2, IL-6, TNF-α, ADAMTS-5 and MMP-13. Western blot analysis was done for expression of proteins and in silico analysis was done by docking studies.
Results:
Pretreatment of RT showed no cytotoxic effect and also ameliorated the AGE mediated inflammatory reaction on human chondrocytes in vitro. Treatment of RT inhibited the levels of COX-2 and iNOS in AGE exposed chondrocytes. RT decreased the AGE mediated up-regulation of IL-6, NO, TNF-α and PGE-2 in a dose dependent manner. Pretreatment of RT decreased the extracellular matrix degradation, inhibited expression of TRAF-6 and BCL-2 the NF-κB/MAPK pathway proteins. The treatment of RT in mice prevented the calcification of cartilage tissues, loss of proteoglycans and also halted the narrowing of joint space is mice subjected to osteoarthritis. The in-silico analysis suggested potential binding affinity of RT with TRAF-6 and BCL-2.
Conclusion:
In brief RT inhibited AGE-induced inflammatory reaction and also degradation of ECM via targeting the NF-κB/MAPK pathway proteins BCL-2 and TRAF-6. RT can be a potential molecule in treating OA.
... Despite the increased expression of pro-apoptotic Bcl family members such as BID in ICOS + CD38 + cTfh, there was a concomitant increase in the expression of pro-survival factors BCL2A1 and MCL1 that strongly correlated with TNF-NF-kB GSVA scores in ICOS + CD38 + cTfh at day 7, suggesting a TNF-NF-kB-dependent survival signal in activated cTfh. These genes have NF-kB binding sites in their respective promoter sequences 70,71 linking TNF signaling to a potential pro-survival circuit in these cells. Moreover, TNFR1 expression in ICOS + CD38 + cTfh at day 7 positively correlated with neutralizing antibody production. ...
Humoral immune responses are dysregulated with aging, but the cellular and molecular pathways involved remain incompletely understood. In particular, little is known about the effects of aging on T follicular helper (Tfh) CD4 cells, the key cells that provide help to B cells for effective humoral immunity. We performed transcriptional profiling and cellular analysis on circulating Tfh before and after influenza vaccination in young and elderly adults. First, whole-blood transcriptional profiling shows that ICOS⁺CD38⁺ cTfh following vaccination preferentially enriches in gene sets associated with youth versus aging compared to other circulating T cell types. Second, vaccine-induced ICOS⁺CD38⁺ cTfh from the elderly had increased the expression of genes associated with inflammation, including tumor necrosis factor-nuclear factor κB (TNF-NF-κB) pathway activation. Finally, vaccine-induced ICOS⁺CD38⁺ cTfh display strong enrichment for signatures of underlying age-associated biological changes. These data highlight the ability to use vaccine-induced cTfh as cellular “biosensors” of underlying inflammatory and/or overall immune health.
... [57] In addition, CYFRA 21-1 is a specific tumour marker associated with poor prognosis and worsening overall survival of NSCLC. [58] To express the mechanism of urethane-induced lung cancer, it may be introduced through persistent activation of Nrf2, [59,60] NF-kB [61] and anti-apoptotic protein Bcl-2 that is indicated by prevention of cellular apoptosis, [62] stimulation of ICAM-1 expression due to activation of NF-kB, [63] leads to activation of MMP-2 [64] and accompanied with induction of cyclin D1 expression due to Bcl-2 in the distinct independent pathway. [65] The results of this study showed no significant changes in animals treated with sildenafil except for a marked increase in cGMP level Figure 5 Effect of sildenafil on lung NF-κB, Caspase-3 and Nrf2 contents in urethane-induced lung cancer in BALB/c mice. ...
Objectives:
Lung cancer is one of the most frequent types of cancers that lead to death. Sildenafil is a potent inhibitor of phosphodiesterase-5 and showed potential anticancer effects, which has not yet been fully evaluated. Thus, this study aims to investigate the potential anticancer effect of sildenafil in urethane-induced lung cancer in BALB/c mice.
Methods:
Five-week-old male BALB/c mice were treated with either (i) normal saline only, (ii) sildenafil only 50 mg kg-1/ P.O every other day for the last four successive weeks, (iii) urethane 1.5 gm kg-1 i.p (at day 1 and day 60), (iv) carboplatin after urethane induction, or (v) sildenafil after urethane induction.
Key findings:
It was shown that sildenafil significantly increased the levels of cGMP and Caspase-3 with a reduction of NF-κB, Bcl-2, Cyclin D1, intercellular adhesion molecule 1, matrix metalloproteinase-2 levels and normalisation of Nrf2 along with pronounced improvement in the histological patterns.
Conclusions:
These results indicated that sildenafil markedly induces cell cycle arrest, apoptosis and inhibits the metastatic activity through activation of cyclic guanosine monophosphate/protein kinase G pathway and down-regulation of cyclin D1 and nuclear factor kappa light chain enhancer of activated B cells with downstream anti-apoptotic gene Bcl-2, which underscores the critical importance of future using sildenafil in the treatment of lung cancer.
... 5,6 NF-κB inhibits cytochrome-c release by provoking B-cell lymphoma 2 (BCL-2) and X-linked inhibitor of apoptosis protein (XIAP) expression, as well as inducing the expression of TNF receptor-associated factor (TRAF)1, TRAF2, a cellular inhibitor of apoptosis protein (c-IAP)1, c-IAP2, and caspase-8 inactivation as a result. 7,8 NF-κB has a key role in the inflammatory responses and regulates lots of proinflammatory genes. Its activation triggers the production of inflammatory cytokines (e.g. ...
The T-cell immunoglobulin and mucin-3 (TIM-3)/galectin-9 (Gal-9) autocrine loop is an indispensable signaling in acute myeloid leukemia (AML) cells, which induces their self-renewal through activation of nuclear factor-kappa b (NF-kB) and β-catenin pathways. In this study, we evaluated the effects of oridonin and doxorubicin on the TIM-3/Gal-9 autocrine loop. We also evaluated oridonin anti-inflammatory and anti-cancer properties on U937 cells, as an AML cell line in comparison to doxorubicin as a common anthracycline drug for AML treatment. Cell counting kit-8 (CCK-8) was applied to evaluate the cytotoxicity of oridonin and doxorubicin on U937 cells and also to determine the impact of galectin-9 (Gal-9) on their proliferation. The effects of oridonin and doxorubicin on Gal-9, TIM-3, and interleukin-1β (IL-1β) gene expression were determined by real-time polymerase chain reaction (RT-PCR). The Gal-9 secretion level was measured by enzyme-linked immunosorbent assay (ELISA) and activation of NF-kB pathway was assessed by western blotting. In a dose-dependent manner, oridonin and doxorubicin were capable to eradicate U937 cells while Gal-9 expanded them. Following the treatment of U937 cells with oridonin, the expression of Gal-9, TIM-3, and IL-1β genes was down-regulated, and the Gal-9 secretion and NF-kB phosphorylation were diminished, whereas doxorubicin increased all of these factors. Doxorubicin is a common treatment agent in AML, but it may induce inflammation and up-regulate the TIM3/Gal-9 autocrine loop, consequently can enhance the possibility of disease relapse. Meanwhile, oridonin is capable to inhibit the essential signaling pathways in AML cells and reduce the inflammation and expansion of tumor cells and postpone AML recurrence.
... Indeed, NF-κB is an ideal candidate for the development of anti-cancer drugs, since the triggering of NF-κB in tumor cells has been shown to block apoptosis and induce proliferation [63,64]. Pro-inflammatory TME can induce the mobilization of NF-κB [65], and the release of NF-κB leads to resistance to chemotherapeutic agents [66,67], and most importantly, molecules involved in tumor initiation, tumor promotion and metastasis are regulated by NF-κB [68]. For this reason, compounds that can block the triggering of the transcription factors NF-κB have the potential to prevent tumor initiation, promotion and metastasis. ...
