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NF-κB and chemoresistance: Potentiation of cancer chemotherapy via inhibition of NF-κB
... NF-κB inhibits tumor cell apoptosis, both the course of the receptor pathway and the mitochondrial pathway, and the mechanisms of this action are very complex and result mainly from the function of this transcription factor in the regulation of gene expression in the pathways of apoptosis [65][66][67]. Because increased gene expression of multidrug resistance proteins and reduced apoptotic response are important reasons for the development of acquired multidrug resistance of tumor cells, attempts to inhibit the function of NF-kB are a promising direction for increasing the sensitivity of tumor cells to cytostatic drugs [65,66,68,69]. Celastrol seems to be a good candidate for chemosensitizing adjuvant medical preparation in cytostatic chemotherapy of cancer, in particular it could enhance the response to the standard cytostatic drugs of these tumors, which contain a large subpopulation of drug-resistant cells. ...
... The rapidly growing number of publications shows that the activity of the NF-κB transcription factor in regulating the expression of many genes also relates to the function and phenotype of tumor stem cells [67,70,71]. It has been shown that in the subpopulation of cancer stem cells NF-κB significantly increases the expression of genes of cellular proteins associated with chemoresistance and invassiveness of cancers, among which intensifies the expression and function of P-glycoprotein, reduces cell susceptibility to apoptosis, enhances repair of DNA damage in cancer cells and increases the ability of new migration and metastasis [65,66,68,69]. NF-κB activation www.oncotarget.com ...
Celastrol (tripterine) a pentacyclic triterpenoid extracted from the roots of Tripterygium wilfordii Hook f., exhibits potent antioxidant and anti-inflammatory activity and also exerts important anti-cancer effects, as induction of apoptosis and lowering the level of drug resistance of several cancers.
Increased level of cellular resistance to cytostatic drugs is typical for colorectal cancers, and largely determines the failure of chemotherapy for this tumor.
The purpose of our research was to evaluate the chemopreventive effect of celastrol on cultures of colon cancer cells resistant to doxorubicin (LOVO/DX). With the use of flow cytometry we have shown that celastrol reduces the cell size of the SP (side population; subpopulation of cancer cells enriched with cancer stem cells), increases frequency of apoptosis and binds to Pgp protein in cell membranes inhibiting its transport function. The inhibition of the Pgp transport function has been shown to increase the accumulation of rhodamine-123 and standard cytostatic- doxorubicin in LOVO/DX cells.
Our results prove that celastrol exhibits significant chemopreventive and chemosensitizing activities on drug resistant colon cancer cells. Celastrol appears to be a good candidate for adjuvant medicine that can improve the effectiveness of standard cytostatic therapy in humans.
... In the study by Tamatani et al, PS-341 enhanced radiosensitivity through inhibiting radiation-induced NF-κB activity and suppressed oral tumor growth (14). Moreover, PS-341 and NPI0052 can enhance chemosensitivity and the tumoricidal response to CPT-11 in colorectal cancer by blocking chemotherapy-induced NF-κB activation and expression of genes involved in cancer cell survival (15)(16)(17)(18). In addition, proteasome inhibition also augments the cancer cell response to chemotherapy and radiation by modulating other NF-κB related proteasome-dependent regulatory proteins involved in treatment resistance such as Bcl2, p53, the caspases and stress response molecules like SAPK/JNK, as well as accumulation of misfolded proteins and anti-angiogenic effects (10,13). ...
... In addition, CFZ induced growth inhibition and apoptosis through inhibiting the NF-κB signaling pathways in mantle cell lymphoma (25), and co-administration abrogated NF-κB activity in vorinostattreated granta cells and HF-4B cells (23). Moreover, synergistic anticancer activity was reported when chemotherapeutic agents such as CPT-11, were combined with NF-κB inhibitors, including proteasome inhibitors (15,18,51). Studies of NF-κB inhibition by proteasome inhibitors, such as NPI-0052, have demonstrated a synergistic response with chemotherapeutic drugs in a colon cancer model (18). ...
Upregulation of nuclear factor-κB (NF-κB) in colorectal carcinoma (CRC) accelerates tumor growth, whereas, irinotecan (CPT-11)-induced NF-κB activation reduces chemosensitivity and weakens the anti-colorectal cancer function itself, while proteasome inhibitors can inhibit NF-κB and improve the effect of chemotherapy. Carfilzomib (CFZ) is a novel proteasome inhibitor that has been recently approved by the FDA and is in clinical use for the treatment of multiple myeloma, but little is known about its activity against CRC. The aim of the present study was to explore whether CFZ alone or in combination with CPT-11 is effective in CRC treatment. We evaluated the novel therapeutic ability and mechanism of action of CFZ in CRC in vitro and in vivo. SW620 cells were incubated with CFZ alone or in combination with CPT-11. Cell proliferation was assessed by WST-1 and clonogenic assays, the cytotoxic interaction was assessed with a combination index (CI). Cell cycle progression was analysed with flow cytometry. Cell apoptosis was evaluated by detecting the Annexin V/propidium iodide (PI) ratio, caspase 3 and CD95 expression, and with TUNEL staining. Cell migration and invasion was determined with a wound-healing assay and a Transwell matrix penetration assay. A CRC xenograft model was established to monitor tumor growth. EMSA was used to analyse NF-κB activation and western blot analysis was used to detect the protein levels of related signaling factors. CFZ significantly inhibited the growth of SW620 cells, and had synergistic inhibitory effects with CPT-11 on survival and colony formation; possibly by inhibition of NF-κB activation, MEK/ERK and PI3K/AKT pathway factor dephosphorylation and survivin downregulation. Co-administration of CFZ and CPT-11 induced G2/M arrest, increased p21WAF1/CIP, and decreased mutant p53 and cdc25c expression. Induction of apoptosis was accompanied by marked increases in PARP cleavage, caspase 3 activation, an increase of CD95 and p-p38, and ATF3 activation. Combination treatment lowered the invasive and migration ability of SW620 cells, reduced MMP and increased TIMP protein expression. Finally, co-administration of CFZ and CPT-11 suppressed tumor growth and increased apoptosis compared with single-agent treatment in SW620 xenograft models correlated with NF-κB downregulation. Carfilzomib alone or in combination with CPT-11 is effective against colorectal cancer through inhibition of multiple mechanisms related to NF-κB, and could be a potential novel therapy for CRC.
... NFκB activity is necessary for paclitaxel and doxorubicin cytotoxicity [49,50], and some traditional chemotherapies activate NFκB signaling [51,52]. Conversely, apoptosis induced by irinotecan, daunorubicin and cisplatin therapies is reportedly enhanced by blocking NFκB [47,49,53]. Once again the complexity of NFκB -modulated apoptosis is revealed, highlighting the importance of the proper balance of NFκB activity in individual tumors treated with specific chemotherapeutic agents. ...
... Yet one must keep in mind that multi-drug therapy has long been the approach to cancer care because of this very issue. Second, blocking NFκB generally enhances the responsiveness of tumors to traditional chemotherapy [47,48]. This, however, is also not straightforward. ...
Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery, whole brain and spine irradiation, and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer.
To test the importance of NFκB to medulloblastoma cell growth, the effects of multiple drugs that inhibit NFκB, pyrrolidine dithiocarbamate, diethyldithiocarbamate, sulfasalazine, curcumin and bortezomib, were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells, xenograft flank tumors, and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB, IκB, was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition.
We report high constitutive activity of the canonical NFκB pathway, as seen by Western analysis of the NFκB subunit p65, in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though, conversely, the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally, expression of a dominant negative form of the endogenous inhibitor of NFκB, dnIκB, resulted in poor xenograft tumor growth, with average tumor volumes 40% smaller than controls.
These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth, and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes.
... Interestingly, in pediatric ALL, the incidence of TP53 mutation is considerably low [6], reflecting the fact that wild-type p53 harboring ALL cells can be targeted by agents that activate p53 signaling pathway [7]. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of nuclear factor-κB (NF-κB) has been shown to promote resistance to apoptosis [8,9]. NF-κB consists of p50 and p65 heterodimer retained in the cytoplasm by inhibitory proteins called IκBs. ...