The interaction between tumor cells and the tumor microenvironment (TME) is an important process for the development of tumor malignancy. Modulation of paracrine cross-talk could be a promising strategy for tumor control within the TME. The exact mechanisms of multi-targeted compound resveratrol are not yet fully understood. Whether resveratrol can modulate paracrine signal transduction-induced malignancy in the multicellular-TME of colorectal cancer cells (CRC) was investigated. An in vitro model with 3D-alginate HCT116 cells in multicellular-TME cultures (fibroblast cells, T-lymphocytes) was used to elucidate the role of TNF-β, Sirt1-ASO and/or resveratrol in the proliferation, invasion and cancer stem cells (CSC) of CRC cells. We found that multicellular-TME, similar to TNF-β-TME, promoted proliferation, colony formation, invasion of CRC cells and enabled activation of CSCs. However, after co-treatment with resveratrol, the malignancy of multicellular-TME reversed to HCT116. In addition, resveratrol reduced the secretion of T-lymphocyte/fibroblast (TNF-β, TGF-β3) proteins, antagonized the T-lymphocyte/fibroblast-promoting NF-κB activation, NF-κB nuclear translocation and thus the expression of NF-κB-promoting biomarkers, associated with proliferation, invasion and survival of CSCs in 3D-alginate cultures of HCT116 cells induced by TNF-β-or multicellular-TME, but not by Sirt1-ASO, indicating the central role of this enzyme in the anti-tumor function of resveratrol. Our results suggest that in vitro multicellular-TME promotes crosstalk between CRC and stromal cells to increase survival, migration of HCT116 and the resveratrol/Sirt1 axis suppresses this loop by modulating paracrine agent secretion and NF-κB signaling. Fibroblasts and T-lymphocytes are promising targets for resveratrol in the prevention of CRC metastasis.
... In this case, the mechanisms o f activation are controversial at the moment (Delhase et al., 2000). However, it seems possible that induction o f antiapoptotic genes such as inhibitor-of-apoptosis proteins (lAPs) might confer a mode o f regulation (Wang et al., 1999;Wang et al., 1998a). ...
Class I PI3-kinases possess an intrinsic protein kinase activity in addition to their lipid kinase activity. The best characterised target of this activity is the Ser608 of the p85 regulatory subunit in the case of the α isoform of the p110 catalytic subunit or the catalytic subunit itself, in the case of the β, γ and δ isoforms, respectively. Furthermore, it has been shown that Ser608 phosphorylation results in downregulation of the lipid kinase activity of the enzyme in vitro. The implications of this finding are very important, as it could provide a mechanism for the regulation of the lipid kinase activity of the enzyme in vivo. By generating phosphospecific antibodies against Ser608, we have been able to demonstrate that Ser608 can be phosphorylated in vivo. We have also showed that mutation of Ser608 to either alanine or glutamic acid interferes with the integrity of the p85α/p110α heterodimer and this likely explains the reduced lipid kinase activity associated with S608 phosphorylation. Also, we have demonstrated that certain class I PI3-kinases phosphorylate novel exogenous substrates, namely the translational regulator 4EBP1 and the small GTPase H-Ras in vitro. In an effort to discriminate between the two activities we have employed two different approaches. First, we have found that certain methylxanthines inhibit the lipid and the protein kinase activities but with very dinstict potencies. Also, a panel of synthetic peptides derived from class II MHC sequences activate the lipid but not the protein kinase activity of heterodimeric PI3-kinases. These findings demonstrate that each one of the two activities can be modulated independently from the other. Second, by mutating certain aminoacids in the activation loop of p110α and p110β, we have created enzyme versions that lack the lipid kinase but retain protein kinase activity. Both of these approaches could be exploited to assign specific functions to each of the lipid and protein kinase activities in cells.
... Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis. Studies have demonstrated that Bcl-2 family proteins can positively and negatively regulate apoptosis by regulating anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax 40,50 . Our RPPA data using patient-derived GBM cells showed that the fold change of Bcl-2 relative to control was 0.71, 1.26, 0.88 times higher in co-treated group, TMZ alone, Bay 11-7082 alone, respectively (Fig. 3b). ...
Glioblastoma (GBM) is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy. GBM tumors show nuclear factor-κB (NF-κB) activity that has been associated with tumor formation, growth, and increased resistance to therapy. We investigated the effect of NF-κB inhibitor BAY 11-7082 with Temozolomide (TMZ) on the signaling pathways in GBM pathogenesis. GBM cells and patient-derived GBM cells cultured in 3D microwells were co-treated with BAY 11-7082 and TMZ or BAY 11-7082 and TMZ alone, and combined experiments of cell proliferation, apoptosis, wound healing assay, as well as reverse-phase protein arrays, western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells. The results revealed that the co-treatment significantly altered cell proliferation by decreasing GBM viability, suppressed NF-κB pathway and enhanced apoptosis. Moreover, it was found that the co-treatment of BAY 11-7082 and TMZ significantly contributed to a decrease in the migration pattern of patient-derived GBM cells by modulating actin cytoskeleton pathway. These findings suggest that in addition to TMZ treatment, NF-κB can be used as a potential target to increase the treatment’s outcomes. The drug combination strategy, which is significantly improved by NF-κB inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatment.
... Caspase-3 plays a vital role to induce apoptosis (Hussein, 2005). The activation of caspase-3 from its zymogen form (procaspase-3) is probably triggered through two mechanisms including the release of cytochrome c (intracellular way) and activation of caspase-8 (extracellular way) (Wang et al., 1999). Induction of apoptosis is considered an anti-cancer mechanism for many bio-active agents. ...
Objective:
Silymarin is a herbal extract containing flavonolignans, and it has inhibitory effects against the growth of different cancer cell lines by inducing apoptosis. Toll-like receptors are suggested as a novel and attractive target to treat cancer. The current study aimed at examining the mechanism of silymarin-induced apoptosis in Ramos cells and investigating its effects on TLR8 expression.
Materials and methods:
The half maximal inhibitory concentration (IC50) of silymarin in Ramos cells was determined via MTT viability test while the type of cell death was tested by annexin V/propidium iodide (PI) double staining method. The activity of caspase-3 and expression of TLR8 were measured in a time-dependent manner (in IC50) by colorimetric assay and real-time polymerase chain reaction (RT-PCR), respectively.
Results:
The results of MTT showed that IC50 of silymarin in Ramos cells was 100 μg/ml after 48 hr treatment (p<0.01). Flow cytometry by annexin V/PI, showed that silymarin induced early/late apoptosis in this cell line (p<0.05 to p<0.01). In addition, the caspase-3 colorimetric method showed that caspase-3 increased in the Ramos cell line after treatment (p<0.01). This treatment led to a reduction in TLR8 mRNA expression in a time-dependent manner (p<0.01).
Conclusion:
The results indicated a new mechanism in the anticancer activity of Toll-like receptor (TLR) signaling after silymarin treatment in Ramos cancer cell line. This plant could be used to develop anticancer agents inhibiting TLRs.
... and BCL2A1.31,32 Importantly, BET inhibitors were shown to downregulate BCL2A1 proteins, which are not inhibited by BCL2, BCLXL and MCL1 inhibitors.33,34 2 | MATERIALS AND METHODS2.1 | Cell linesThe panel of human melanoma cell lines used in the drug screen(Table S1)were generated by Professor Nicholas Hayward, QIMR Berghofer, Queensland. ...
The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti‐apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra‐terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti‐apoptotic targets regulated by NF‐kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro‐apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I‐BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3‐only protein, BIM, and downregulated anti‐apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT‐199 or ABT‐263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I‐BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.
... 31 It has been reported that Bcl-2 is directly transcriptionally targeted by NF-κB, leading to suppression of apoptosis. 32,33 Moreover, it has been found that the activation of NF-κB suppresses caspase-8 activation through the induction of cIAP1 and cIAP2. 34,35 At this point, the Ras signaling pathway is connected to the anti-apoptosis pathway, which might be the major mechanism of fibrous epulis. ...
Background
Epulis has a tumor‐like appearance but is considered to be a massive reactive lesion rather than a true neoplasia. Limited information about the pathogenesis of epulis is available. The purpose of our study was to identify potential signaling pathways in fibrous epulis through transcriptome profiling.
Methods
Differentially expressed genes (DEGs) between fibrous epulis lesions and normal gingival tissues were detected using RNA sequencing (RNAseq). The expression levels of eighteen genes were validated using quantitative real‐time PCR (qRT‐PCR).