bstract B cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common type of cancer in children. Dramatic improvements in primary therapy for childhood ALL have led to an overall cure rate of 80 %, providing opportunities for innovative combined-modality strategies that would increase cure rates while reducing the toxic side effects of current intensive regimens. In this study, we report that indole-3-carbinol (I3C), a natural phytochemical found in cruciferous vegetables, had anti-leukemic properties in BCPALL NALM-6 cells. I3C induced cell growth inhibition by G1 cell cycle arrest and triggered apoptosis in a dose-and time-dependent manner. p53, p21, and Bax proteins showed increased expression after I3C treatment. Real-time PCR analysis of pro-apoptotic p53 target genes revealed up-regulation of PUMA,NOXA,andApaf-1.I3Calsosuppressed constitutive nuclear factor-κB(NF-κB) activation and inhibited the protein expression of NF-kappa B-regulated antiapoptotic (IAP1, Bcl-xL, Bcl-2, XIAP) and proliferative (c-Myc) gene products. Coadministration of I3C with the topoisomerase II inhibitor, doxorubicin, potentiates cytotoxic effects compared with either agent alone. Apoptosis induction by the drug combination was associated with enhanced caspase-9 activation and PARP cleavage. Furthermore, I3C abolished doxorubicin-induced NF-κB activity as evidenced by decreased nuclear accumulationofp65,inhibition ofIκBα phosphorylation and its degradation, and decreased NF-κBDNAbinding activity. Western blot analysis revealed that doxorubicin-induced Bcl-2 protein expression was inhibited by I3C. Overall, our results indicated that using nontoxic agents, such as I3C, in combination with anthracyclines might provide a new insight into the development of novel combination therapies in childhood BCP-ALL.
... Recently, numerous mechanisms have been identified to be responsible for the development of resistance to first-line chemotherapeutic drugs. The initial response to the first-line chemotherapy drug may vary as tumor cells reemerge at a relatively high frequency during relapse in a sensitive population after subsequent treatment failures with various anticancer drugs [2][3][4][5][6][7][8]. Drug resistance is widely observed in various cancers because of their ability to survive through crosstalk with factors in multiple signaling pathways [9][10][11]. ...
Guanine nucleotide-binding protein-like-3-like (GNL3L) is a crucial regulator of NF-κB signaling that is aberrantly activated during diverse chemoresistance-associated cellular processes. However, the molecular mechanisms of GNL3L tumor initiation and resistant state are largely unknown. Moreover, the identification of predictive biomarkers is necessary to effectively generate therapeutic strategies for metastatic human colorectal cancer (CRC). This study aims to identify how cells acquire resistance to anticancer drugs and whether the downregulation of miR-4454 is associated with the progression of CRC. Here, we have shown that the overexpression of miR-4454 in resistant tumors is a crucial precursor for the posttranscriptional repression of GNL3L in human chemoresistant CRC progression, and we used doxycycline induced miR-4454 overexpression that significantly reduced tumor volume in a subcutaneous injection nude mice model. Together, these observations highlight that the downregulation of miR-4454 in resistant clones is prominently responsible for maintaining their resistance against anticancer drug therapy. Our study indicates that the development of miR-4454 as a microRNA-based therapeutic approach to silence GNL3L may remarkably reduce oncogenic cell survival that depends on GNL3L/NF-κB signaling, making miR-4454 a candidate for treating metastatic human CRC.
... Daunorubicin, another member of anthracyclines family, has been shown to activate NF-κB and inhibition of NF-κB has been reported to lead to dramatically improved cell death response as compared to the parental cell line HT1080 [54,58]. This phenomenon was observed in this work as well; an increase in the P65 level was observed in the cells that were treated with P-SSM in comparison with control. ...
This study aims to investigate the effects of co-administration of nano-Curcumin on anticancer effect of nano-Pirarubicin considering the fact that the clinical use of Pirarubicin and other anthracyclines has been limited by development of multidrug resistance (MDR) and based on previous data which have shown that Curcumin can down-regulate MDR proteins and suppress the activity of Nuclear factor-kappa B (NF-κB) a protein involved in chemoresistance. Nano micellar formulations of both Curcumin and Pirarubicin were separately synthesized using 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-[poly(ethylene glycol); PEG MW 2,000] (DSPE-PEG2000). The produced sterically stabilized micelles (SSM) are very well known drug delivery systems in terms of their enhanced efficacy and good toxicity profile. We measured the differences of NF-κB levels in two different situations: when Pirarubicin is used alone and when it is co-administered with Curcumin. This study showed that co-administration of SSM-Curcumin (CSSM) and SSM-Pirarubicin (PSSM) with size of 12.81 nm enhances the efficacy of Pirarubicin by suppressing P65, an NF-κB subunit. Furthermore, we showed that SSM is able to successfully enhance water solubility and therapeutic stability of curcumin for using as a complementary agent.
... In addition to its roles in causing cancer through inflammation, NF-κB is known to regulate a range of downstream anti-apoptotic genes, including cellular inhibitor of apoptosis 2 and Bcl-extra large (38,39). Owing to this anti-apoptotic function, NF-κB has been demonstrated to actively participate in the development of resistance to chemotherapy and radiotherapy in numerous neoplasms (40)(41)(42). Notably, it has been established that NF-κB is activated in response to treatment with chemotherapeutic drugs or irradiation (43,44). More specifically, inhibiting NF-κB activation enhances the cytotoxic effects of apoptotic inducers (26). ...
Nuclear factor κ-light-chain-enhancer of B cells (NF-κB) is one of the most important tumorigenic factors. Although it has been established that NF-κB is overly activated in human glioma cells, the molecular mechanisms that lead to the signal transduction to NF-κB and thereby the induction of resistance to apoptosis remain poorly understood. The present study demonstrated that mRNA and protein levels of E3 ubiquitin-protein ligase 2 (MIB2) were markedly upregulated in glioma cell lines and clinical samples. Immunohistochemical analysis also revealed high levels of MIB2 expression in glioma specimens. Ectopic overexpression of MIB2 was established in glioma cell lines to investigate its fundamental roles in the response of human glioma to apoptotic inducers. The results indicated that ultraviolet irradiation-induced cell apoptosis was inhibited with MIB2 overexpression in glioma cells. Notably, knockdown of MIB2 using RNA interference was able to increase the sensitivity of glioma cells to the pro-apoptotic agents. The present study identified that MIB2 induces NF-κB activation and facilitates the resistance of glioma cell to apoptosis. It was proposed that MIB2 may not only be an important hallmark to glioma disease progression, but that it may also offer novel clinical strategies to overcome resistance to cancer therapies.
... Interestingly, in pediatric ALL, the incidence of TP53 mutation is considerably low [6], reflecting the fact that wild-type p53 harboring ALL cells can be targeted by agents that activate p53 signaling pathway [7]. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of nuclear factor-κB (NF-κB) has been shown to promote resistance to apoptosis [8,9]. NF-κB consists of p50 and p65 heterodimer retained in the cytoplasm by inhibitory proteins called IκBs. ...
Background and Objective: The use of low-molecular-weight, nonpeptidic molecules that degrade the
interaction between the p53 protein and its negative regulator MDM2 (Murine- double minute colon 2) is a
new therapeutic strategy for treatment of various types of cancer. One of these agents is RITA (reactivation
of p53 and induction of tumor cell apoptosis) which binds to p53 protein and inhibits formation of p53- MDM2 complex while induces apoptosis in tumor cells by increasing p53 protein levels. The aim of this
study was to assess the possible in vitro apoptotic effects of RITA on pre-B ALL NALM-6 cells. Materials and Methods: NALM-6 cells were treated with different concentrations of RITA at different time
intervals. The viability of NALM-6 cells and apoptosis was measured by MTT assay and PI staining, respectively. The level of p53, acetylated p53 as well as cleaved PARP and procaspase-3 were determined by
Western blot in RITA-treated cells. The results were analyzed using Paired t test. Results: RITA has cytotoxic effects on NALM-6 cells. Flow cytometry analysis indicated increased
apoptotic cells in sub G1 region in treated cells (P< 0.05). The Western blot analysis revealed that protein
expression levels of p53, and its acetylation increased in response to RITA. In addition, RITA-induced
apoptosis associated with activation of caspase-3 and PARP cleavage. Conclusion: The results of this study showed that RITA induced p53-dependent apoptosis in pre-B ALL
NALM-6 cells.
... Interestingly, in pediatric ALL, the incidence of TP53 mutation is considerably low [6], reflecting the fact that wild-type p53 harboring ALL cells can be targeted by agents that activate p53 signaling pathway [7]. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of nuclear factor-κB (NF-κB) has been shown to promote resistance to apoptosis [8,9]. NF-κB consists of p50 and p65 heterodimer retained in the cytoplasm by inhibitory proteins called IκBs. ...
Background: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children. Currently, chemotherapy is the most effective method of leukemia cancer treatment which has many side effects. New strategies in cancer therapy utilize compounds that specifically target aberrant signaling pathway in order to reduce toxic side effect Indol-3-carbonil (I3C) found in vegetable has multiple anti-cancer properties because of its ability to modulate multiple cellular signaling pathways. In this study the molecular mechanism of the action of indol-3-carbonil on pre-B ALL cells was investigated.