Results
RNAseq identified 533 upregulated genes and 732 downregulated genes. The top 10 upregulated genes were IL11, OSM, MMP3, KRT75, MMP1, IL6, IL1B, IL24, SP7, and ADGRG3. The top 10 downregulated genes were BCHE, TYR, DCT, KRT222, RP11‐507K12.1, COL6A5, PMP2, GFRA1, SCN7A, and CDH19. KEGG pathway analysis further indicated that the DEGs were enriched in “Pathways in cancer” and the “Ras signaling pathway”. quantitative real‐time PCR verified that the expression levels of SOS1, HRAS, PIK3CA, AKT3, IKBKA, IKBKB, NFKB1, BCL2, BCL2L1, XIAP, BIRC2, and BIRC3 were increased significantly.
Conclusions
The current transcriptomic profiling study reveals that in fibrous epulis, RAS‐PI3K‐AKT‐NF‐κB pathway transcriptionally regulates the expression of BCL2 family and IAP family genes, leading to increased proliferation and apoptosis inhibition.
... Bfl-1 protein is a member of the six characterized antiapoptotic Bcl-2 family including Bcl-2, Bcl-X L , Mcl-1, Bcl-W, Bfl-1, and Bcl-B ( Wang et al., 2000, Reed., 1998. Bfl-1 is a direct transcriptional target of nuclear factor-kappa B (NF-kB) ( Wang et al., 1999;Zong et al., 1999), Researchers ( Cheng et al., 2000, Kitada et al., 2003 have shown that the anti-apoptotic Bcl-2 family proteins including Bfl-1 are over expressed in many cancers. This over expression is responsible for tumor progression and resistance to chemotherapeutic drugs ( Morales et al., 2005). ...
The release of cytochrome c into the cytoplasm via the mitochondrial membrane represents a significant step in programmed cell death. Bcl-2 family proteins play a crucial role as they regulate mitochondrial membrane permeability to cytochrome c. Bfl-1 belongs to the antiapoptotic subfamily of Bcl-2 protein which forms inhibitory heterodimeric complex with Bid thereby preventing the role of Bid in the disruption of Bax/Bcl-X L and Bak/Bcl-X L heterodimeric assembly critical for Bax and Bak dimerization in membrane permeability to cytochrome c. This Bfl-1 intracellular attitude connotes one major mechanism of cellular immortality in cancer cells. Other role played by Bfl-1 includes regulation of immunity, neutrophil development, maturation of B-cells and in post allergic mast cell. Cancer cells overexpress Bfl-1. Here, we hypothesize that cancer cells also overexpress Bid due to accumulated mutation but not to a threshold commensurate with the overexpressed Bfl-1. Therefore, using inhibitors of Bfl-1 and mimetic of Bid/BH3-only domain may represent one way to subvert apoptotic resistance in cancer. Sadly, Bfl-1 selective inhibitors remained undrugged. However, synergistic inhibition of Bfl-1 and other co-dependent proteins may also represent a major breakthrough in overcoming apoptotic resistance.
... c-FLIP is a short-lived protein and if it is not replenished by NFκB-dependent transcription, unprocessed CASPASE 8 undergoes autocatalysis to generate a p18/p10 tetrameric complex that initiates the apoptotic cascade. Other pro-survival molecules induced by NFκB includes members of the BCL2 family and several components of the TNF receptor 1 (TNFR1) signaling complex such as cIAP1/2, TRAF2 and A20 (Wang et al., 1998(Wang et al., , 1999Lee et al., 2000;He and Ting, 2002). Another checkpoint was discovered in 2007 and this was shown to be dependent on the non-degradative ubiquitination of the TNF signaling molecule RIPK1 but did not depend on NFκB-mediated transcription (O'Donnell et al., 2007). ...
The clinical success of biologics that inhibit TNF (Tumor Necrosis Factor) in inflammatory bowel diseases (IBD), psoriasis and rheumatoid arthritis (RA) has clearly established a pathogenic role for this cytokine in these inflammatory disorders. TNF binding to its receptors activates NFκB and MAPK signaling, inducing the expression of downstream pro-inflammatory genes. This is thought to be the primary mechanism by which TNF elicits inflammation. TNF is also a well-known trigger of caspase-dependent apoptosis or caspase-independent necroptosis. Whether cell death has any role in TNF-mediated inflammation has been less clear. Emerging data from animal models now suggest that cellular demise caused by TNF may indeed provoke inflammation. The default response of most cells to TNF stimulation is survival, rather than death, due to the presence of two sequential cell death checkpoints. The early checkpoint is transcription-independent involving the non-degradative ubiquitination of RIPK1 to prevent RIPK1 from becoming a death-signaling molecule. The later checkpoint requires the induction of pro-survival genes by NFκB-mediated transcription. When the early checkpoint is disrupted, RIPK1 initiates cell death and we suggest the term ripoptocide to describe this manner of death (encompassing both apoptosis and necroptosis). The sensitivity of a cell to ripoptocide is determined by the balance between regulatory molecules that enforce and those that disassemble the early checkpoint. As there is evidence suggesting that ripoptocide is inflammatory, individuals may develop inflammation due to ripoptocide brought about by genetic, epigenetic or post-translational alteration of these checkpoint regulators. For these individuals, drugs that reinforce the early checkpoint and inhibit ripoptocide could be useful in ameliorating inflammation.
Periodontitis ve osteoporoz, yaşlanan nüfus için önemli halk sağlığı sorunları oluşturan yaygın inflamasyonla ilişkili durumlardır. Periodontal hastalıklar, ağırlıklı olarak dento-gingival dokulardaki disbiyozis ve inflamatuvar nitelikli bir dizi bozukluktan oluşur. Osteoporoz, artan kırık riski ile birlikte kemik mineral yoğunluğu (KMY)’ nun ve yapısının bozulması ile karakterize, yaşa bağlı bir kemik hastalığıdır. Bu derlemede, osteoporoz ve periodontitis arasındaki ilişki hakkında bir güncelleme sağlamak için son 25 yıldaki literatür değerlendirilip, bunların mekanistik bağlantılarının, ortak risk faktörlerinin ve terapötik etkilerinin tartışılması amaçlanmaktadır. Sistemik ve alveolar kemik kaybı (AKK) arasındaki ilişkiyi inceleyen klinik çalışmalarda, 1996-2020 yılları arasındaki 10 araştırma, KMY ile AKK arasında ters bir korelasyon olduğunu ortaya koymuştur. Alveolar kemik kaybının radyografik olarak değerlendirilmesi önemli bir kriter olsa da, klinik ataçman kaybı (KAK); hastanın yaşamı boyunca periodontitis prognozunu yansıtan, teşhisi ve evrelendirilmesi için kritik bir ölçümdür. 1995-2020 yılları arasındaki 23 çalışmadan 17'si KAS ile osteoporoz arasında anlamlı bir ilişki olduğunu ortaya koymuştur. Her iki hastalıkta yaşa bağlı oksidatif stres ve yaşlanma, proinflamatuvar doku mikroçevresini yönlendiren ve kemik yeniden şekillenme sürecinin bozulmasına neden olan altta yatan mekanizmalardır. D vitamini eksikliği ve sigara kullanımı gibi ortak risk faktörleri bu mekanistik ilişkide önemli rol oynamaktadır. Bu faktörlerin ve aralarındaki etkileşimin anlaşılması, disiplinler arası yönetimi ve her iki hastalığa yönelik potansiyel terapötikleri incelemek için iyi kontrollü uzun dönem çalışmalar yapılmasını gerektirmektedir.
Immune checkpoint inhibitors (ICIs) are specialized monoclonal antibodies (mAbs) that target immune checkpoints and their ligands, counteracting cancer cell-induced T-cell suppression. Approved ICIs like cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), its ligand PD-L1, and lymphocyte activation gene-3 (LAG-3) have improved cancer patient outcomes by enhancing anti-tumor responses. However, some patients are unresponsive, and others experience immune-related adverse events (irAEs), affecting organs like the lung, liver, intestine, skin and now the cardiovascular system. These cardiac irAEs include conditions like myocarditis, atherosclerosis, pericarditis, arrhythmias, and cardiomyopathy. Ongoing clinical trials investigate promising alternative co-inhibitory receptor targets, including T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT). This review delves into the mechanisms of approved ICIs (CTLA-4, PD-1, PD-L1, and LAG-3) and upcoming options like Tim-3 and TIGIT. It explores the use of ICIs in cancer treatment, supported by both preclinical and clinical data. Additionally, it examines the mechanisms behind cardiac toxic irAEs, focusing on ICI-associated myocarditis and atherosclerosis. These insights are vital as ICIs continue to revolutionize cancer therapy, offering hope to patients, while also necessitating careful monitoring and management of potential side effects, including emerging cardiac complications.