... TNF-α is known to activate NF-kβ to protect tumor cells, and its upregulation leads to resistance to apoptosis and induces drug resistance in certain cancers [55]. Nevertheless, their expression and prognostic role in CRC remains unclear [56]. ...
Background:
Colorectal cancer (CRC) remains a deadly disease, afflicting the lives of millions worldwide. The prognosis of CRC patients is best predicted by surgical resection and pathological analysis of specimens. Emerging evidence has attributed a significant role to inflammatory markers and microRNAs (miRNAs) in the prognosis and survival of CRC patients.
Aim:
Here, we review the literature on inflammatory markers and miRNAs with an established role on survival rates, response to systemic chemotherapy, and other clinic-pathological parameters in CRC patients.
Results:
Our literature review revealed a critical role of inflammatory markers—specifically, the acute-phase proteins, inflammatory cytokines, and blood cell ratios—on prognostic outcomes in CRC patients. MiRNAs, on the other hand, were useful in predicting prognosis and clinical response and accordingly stratifying CRC patients for optimal drug selection.
Conclusion:
These biomarkers are easily measured in routine blood exams and can be used in adjunct to the tumor-node-metastasis (TNM) staging system to identify high-risk patients and those who are more likely to benefit from chemotherapy and other targeted therapies. However, more prospective studies are needed for the validation of these discussed prognostic and predictive biomarkers.
... Activated NF-κB induces the expression of various antiapoptotic genes that block cellular apoptosis, leading to drug resistance and accounting for the phenomenon of acquired chemoresistance, the main cause of treatment failure in cancer chemotherapy [9,10]. In light of these findings, a large number of studies in vitro and in vivo have shown that inhibiting the activation of NF-κB and its signaling pathway offers a potential cancer therapeutic strategy [9,[11][12][13][14]. However, only a few potential inhibitory compounds have been applied 1 3 to clinical use. ...
PurposeTreatment for high-risk neuroblastoma is still challenging. The purpose of the present study was to determine whether thalidomide suppresses etoposide-induced NF-κB activation and thus potentiates apoptosis in murine neuroblastoma. MethodsA murine neuroblastoma cell line, C1300, and A/J mice were used in this study. We evaluated NF-κB activation after using etoposide with or without thalidomide by quantitative analysis of NF-κB by ELISA and by Western blot analysis of IκB phosphorylation in vitro and in vivo. Induction of apoptosis was evaluated by Western blot analysis of the apoptotic signals caspase-3, 8, and 9 in vitro and by TUNEL assays in vivo. We also evaluated the efficacy of the combination of etoposide and thalidomide by assessing tumor growth and mouse survival in vivo. ResultsEtoposide activated NF-κB in C1300 cells. This activation was suppressed by thalidomide and IκB was re-upregulated. The apoptotic signals were enhanced by the combination of thalidomide and etoposide compared with etoposide alone in vitro, which was consistent with TUNEL assays. The combination of etoposide and thalidomide also slowed tumor growth and mouse survival. Conclusion
Thalidomide potentiates etoposide-induced apoptosis in murine neuroblastoma by suppressing NF-κB.
... We furthermore observed that attenuation of the CXCR2 signaling axis sensitized mammary tumor cells towards chemotherapy both in vitro and in vivo [6]. In addition, earlier reports have demonstrated the role of NF-kB in chemotherapy resistance and CXCR2 signaling [16,17]. ...
CXCR2 and its ligands have been shown to play an important role in tumor angiogenesis, therapy resistance and progression. In this study, we investigated whether CXCR2 ligands are responsible for the survival advantage and metastasis of drug-resistant cells and examined the underlying mechanism(s) doxorubicin or paclitaxel resistant mammary tumor cells. Our results demonstrated that drug-resistant Cl66 cells upregulated CXCR2 ligands but downregulated expression of CXCR2. We observed delayed tumor growth but increased metastasis in mice using these drug-resistant cells. Furthermore, we observed differential upregulation of stem cell and mesenchymal markers in the doxorubicin and paclitaxel-resistant tumor cells. Abrogation of the CXCR2 signaling axis using CXCR2 ligand neutralization resulted in significant inhibition of drug-resistant cell growth. Together, our data suggest chemotherapy-specific differential regulation of CXCR2 ligands, stem cell-like and mesenchymal phenotypes, and enhanced metastasis in drug-resistant cells and targeting CXCR2 signaling, may help circumvent therapy resistance in breast cancer.
... Hyperactivation of NF-B in cancer cells promotes cancer cell survival by inducing the upregulation of antiapoptotic proteins such as MnSOD and Bcl-2 family members and the inhibition of proapoptotic proteins and is linked directly to the inflammation-induced chemoresistance. NF-B protects against oxidative stress and activates transcription factor c-myc, MMP gene expression, and tumour angiogenesis and remodels extracellular matrix, while NF-B inhibition blocks cell proliferation [95,[103][104][105][106]. NF-B is associated with aberrant growth, resistance to apoptosis, and overexpression of the genes involved in cell cycle promotion in cancer cells. ...
Clinical efficacy of anticancer chemotherapies is dramatically hampered by multidrug resistance (MDR) dependent on inherited traits, acquired defence against toxins, and adaptive mechanisms mounting in tumours. There is overwhelming evidence that molecular events leading to MDR are regulated by redox mechanisms. For example, chemotherapeutics which overrun the first obstacle of redox-regulated cellular uptake channels (MDR1, MDR2, and MDR3) induce a concerted action of phase I/II metabolic enzymes with a temporal redox-regulated axis. This results in rapid metabolic transformation and elimination of a toxin. This metabolic axis is tightly interconnected with the inducible Nrf2-linked pathway, a key switch-on mechanism for upregulation of endogenous antioxidant enzymes and detoxifying systems. As a result, chemotherapeutics and cytotoxic by-products of their metabolism (ROS, hydroperoxides, and aldehydes) are inactivated and MDR occurs. On the other hand, tumour cells are capable of mounting an adaptive antioxidant response against ROS produced by chemotherapeutics and host immune cells. The multiple redox-dependent mechanisms involved in MDR prompted suggesting redox-active drugs (antioxidants and prooxidants) or inhibitors of inducible antioxidant defence as a novel approach to diminish MDR. Pitfalls and progress in this direction are discussed.
... Several types of cancer cells constitutively express NF-B, which contribute to their resistance to apoptosis induced by chemotherapeutic drugs [39]. Extensive studies reported in literature indicate that the anti-apoptotic protein Bcl-2 inhibits the release of apoptogenic cytochrome C from mitochondria into the cytosol [40,41], suggesting that Bcl-2 may participate in stabilizing mitochondria [42]. ...
... P53 is an important cellular signaling molecule that plays a significant role in apoptosis, genomic stability and inhibition of angiogenesis [49]. On the other hand, activation of NF-jB is known to inhibit apoptosis [50][51][52]. Thus, to explore the involvement of p53 and NF-jB, we measured the expression of these gene at the mRNA levels. ...
Carfilzomib is a proteasome inhibitor, commonly used in multiple myeloma, but its clinical use may be limited due to cardiotoxicity. This study was aimed to evaluate the influence of rutin in carfilzomib-induced cardiotoxicity in rats. Wistar albino male rats weighing 200-250 g (approximately 10 weeks old) were taken for this study. Animals were divided into four groups of six animals each. Group 1 served as normal control (NC), received normal saline; group 2 animals received carfilzomib (dissolved in 1 % DMSO) alone; group 3 animals received rutin (20 mg/kg) + carfilzomib; and group 4 animals received rutin (40 mg/kg) + carfilzomib. Hematological changes, biochemical changes, oxidative stress, hypertrophic gene expression, apoptotic gene expression, NFκB and IκB-α protein expression and histopathological evaluation were done to confirm the finding of carfilzomib-induced cardiotoxicity. Treatment with rutin decreased the carfilzomib-induced changes in cardiac enzymes such as lactate dehydrogenase, creatine kinase (CK) and CK-MB. For the assessment of cardiotoxicity, we further evaluated cardiac hypertrophic gene and apoptotic gene expression such as α-MHC, β-MHC and BNP and NF-κB and p53 gene expression, respectively, using RT-PCR. Western blot analysis showed that rutin treatment prevented the activation of NF-κB by increasing the expression of IκB-α. Rutin also attenuated the effects of carfilzomib on oxidant-antioxidant including malondialdehyde and reduced glutathione. Histopathological study clearly confirmed that rutin attenuated carfilzomib-induced cardiotoxicity in rats.
... NF-B activity also induces chemotherapy resistance and the inhibition of its activity could reverse the chemotherapy-resistant phenotype (Cusack et al., 1999). Arsenite has been shown to be a potent NF-B inhibitor (Kapahi et al., 2000). ...