Myosin heavy chain‐perinatal (MyHC‐perinatal) is one of two development‐specific myosin heavy chains expressed exclusively during skeletal muscle development and regeneration. The specific functions of MyHC‐perinatal are unclear, although mutations are known to lead to contracture syndromes such as Trismus‐pseudocamptodactyly syndrome. Here, we characterize the functions of MyHC‐perinatal during skeletal muscle differentiation and regeneration. Loss of MyHC‐perinatal function leads to enhanced differentiation characterized by increased expression of myogenic regulatory factors and differentiation index as well as reduced reserve cell numbers in vitro . Proteomic analysis revealed that loss of MyHC‐perinatal function results in a switch from oxidative to glycolytic metabolism in myofibers, suggesting a shift from slow type I to fast type IIb fiber type, also supported by reduced mitochondrial numbers. Paracrine signals mediate the effect of loss of MyHC‐perinatal function on myogenic differentiation, possibly mediated by non‐apoptotic caspase‐3 signaling along with enhanced levels of the pro‐survival apoptosis regulator Bcl2 and nuclear factor kappa‐B (NF‐κB). Knockdown of MyHC‐perinatal during muscle regeneration in vivo results in increased expression of the differentiation marker myogenin (MyoG) and impaired differentiation, evidenced by smaller myofibers, elevated fibrosis and reduction in the number of satellite cells. Thus, we find that MyHC‐perinatal is a crucial regulator of myogenic differentiation, myofiber oxidative phenotype and regeneration.
Background:
Bunium persicum seeds, a member of the Apiaceae family, have historically been consumed as part of the Iranian diet.
Objective:
While many of this herb's biological properties have been fully investigated, there is currently no reliable information about its anticancer/cytotoxic properties.
Methods:
Herein, we first determined the major bioactive compounds of B. persicum seed extract (BPSE) via GC-Mass analysis. We evaluated the cytotoxicity of the extract alone as well as in combination with vincristine (VCR), a commonly used chemotherapy drug, using MTT assays on two breast cancer cell lines, MCF-7 and MDA-MB-231, as well as a normal breast cancer cell line, MCF-10A. Moreover, these compounds were evaluated in vitro for their anticancer activity using ROS assays, Real-Time PCR, Western blots, flow cytometry, and cell cycle assays.
Results:
As a result of our investigation, it was determined that the extract significantly reduced the viability of cancerous cells while remaining harmless to normal cells. The combination of BPSE and VCR also resulted in synergistic effects. BPSE and/or BPSE-VCR treatment increased the intracellular ROS of MCF-7 cells by over twofold. Moreover, the IC30 of BPSE (100 μg/ml) significantly increased the BAX/BCL-2 and P53 gene expression while reducing the expression of the MYC gene. Moreover, treated cells were arrested in the G2 phase of the cell cycle. The BPSE-VCR combination synergistically reduced the NF-κB and increased the Caspase-7 proteins’ expression. The percent of apoptosis in the cells treated with the extract, VCR, and their combination was 27, 11, and 50, respectively.
Conclusions:
The present study demonstrated the anticancer activity of the BPSE and its potential for application in combination therapy with VCR.
Fenamic acid-derived NSAIDs contain N-phenyl anthranilic acid as a pharmacophore with pKa ~ 4, which is completely ionized at the physiological pH and is mainly excreted in the urine by hydroxylation or glucuronidation. The acid (-COOH) functionality in fenamic acid provides a template for their fenamate derivatization for the development of analogues based on amidation, esterification, and etherification. Besides the repurposing of fenamates as neuroprotective agents in unmodified form, several of their derivatives have been reported for the management of disease pathogenesis by regulating the responsible pathways. In this review, we discuss the chemical modification of fenamic acid and its medicinal chemistry thereof.
Nitric oxide synthase (NOS) was initially discovered to participate in the generation of nitric oxide as a defense mechanism against pathogenic infections. In recent years, it has been found that NOS plays a pivotal role in regulating apoptosis and inflammation in mammals. However, the mechanisms underlying NOS-mediated apoptosis in invertebrates remain largely unclear. In this study, we found that the Apostichopus japonicus NOS (AjNOS) expression levels were upregulated by 2.20-fold and 3.46-fold after being challenged with Vibrio splendidus at concentrations of 107 CFU mL-1 and 108 CFU mL-1 for 12 h compared to the control group, respectively. Under these conditions, the rates of coelomocytes apoptosis were increased from 14.7% to 32.7% and 45.4%, respectively. Treatment with NOS inhibitor (l-NAME) resulted in a reduction of coelomocytes apoptosis rates from 32.6% to 26.5% in V. splendidus (107 CFU mL-1) groups and from 42.3% to 33.3% in V. splendidus (108 CFU mL-1) groups, respectively. NOS has been reported to regulate apoptosis through IκBα phosphorylation. Simultaneously, exposure to V. splendidus in conjunction with l-NAME resulted in down-regulation of AjIκBα phosphorylation levels compared to the group infected solely with V. splendidus. Furthermore, immunofluorescence analysis revealed that treatment with l-NAME or interference of AjNOS using siRNA inhibited translocation of AjNF-κB/p65 (RelA) into the nucleus. Previous studies have shown that NF-κB can down-regulate expression levels of Bcl-2 family members, which is an important pathway for regulating apoptosis. In the present study, treatment with l-NAME was found to promote anti-apoptotic AjBcl-2 mRNA increase to 1.41-fold and protein expression increase to 1.86-fold at 12 h post V. splendidus challenge. However, these effects were suppressed by PMA (an NF-κB activator). Overall, our findings demonstrate that AjNOS regulates coelomocytes apoptosis induced by V. splendidus through activation of the AjNF-κB signaling pathway and down-regulation of AjBcl-2 in A japonicus.
This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.
The strength and duration of the NF-κB signaling response must be tightly modulated to avoid an inadequate or excessive immune response. Relish, a core NF-κB transcription factor of the Drosophila Imd pathway, can control the expression of antimicrobial peptides, including Dpt and AttA, to defend against Gram-negative bacterial infections, but whether Relish may regulate miRNA expression to participate in the immune response remains unclear. In this study, taking advantage of Drosophila S2 cells and different overexpression/knockout/knockdown flies, we first found that Relish could directly activate the expression of miR-308 to negatively regulate the immune response and promote the survival of Drosophila during Enterobacter cloacae infection. Second, our results demonstrated that Relish-mediated expression of miR-308 could suppress target gene Tab2 to attenuate the Drosophila Imd pathway signal during the middle and late stages of the immune response. Third, we detected the dynamic expression patterns of Dpt, AttA, Relish, miR-308, and Tab2 in wild-type flies after E. coli infection, which further revealed that the feedback regulatory loop of Relish-miR-308-Tab2 plays a crucial role in the immune response and homeostasis maintenance of the Drosophila Imd pathway. Overall, our present study not only illustrates an important mechanism by which this Relish-miR-308-Tab2 regulatory axis can negatively control the Drosophila immune response and participate in homeostasis maintenance but also provides new insights into the dynamic regulation of the NF-κB/miRNA expression network of animal innate immunity.
Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is the most aggressive form of DLBCL, with a significantly inferior prognosis due to resistance to the standard R-CHOP immunochemotherapy. Survival of ABC-DLBCL cells addicted to the constitutive activations of both canonical and noncanonical NF-κB signaling makes them attractive therapeutic targets. However, a pharmaceutical approach simultaneously targeting the canonical and noncanonical NF-κB pathway in the ABC-DLBCL cell is still lacking. Peptide-conjugated gold nanoclusters (AuNCs) have emerged unique intrinsic biomedical activities and possess a great potential in cancer theranostics. Here, we demonstrated a Au25 nanocluster conjugated by cell-penetrating peptides that can selectively repress the growth of ABC-DLBCL cells by inducing efficient apoptosis, more efficiently than glutathione (GSH)-conjugated AuNCs. The mechanism study showed that the cell-penetrating peptides enhanced the cellular internalization efficiency of AuNCs, and the selective repression in ABC-DLBCL cells is due to the inhibition of inherent constitutive canonical and noncanonical NF-κB activities by AuNCs. Several NF-κB target genes involved in chemotherapy resistance in ABC-DLBCL cells, including anti-apoptotic Bcl-2 family members and DNA damage repair proteins, were effectively down-regulated by the AuNC. The emerged novel activity of AuNCs in targeting both arms of NF-κB signaling in ABC-DLBCL cells may provide a promising candidate and a new insight into the rational design of peptide-conjugated Au nanomedicine for molecular targeting treatment of refractory lymphomas.
Apoptosis is an evolutionarily conserved sequential process of cell death to maintain a homeostatic balance between cell formation and cell death. It is a vital process for normal eukaryotic development as it contributes to the renewal of cells and tissues. Further, it plays a crucial role in the elimination of unnecessary cells through phagocytosis and prevents undesirable immune responses. Apoptosis is regulated by a complex signaling mechanism, which is driven by interactions among several protein families such as caspases, inhibitors of apoptosis proteins, B-cell lymphoma 2 (BCL-2) family proteins, and several other proteases such as perforins and granzyme. The signaling pathway consists of both pro-apoptotic and pro-survival members, which stabilize the selection of cellular survival or death. However, any aberration in this pathway can lead to abnormal cell proliferation, ultimately leading to the development of cancer, autoimmune disorders, etc. This review aims to elaborate on apoptotic signaling pathways and mechanisms, interacting members involved in signaling, and how apoptosis is associated with carcinogenesis, along with insights into targeting apoptosis for disease resolution.
Periodontitis and osteoporosis are prevalent inflammation‐associated skeletal disorders that pose significant public health challenges to our aging population. Both periodontitis and osteoporosis are bone disorders closely associated with inflammation and aging. There has been consistent intrigue on whether a systemic skeletal disease such as osteoporosis will amplify the alveolar bone loss in periodontitis. A survey of the literature published in the past 25 years indicates that systemic low bone mineral density (BMD) is associated with alveolar bone loss, while recent evidence also suggests a correlation between clinical attachment loss and other parameters of periodontitis. Inflammation and its influence on bone remodeling play critical roles in the pathogenesis of both osteoporosis and periodontitis and could serve as the central mechanistic link between these disorders. Enhanced cytokine production and elevated inflammatory response exacerbate osteoclastic bone resorption while inhibiting osteoblastic bone formation, resulting in a net bone loss. With aging, accumulation of oxidative stress and cellular senescence drive the progression of osteoporosis and exacerbation of periodontitis. Vitamin D deficiency and smoking are shared risk factors and may mediate the connection between osteoporosis and periodontitis, through increasing oxidative stress and impairing host response to inflammation. With the connection between systemic and localized bone loss in mind, routine dental exams and intraoral radiographs may serve as a low‐cost screening tool for low systemic BMD and increased fracture risk. Conversely, patients with fracture risk beyond the intervention threshold are at greater risk for developing severe periodontitis and undergo tooth loss. Various Food and Drug Administration‐approved therapies for osteoporosis have shown promising results for treating periodontitis. Understanding the molecular mechanisms underlying their connection sheds light on potential therapeutic strategies that may facilitate co‐management of systemic and localized bone loss.
Objectives
Epulis is considered to be a massive reactive lesion rather than a true neoplasia. AhR is thought to be associated with inflammation and development of neoplasms. Here, we aimed to observe the expression of AhR in fibrous epulis and explore its role and possible mechanism in the pathogenesis of epulis.
Materials and Methods
Epulis and normal gingival tissues were collected, and AhR expression was detected at the mRNA and protein levels by quantitative polymerase chain reaction (qPCR) and immunohistochemistry, respectively. The expression levels of proinflammatory cytokines and apoptosis-related factor genes in human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs) transfected with AhR short interfering RNA (siRNA) or negative control siRNA, upon stimulation with lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) were then examined. Finally, the expression levels of the proinflammatory cytokines and apoptosis-related factor genes in the epulis tissues were observed by qPCR.
Results
AhR expression in fibrous epulis was significantly increased at both the mRNA and protein levels. The expression of proinflammatory cytokines and apoptosis-related factor genes in hPDLCs transfected with AhR siRNA was significantly decreased when stimulated with Pg-LPS. The same trends were observed for hGFs. The opposite trend was detected in the epulis tissues.
Conclusion
AhR may be a key factor in fibrous epulis pathogenesis that acts by regulating the expression of BCL2 family genes and inflammatory factor-related genes.
The Bcl-2 family members rigorously regulate cell endogenous apoptosis, and targeting anti-apoptotic members is a hot topic in design of anti-cancer drugs. At present, FDA and EMA have approved Bcl-2 inhibitor Venetoclax (ABT-199) for treating chronic lymphocytic leukemia (CLL). However, inhibitors of anti-apoptotic protein BCL2A1/Bfl-1 have not been vigorously developed, and no molecule with ideal activity and selectivity has been found yet. Here we review the biological function and protein structure of Bfl-1, discuss the therapeutic potential and list the currently reported inhibitory peptides and small molecules. This will provide a reference for Bfl-1 targeting drug discovery in the future.
Cerebral ischemia is a devastating disease that affects many people worldwide every year. The neurodegenerative damage as a consequence of oxygen and energy deprivation, to date, has no known effective treatment. The ischemic insult is followed by an inflammatory response that involves a complex interaction between inflammatory cells and molecules which play a role in the progression towards cell death. However, there is presently a matter of controversy over whether inflammation could either be involved in brain damage or be a necessary part of brain repair. The inflammatory response is triggered by inflammasomes, key multiprotein complexes that promote secretion of pro-inflammatory cytokines. An early event in post-ischemic brain tissue is the release of certain molecules and reactive oxygen species (ROS) from injured neurons which induce the expression of the nuclear factor-kappaB (NF-κB), a transcription factor involved in the activation of the inflammasome. There are conflicting observations related to the role of NF-κB. While some observe that NF-κB plays a damaging role, others suggest it to be neuroprotective in the context of cerebral ischemia, indicating the need for additional investigation. Here we discuss the dual role of the major inflammatory signaling pathways and provide a review of the latest research aiming to clarify the relationship between NF-κB mediated inflammation and neuronal death in cerebral ischemia.
Breast cancer is the leading cause of death in women worldwide and these deaths are mostly attributed to metastasis and tumour recurrence following initially successful therapy. Metastasis refers to the development of invasive disease, wherein malignant cells dissociate from primary tumours, infiltrating other organs and tissues to give rise to secondary outgrowths. Previously, metastasis was thought to be initiated in advanced tumours, but breast cancer cellsh with metastatic potential have now been shown to disseminate very early from the primary site via largely unknown mechanisms. These early interactions of tumour cells with their cellular micro-environment and normal neighbours also results in early tumour cell heterogeneity and must therefore be elucidated such that we can prevent metastatic spread in the patient situation and better treat the resulting heterogenous tumours. However, studying tumour initiation is not possible in patients because it happens on a cellular level not detectable by current technology. Tumour recurrence is another major cause of breast cancer related death and is believed to be caused by residual disease cells that survive initial therapy. These are a reservoir of refractory cells that can lay dormant for many years (sometimes decades) before resulting in relapse tumours. They are also difficult to obtain from human patients, since they are very few and cannot be detected easily, and thus their molecular mechanisms have not been fully explored. In addition to the unavailability of human tissue, mouse models of breast cancer also fall short in helping us study early cancer initiation, because they allow oncogenic expression in all cells of the tissue instead of initiating cancer like in the human situation|one neoplastic transformed cell proliferating unchecked in a normal epithelium. To address this issue, we used primary organoids from an inducible mouse model of breast cancer and lentivirally transduced single cells within these organoids to express oncogenes. We further optimized parameters for long term imaging using light sheet microscopy and developed big data analysis pipelines that lead us to discern that single transformed cells had a lower chance at establishing tumorigenic foci, when compared to clusters of cells. Thus, we postulate a proximity-controlled signalling that is imperative to tumour initiation within epithelial tissues using the first ever in vitro stochastic breast tumorigenesis model system. This new stochastic tumorigenesis system can be further used to identify the molecular interactions in the early breast cancer cells. Our group has already revealed distinct characteristics, such as dysregulated lipid metabolism, of the residual disease correlate obtained from an inducible mouse model. As survival mechanisms invoked by residual cells remain largely unknown, we analysed the dynamic transcriptome of regressing tumours at important timepoints during the establishment of residual disease. Key molecular players upregulated during regression {like c-Jun and BCL6 { were identified and the inflammatory arm of the Nf-kB cascade was found to be dysregulated among others. Further validation of these molecular targets as potentially synthetic lethal interactors remains to be performed so that they can be used to limit the residual disease reservoir and eventually tumour recurrence.
Background
Melanoma causes more than 80% of deaths from all dermatologic cancers. Hence, screening and identifying effective compounds to inhibit the growth of melanoma have crucial importance in basic and clinical treatment.
Methods
High throughput screening was performed to screen and identify compounds which have anti-melanoma ability. Melanoma cell and mouse allograft models was used to examine the anti-tumor effects of Nuciferine (NCFR). Western blot, qPCR, and lentivirus overexpression were applied to detect the activation of TLR4/NF-κB signaling pathway.
Results
NCFR administration significantly suppressed melanoma cell growth and tumor size by inhibiting the phosphorylation of p65. NCFR treatment also could suppress TNF-α-induced activation of NF-κB signaling. The anti-tumor effect of NCFR might be mediated by targeting Toll-like receptors 4.
Conclusion
NCFR inhibits melanoma cell growth and suppresses tumor size, which provides a potential therapeutic strategies of melanoma treatment.
Objective: Use of methamphetamine (METH) is prevalent among HIV-infected individuals. Previous research has shown that both METH and HIV protease inhibitors exert influences on mitochondrial respiratory metabolism and hepatic nervous system. This study aims to study the joint effect of METH and HIV protease inhibitors on hepatic immune function.
Materials and methods: Based on the differentially expressed genes obtained from RNA-seq of the liver from mouse model, the expression levels of CD48 and Macrophage Receptor with Collagenous Structure (MARCO) were examined using qRT-PCR and flow cytometry, and the expression and secretion of cytokines IL-1β, IL-6, IL-8, IL-10, IFN-γ, IFN-β, and TNF-α were determined using qRT-PCR and ELISA in THP-1-derived macrophages.
Results: Our results indicated that compared with the control group, CD48 molecules were significantly down-regulated by METH–atazanavir co-treatment, and the expression level of CD48 decreased as METH concentration increases. MARCO molecules were increased, especially at larger doses of METH and atazanavir treatment. In addition, in the presence of METH–atazanavir, the expression and secretion of a series of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 increased while the expression and secretion of anti-inflammatory cytokine IL-10 decreased.
Conclusion: These results demonstrated that METH and atazanavir had a combined impact on the liver immunity, suggesting that the co-treatment could enhance inflammatory response and suppress NK cell activation via CD48.
As a multifunctional protein, cyclophilin A (CypA) plays an important role in cell apoptosis. In our previous work, we found that CypA from Apostichopus japonicus (AjCypA), as a cofactor, could modulate nuclear translocation of NF-κB. However, the immune function of AjCypA is largely unknown. In the present study, we found that siRNA-mediated AjCypA knockdown in vivo significantly increased the coelomocyte apoptosis rate. In addition, the expression of B-cell lymphoma-2 (AjBcl-2, an anti-apoptosis gene) was synchronously downregulated. To better understand the connection between AjCypA and AjBcl-2 expression, we cloned the promoter of AjBcl-2 via genomic walking, which spanned 1870 bp and contained four potential binding sites of NF-κB. Dual-luciferase reporter assay revealed that the full-length sequence and all truncated fragments exhibited high transcriptional activity. Moreover, 1 μg/mL LPS exposure significantly increased the luciferase activity of P1 (-1870/+57) by 2.31-fold and 3.15-fold at 12 and 24 h, respectively. Furthermore, the four potential NF-κB binding sites and pCMV-Flag2C-AjNF-κB co-transfection assay demonstrated that NF-κB could regulate the expression of AjBcl-2 via the NF-κB binding sites of AjBcl-2 promoter. All results supported that AjCypA mediates coelomocyte apoptosis via NF-κB/AjBcl-2 signaling pathway in A. japonicus.
Cancer is a worldwide endemic and continues to be one of the most difficult diseases to treat and manage. It generally exhibits considerable genetic complexity and aberrant cell death and survival signaling pathways. Resistance to cell death induction has long been recognized as a hallmark of cancer. Thus, understanding of the underlying molecular events regulating different cell death mechanisms such as apoptosis has provided new possibilities for targeted interference of these pathways. This chapter highlights the significant signaling pathways of apoptosis and its relevant therapeutic targets and summarizes the current state of development of specific modulators of cell death and the overall outcome of this group of novel therapeutics in various phases of clinical trials.
Members of the nuclear factor (NF)-κB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-κB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IκBα that constitutively represses the activity of multiple NF-κB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4⁺ T cells. Taken together, these in vivo findings indicate that the NF-κB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability
transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and
PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1β
converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential
(ΔΨm). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear
apoptosis only occur in cells in which the ΔΨm is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the ΔΨm collapse and subsequent apoptosis. Cytosols from anti-Fas–treated human lymphoma cells accumulate an activity that induces
PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to
cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their ΔΨm. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin
condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator)
that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of
CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + ΔΨm collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced
mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis
regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis
in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.
Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.
Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.
Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.
We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).
Resistance against the cytotoxic actions of tumor necrosis factor alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc finger protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.
A1, a bcl-2 family member, has been identified as a hematopoietic-specific, early inducible gene. In this study it is shown that stable transfection of A1 into an interleukin-3 (IL-3)-dependent myeloid precursor cell line, 32D c13, leads to a retardation of IL-3 withdrawal-induced cell death similar to that observed with transfection of bcl-2. However, unlike bcl-2. A1 expression permits the accumulation of differentiated myeloid cells both before and after IL-3 withdrawal. Total cell accumulation, on the other hand, is considerably greater after IL-3 deprivation in the bcl-2 transfectant than in A1-expressing cells. Cells cotransfected with the two genes behave similarly to cells singly transfected with bcl-2, except that viability following IL-3 withdrawal is somewhat further enhanced. These results suggest that these two proteins have distinct roles that may be related to the divergent regulation of their expression during myeloid differentiation.
The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. Here we show that iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, we have isolated Drosophila and human genes which encode IAP-like proteins (dILP and hILP). Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis.
Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.
The response of human myeloid leukemia cells to treatment with 1-beta-arabinofuranosylcytosine (ara-C) includes the induction of apoptosis. Ara-C induced apoptosis is associated with proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and protein kinase C (PKC) delta. However, the signals involved in this response are unknown. The present studies show that ara-C treatment of U-937 cells is associated with induction of a protease activity that cleaves the tetrapeptides Ac-DEVD-pNA and Ac-DMOD-pNA found at the cleavage sites of PARP and PKC delta, respectively. The ara-C-induced protease activity was sensitive to overexpression of the anti-apoptotic protein Bcl-xL and the baculovirus protein p35. By contrast, overexpression of the cowpox virus protein CrmA blocked apoptosis induced by engagement of the Fas receptor but not that induced by ara-C. CrmA overexpression also had no detectable effect on ara-C-induced cleavage of PKC delta. The results further show that ara-C induces activation of the CPP32 protease by a CrmA-insensitive and p35-sensitive mechanism. Similar results were obtained with cisplatinum, etoposide, and camptothecin. These findings indicate that ara-C and other DNA-damaging agents activate a CrmA-insensitive apoptotic pathway involving CPP32 and that these signals differ from those associated with apoptosis induced by the Fas receptor.
Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti-IgM) is preceded by dramatic changes in Nuclear Factor-kappaB (NF-kappaB)/ Rel binding activities. An early transient increase in NF-kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel-related factors in B cell apoptosis. Treatment of WEH1 231 cells with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF-kappaB (IkappaB)-alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF-kappaB/Rel factor binding and induced apoptosis. Bcl-XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB-alpha-GST protein or a c-Rel affinity-purified antibody induced apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TPCK or anti-IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF-kappaB/Rel binding following anti-IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF-kappaB/Rel family in control of apoptosis of normal and transformed B cells.
Endothelial cells play a central role in the inflammatory process. Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine which elicits many of the inflammatory responses of endothelial cells. While TNF directly causes apoptosis of tumor cells and virally infected cells, normal cells are generally resistant. However, most resistant cells, including human endothelial cells, can be rendered susceptible to TNF by inhibiting RNA or protein synthesis. This finding suggests that TNF provides a cell survival signal in addition to a death signal. We have previously cloned a human Bcl-2 homologue, A1, and shown that it is specifically induced by proinflammatory cytokines but not by endothelial growth factors. In this study, we show that retroviral-mediated transfer of the A1 cDNA to a human microvascular endothelial cell line provides protection against cell death initiated by TNF in the presence of actinomycin D. The induction of A1 by TNF in this system is mediated via a protein kinase C pathway. Since TNF signaling has also been shown to proceed via ceramides, we tested whether exogenous ceramides could induce A1. Our findings indicate that ceramides do not induce A1 but do up-regulate c-jun and induce endothelial death. Ceramide-activated endothelial death is also inhibited by A1, suggesting that TNF may initiate divergent survival and death pathways via separate lipid second messengers.
In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases,
leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of
cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked
by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2.
Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome
c translocation, thereby blocking caspase activation and the apoptotic process.
Members of the nuclear factor (NF)-kappaB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-kappaB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IkappaBalpha that constitutively represses the activity of multiple NF-kappaB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells. Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.
The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.
Studies on mice deficient in nuclear factor kappa B (NF-κB) subunits have shown that this transcription factor is important for lymphocyte responses to antigens and cytokine-inducible gene expression. In particular, the RelA (p65) subunit is required for induction of tumor necrosis factor-α (TNF-α)-dependent genes. Treatment of RelA-deficient (RelA^(−/−)) mouse fibroblasts and macrophages with TNF-α resulted in a significant reduction in viability, whereas RelA^(+/+) cells were unaffected. Cytotoxicity to both cell types was mediated by TNF receptor 1. Reintroduction of RelA into RelA^(−/−) fibroblasts resulted in enhanced survival, demonstrating that the presence of RelA is required for protection from TNF-α. These results have implications for the treatment of inflammatory and proliferative diseases.
The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. Here we show that iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, we have isolated Drosophila and human genes which encode IAP-like proteins (dILP and hILP). Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis.
Bcl-2 family proteins serve as critical regulators of pathways involved in apoptosis, acting to either inhibit or promote cell death. Because chemotherapeutic drugs typically exert their cytotoxic actions by inducing apoptosis, the ultimate efficacy of most anticancer drugs can be heavily influenced by the relative levels and state of activation of members of the Bcl-2 family. But how important are Bcl-2 family proteins in the overall complex picture of chemoresponses in cancer?
Taking into consideration that such knowledge is far from complete, this chapter attempts to critically examine this question. It presents a discussion of some of the experimental and clinical observations that provide insights into the complexities of chemotherapeutic drug mechanisms and the role of Bcl-2 family proteins as modulators of tumor cell susceptibility to apoptosis.
Resistance against the cytotoxic actions of tumor necrosis factor-alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc ringer protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.
Extracellular survival factors alter a cell's susceptibility to apoptosis, often through posttranslational mechanisms. However, no consistent relationship has been established between such survival signals and the BCL-2 family, where the balance of death agonists versus antagonists determines susceptibility. One distant member, BAD, heterodimerizes with BCL-X(L) or BCL-2, neutralizing their protective effect and promoting cell death. In the presence of survival factor IL-3, cells phosphorylated BAD on two serine residues embedded in 14-3-3 consensus binding sites. Only the nonphosphorylated BAD heterodimerized with BCL-X(L) at membrane sites to promote cell death. Phosphorylated BAD was sequestered in the cytosol bound to 14-3-3. Substitution of serine phosphorylation sites further enhanced BAD's death-promoting activity. The rapid phosphorylation of BAD following IL-3 connects a proximal survival signal with the BCL-2 family, modulating this checkpoint for apoptosis.
The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-κB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-κB. These results provide an explanation for the requirement of NF-κB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.
In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.
Almost exactly ten years following the first publication on NF-κB (Sen and Baltimore 1986), researchers working on transcriptional regulation by NF-κB/Rel and IκB proteins gathered for the third time to discuss recent developments in the field (Madrid, July 8-10, 1996). The first meeting of its kind was a Howard Hughes workshop at the NIH in November 1992 and the second one a Banbury Conference held at Cold Spring Harbor in October 1993. This year's meeting was organized by R. Bravo (Bristol-Myers Squibb, Princeton) and P. S. Lazo (Universidad de Oviedo) and held at the Juan March Foundation in Madrid, Spain.
Members of the NF-kappa B/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor alpha (TNF). Although NF-kappa B appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-kappa B is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-kappa B and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of Ikappa B that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-kappa B induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-kappa B via an Ikappa B targeting mechanism. Functional coupling of NF-kappa B and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.
In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Although
much is known about the control of cell proliferation, less is known about the control of cell death. Physiologic cell death
occurs primarily through an evolutionarily conserved form of cell suicide termed apoptosis. The decision of a cell to undergo
apoptosis can be influenced by a wide variety of regulatory stimuli. Recent evidence suggests that alterations in cell survival
contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative
disorders, and AIDS (acquired immunodeficiency syndrome). Treatments designed to specifically alter the apoptotic threshold
may have the potential to change the natural progression of some of these diseases.
Bcl-2 is an intracellular membrane-associated protein that functions to block programmed cell death. Despite recurrent exposure to cellular toxins from the circulation and tissue, endothelial cells are remarkably resistant to cell death. Because Bcl-2 protein levels are low or undetectable in endothelial cells, we postulated that other members of the growing Bcl-2 family would be present in endothelial cells to provide protection against apoptosis. Degenerate primers to two conserved regions of the Bcl-2 family were used to amplify potential homologues in endothelial cells. This strategy resulted in the isolation of a human Bcl-2 homologue related to murine Al, a recently identified member of this family. We show here that, in endothelial cells, human Al is rapidly inducible by phorbol ester and the inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta, but not by the growth factors, basic fibroblast growth factor or vascular endothelial growth factor. Al is the only known Bcl-2 family member that is inducible by inflammatory cytokines, suggesting that it may play a protective role during inflammation. Additionally, vascular smooth muscle cells and various nonhematopoietic tissues express human Al, indicating that human Al is a widely expressed Bcl-2 homologue.
A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.
The transcription factor NF-kappa B has attracted widespread attention among researchers in many fields based on the following: its unusual and rapid regulation, the wide range of genes that it controls, its central role in immunological processes, the complexity of its subunits, and its apparent involvement in several diseases. A primary level of control for NF-kappa B is through interactions with an inhibitor protein called I kappa B. Recent evidence confirms the existence of multiple forms of I kappa B that appear to regulate NF-kappa B by distinct mechanisms. NF-kappa B can be activated by exposure of cells to LPS or inflammatory cytokines such as TNF or IL-1, viral infection or expression of certain viral gene products, UV irradiation, B or T cell activation, and by other physiological and nonphysiological stimuli. Activation of NF-kappa B to move into the nucleus is controlled by the targeted phosphorylation and subsequent degradation of I kappa B. Exciting new research has elaborated several important and unexpected findings that explain mechanisms involved in the activation of NF-kappa B. In the nucleus, NF-kappa B dimers bind to target DNA elements and activate transcription of genes encoding proteins involved with immune or inflammation responses and with cell growth control. Recent data provide evidence that NF-kappa B is constitutively active in several cell types, potentially playing unexpected roles in regulation of gene expression. In addition to advances in describing the mechanisms of NF-kappa B activation, excitement in NF-kappa B research has been generated by the first report of a crystal structure for one form of NF-kappa B, the first gene knockout studies for different forms of NF-kB and of I kappa B, and the implications for therapies of diseases thought to involve the inappropriate activation of NF-kappa B.
The bcl-2 family of genes code for proteins that contain anti-apoptotic or pro-apoptotic activity. The human bfl-1 gene contains an open reading frame for a 175-amino acid Bcl-2 family protein. Among the various Bcl-2 family members, the Bfl-1 protein shares the highest homology with the mouse A1 protein. These two proteins share three conserved domains, Bcl homology (BH)1, BH2, and BH3, with other Bcl-2 family proteins. Unlike other Bcl-2 family members, Bfl-1 contains a GIn-rich NH2-terminal region and lacks an NH (19K homology) domain 1. We demonstrate that the Bfl-1 protein suppresses apoptosis induced by the p53 tumor suppressor protein in a manner similar to other Bcl-2 family members such as Bcl-2, Bcl-xL and EBV-BHRF1. In addition, the bfl-I gene cooperates efficiently with the Ela oncogene in transformation of primary rodent epithelial cells. Our results suggest that the human bfl-1 gene may play an important role in carcinogenesis.
Tumor necrosis factor α (TNF-α) signaling gives rise to a number of events, including activation of transcription factor NF-κB
and programmed cell death (apoptosis). Previous studies of TNF-α signaling have suggested that these two events occur independently.
The sensitivity and kinetics of TNF-α-induced apoptosis are shown to be enhanced in a number of cell types expressing a dominant-negative
IκBα (IκBαM). These findings suggest that a negative feedback mechanism results from TNF-α signaling in which NF-κB activation
suppresses the signals for cell death.
Many cells are resistant to stimuli that can induce apoptosis, but the mechanisms involved are not fully understood. The activation
of the transcription factor nuclear factor-kappa B (NF-κB) by tumor necrosis factor (TNF), ionizing radiation, or daunorubicin
(a cancer chemotherapeutic compound), was found to protect from cell killing. Inhibition of NF-κB nuclear translocation enhanced
apoptotic killing by these reagents but not by apoptotic stimuli that do not activate NF-κB. These results provide a mechanism
of cellular resistance to killing by some apoptotic reagents, offer insight into a new role for NF-κB, and have potential
for improvement of the efficacy of cancer therapies.
Studies on mice deficient in nuclear factor kappa B (NF-kappaB) subunits have shown that this transcription factor is important for lymphocyte responses to antigens and cytokine-inducible gene expression. In particular, the RelA (p65) subunit is required for induction of tumor necrosis factor-alpha (TNF-alpha)-dependent genes. Treatment of RelA-deficient (RelA-/-) mouse fibroblasts and macrophages with TNF-alpha resulted in a significant reduction in viability, whereas RelA+/+ cells were unaffected. Cytotoxicity to both cell types was mediated by TNF receptor 1. Reintroduction of RelA into RelA-/- fibroblasts resulted in enhanced survival, demonstrating that the presence of RelA is required for protection from TNF-alpha. These results have implications for the treatment of inflammatory and proliferative diseases.
Through its type 1 receptor (TNFR1), the cytokine TNF elicits an unusually wide range of biological responses, including inflammation, tumor necrosis, cell proliferation, differentiation, and apoptosis. We investigated how TNFR1 activates different effector functions; the protein kinase JNK, transcription factor NF-kappaB, and apoptosis. We found that the three responses are mediated through separate pathways. Recruitment of the signal transducer FADD to the TNFR1 complex mediates apoptosis but not NF-kappaB or JNK activation. Two other signal transducers, RIP and TRAF2, mediate both JNK and NF-kappaB activation. These two responses, however, diverge downstream to TRAF2. Most importantly, JNK activation is not involved in induction of apoptosis, while activation of NF-kappaB protects against TNF-induced apoptosis.
A green fluorescent protein (GFP)-Raf-1 fusion protein was used to show that Bcl-2 can target this kinase to mitochondria. Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD, a proapoptotic Bcl-2 homolog. Plasma membrane-targeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-1 and ERK-2. Untargeted active Raf-1 improved Bcl-2-mediated resistance to apoptosis, whereas a kinase-inactive Raf-1 mutant abrogated apoptosis suppression by Bcl-2. Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD or possibly other protein substrates involved in apoptosis regulation.
Extracellular survival factors alter a cell's susceptibility to apoptosis, often through posttranslational mechanisms. However, no consistent relationship has been established between such survival signals and the BCL-2 family, where the balance of death agonists versus antagonists determines susceptibility. One distant member, BAD, heterodimerizes with BCL-X(L) or BCL-2, neutralizing their protective effect and promoting cell death. In the presence of survival factor IL-3, cells phosphorylated BAD on two serine residues embedded in 14-3-3 consensus binding sites. Only the nonphosphorylated BAD heterodimerized with BCL-X(L) at membrane sites to promote cell death. Phosphorylated BAD was sequestered in the cytosol bound to 14-3-3. Substitution of serine phosphorylation sites further enhanced BAD's death-promoting activity. The rapid phosphorylation of BAD following IL-3 connects a proximal survival signal with the BCL-2 family, modulating this checkpoint for apoptosis.
Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents
cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation
of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the
mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol
and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria
and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release
from mitochondria.
Programmed cell death or apoptosis provides an irreversible mechanism for the elimination of excess or damaged cells. Several recent studies have implicated the activation of the interleukin 1beta-converting enzyme/Ced-3 (ICE/Ced-3) family of proteases as the "point of no return" in apoptotic cell death, while others have suggested that loss of mitochondrial membrane potential (delta psi(m)) is the ultimate determinant of cell death. The temporal relationship of these two events during apoptosis and the role of Bcl-2 proteins in inhibiting these steps has not been defined. To examine these issues, control and Bcl-x(L)-transfected Jurkat T cells were treated with Fas antibodies in the presence and absence of the ICE protease inhibitor zVAD-FMK. ICE/Ced-3 protease activity was monitored by following the cleavage of poly(ADP-ribose) polymerase (PARP) and delta psi(m) was followed by rhodamine 123 fluorescence. Although Bcl-x(L) expression did not block Fas-induced protease activation, it substantially inhibited the subsequent loss of delta psi(m) and cell death in Fas-treated cells. In contrast, zVAD-FMK blocked PARP cleavage as well as loss of delta psi(m) and cell death. Together these data demonstrate that Bcl-x(L) can maintain cell viability by preventing the loss of mitochondrial membrane potential that occurs as a consequence of ICE/Ced-3 protease activation.
We have identified and purified from HeLa cytosol a protein that induces DNA fragmentation in coincubated nuclei after it is activated by caspase-3. This protein, designated DNA Fragmentation Factor (DFF), is a heterodimer of 40 kDa and 45 kDa subunits. The amino acid sequence of the 45 kDa subunit, determined from its cDNA sequence, reveals it to be a novel protein. Caspase-3 cleaves the 45 kDa subunit at two sites to generate an active factor that produces DNA fragmentation without further requirement for caspase-3 or other cytosolic proteins. In cells undergoing apoptosis, the 45 kDa subunit is cleaved in the same pattern as it is cleaved by caspase-3 in vitro. These data delineate a direct signal transduction pathway during apoptosis: caspase-3 to DFF to DNA fragmentation.
Bcl-2 is an oncogenic protein that acts by inhibiting programmed cell death. The mechanisms used by this and related anti-apoptotic proteins to protect cells from cytotoxic stimuli are now emerging, with the discovery that Bcl-2 can function both as an ion channel and as an adaptor or docking protein.
We report here the purification and cDNA cloning of Apaf-1, a novel 130 kd protein from HeLa cell cytosol that participates in the cytochrome c-dependent activation of caspase-3. The NH2-terminal 85 amino acids of Apaf-1 show 21% identity and 53% similarity to the NH2-terminal prodomain of the Caenorhabditis elegans caspase, CED-3. This is followed by 320 amino acids that show 22% identity and 48% similarity to CED-4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of Apaf-1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3, leading to apoptosis.
Caspases (cysteinyl aspartate-specific proteinases) mediate highly specific proteolytic cleavage events in dying cells, which collectively manifest the apoptotic phenotype. The key and central role that these enzymes play in a biochemical cell-suicide pathway has been conserved throughout the evolution of multicellular eukaryotes.
The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-kappaB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-kappaB. These results provide an explanation for the requirement of NF-kappaB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.
We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.