Multiple myeloma (MM), a neoplasm of plasma cells, is the second most common hematological malignancy. Incidance rates increase after age 40. MM is most commonly seen in men and African-American population. There are several factors to this, such as obesity, environmental factors, family history, genetic factors and monoclonal gammopathies of undetermined significance (MGUS) that have been implicated as potentially etiologic. Development of MM involves a series of complex molecular events, including chromosomal abnormalities, oncogene activation and growth factor dysregulation. Chemotherapy is the most commonly used treatment strategy in MM. However, MM is a difficult disease to treat because of its marked resistance to chemotherapy. MM has been shown to be commonly multidrug resistance (MDR)-negative at diagnosis and associated with a high incidence of MDR expression at relapse. This review deals with the molecular aspects of MM, drug resistance mechanisms during treatment and also possible new applications for overcoming drug resistance.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
... Following cellular stimulation, IκB and NF-κB/p65 are phosphorylated by IκB-α kinase (IKK), leading to IκB degradation [33] and the DNA binding of NF-κB/p65, which initiates the transcription of its target genes [33,48]. Doxorubicin inhibits NF-κB activation in various cell lines [49]. In our previous study, we found that isomorellin inhibits NF-κB activation in KKU-100 and KKU-M156 cells by increasing the level of IκB-α in the cytosol and decreasing the expression and nuclear translocation of NF-κB/p65 protein [30]. ...
Background
Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous studies, isomorellin and forbesione, caged xanthones isolated from Garcinia hanburyi, were found to induce cell cycle arrest and apoptosis in CCA cell lines. The subject of our inquiry is the synergistic effect(s) of these caged xanthones with doxorubicin on growth inhibition and apoptosis induction in human CCA cell lines.
Methods
KKU-100, KKU-M139 and KKU-M156 cell lines and Chang cells were treated with either isomorellin or forbesione alone or in combination with doxorubicin. Cell viability was determined using the sulforhodamine B assay. The combined effects of plant compounds with doxorubicin were analyzed using the isobologram and combination index method of Chou-Talalay. Apoptosis was determined by ethidium bromide/acridine orange staining. Protein expressions were determined by Western blot analysis.
Results
Isomorellin or forbesione alone inhibited the growth of these CCA cell lines in a dose-dependent manner and showed selective cytotoxicity against CCA cells but not against Chang cells. Isomorellin/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-M139 and KKU-M156 cells, while the forbesione/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-100 and KKU-M139 cells. The percentages of apoptotic cells were significantly higher in the combined treatments than in the respective single drug treatments. The combined treatments strongly enhanced the expression of Bax/Bcl-2, activated caspase-9 and caspase-3, while suppressing the expression of survivin, procaspase-9 and procaspase-3, compared with single drug treatments. The degree of suppression of NF-κB activation mediated by a decrease in the expression of NF-κB/p65, a reduction of the pIκB-α level and an increase in the IκB-α protein level, was significantly higher in the combined treatment groups than in the single drug treatment groups. The degree of suppression of MRP1 protein expression was also significantly higher in the combined treatment than in the single drug treatment groups.
Conclusion
The combinations of isomorellin/doxorubicin and forbesione/doxorubicin showed significant synergistic effects on the growth inhibition and apoptosis induction in KKU-M156 and KKU-100 cells. Caged xanthones may be useful adjunct treatments with chemotherapy for Opisthorchis viverrini (OV)-associated CCA.
... Interestingly, in pediatric ALL, the incidence of TP53 mutation is considerably low [6], reflecting the fact that wild-type p53 harboring ALL cells can be targeted by agents that activate p53 signaling pathway [7]. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of nuclear factor-κB (NF-κB) has been shown to promote resistance to apoptosis [8,9]. NF-κB consists of p50 and p65 heterodimer retained in the cytoplasm by inhibitory proteins called IκBs. ...
B cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common type of cancer in children. Dramatic improvements in primary therapy for childhood ALL have led to an overall cure rate of 80 %, providing opportunities for innovative combined-modality strategies that would increase cure rates while reducing the toxic side effects of current intensive regimens. In this study, we report that indole-3-carbinol (I3C), a natural phytochemical found in cruciferous vegetables, had anti-leukemic properties in BCP-ALL NALM-6 cells. I3C induced cell growth inhibition by G1 cell cycle arrest and triggered apoptosis in a dose- and time-dependent manner. p53, p21, and Bax proteins showed increased expression after I3C treatment. Real-time PCR analysis of pro-apoptotic p53 target genes revealed up-regulation of PUMA, NOXA, and Apaf-1. I3C also suppressed constitutive nuclear factor-κB (NF-κB) activation and inhibited the protein expression of NF-kappa B-regulated antiapoptotic (IAP1, Bcl-xL, Bcl-2, XIAP) and proliferative (c-Myc) gene products. Coadministration of I3C with the topoisomerase II inhibitor, doxorubicin, potentiates cytotoxic effects compared with either agent alone. Apoptosis induction by the drug combination was associated with enhanced caspase-9 activation and PARP cleavage. Furthermore, I3C abolished doxorubicin-induced NF-κB activity as evidenced by decreased nuclear accumulation of p65, inhibition of IκBα phosphorylation and its degradation, and decreased NF-κB DNA-binding activity. Western blot analysis revealed that doxorubicin-induced Bcl-2 protein expression was inhibited by I3C. Overall, our results indicated that using nontoxic agents, such as I3C, in combination with anthracyclines might provide a new insight into the development of novel combination therapies in childhood BCP-ALL.
... In addition, the CPT-11 resistant strain also had significantly lower expression of Wnt/b-catenin pathway than the parental cells. NF-jB is known to mediate chemoresistance [42][43][44] as it is constitutionally activated by massive genetic mutations introduced by chemo-or radiotherapy [39]. In previous studies, NF-jB was found to be downstream to EGFR and IKKa/b [26,27], which is similar to the present study. ...
Chemotherapy is usually applied to treat colon cancer but leads to chemoresistance, and increased metastasis and invasion. The main focus of this study is to observe effects of resistance to irinotecan (CPT-11) on metastasis, invasion and autophagy in CPT-11 resistant (CPT-11-R) LoVo colon cancer cells. CPT-11, a topoisomerase I inhibitor and a first-line chemotherapeutic drug, is used to treat colon cancer. CPT-11-R cells were constructed in a step-wise fashion with increasing CPT-11 doses. The CPT-11-R strain had a significantly lower expression of Wnt/β-catenin pathway, but induced an EGFR/IKKα/β/NF-κB pathway with elevated cell cycle, metastasis and basal autophagy.
... Inhibition of the IKK/NF-κB activation pathway is considered an effective target for many anticancer drugs [43]. NF-kB inhibition in cancer cells has been shown to enhance chemotherapeutic response [44,45]. BT has also been reported to inhibit NF-kB signalling via inhibition of IkBα phosphorylation in vitro [14]. ...
Drug resistance is a cause of ovarian cancer recurrence and low overall survival rates. There is a need for more effective treatment approaches because the development of new drug is expensive and time consuming. Alternatively, the concept of 'drug repurposing' is promising. We focused on Bithionol (BT), a clinically approved anti-parasitic drug as an anti-ovarian cancer drug. BT has previously been shown to inhibit solid tumor growth in several preclinical cancer models. A better understanding of the anti-tumor effects and mechanism(s) of action of BT in ovarian cancer cells is essential for further exploring its therapeutic potential against ovarian cancer.
The cytotoxic effects of BT against a panel of ovarian cancer cell lines were determined by Presto Blue cell viability assay. Markers of apoptosis such as caspases 3/7, cPARP induction, nuclear condensation and mitochondrial transmembrane depolarization were assessed using microscopic, FACS and immunoblotting methods. Mechanism(s) of action of BT such as cell cycle arrest, reactive oxygen species (ROS) generation, autotaxin (ATX) inhibition and effects on MAPK and NF-kB signalling were determined by FACS analysis, immunoblotting and colorimetric methods.
BT caused dose dependent cytotoxicity against all ovarian cancer cell lines tested with IC50 values ranging from 19 muM - 60 muM. Cisplatin-resistant variants of A2780 and IGROV-1 have shown almost similar IC50 values compared to their sensitive counterparts. Apoptotic cell death was shown by expression of caspases 3/7, cPARP, loss of mitochondrial potential, nuclear condensation, and up-regulation of p38 and reduced expression of pAkt, pNF-kappaB, pIkappaBalpha, XIAP, bcl-2 and bcl-xl. BT treatment resulted in cell cycle arrest at G1/M phase and increased ROS generation. Treatment with ascorbic acid resulted in partial restoration of cell viability. In addition, dose and time dependent inhibition of ATX was observed.
BT exhibits cytotoxic effects on various ovarian cancer cell lines regardless of their sensitivities to cisplatin. Cell death appears to be via caspases mediated apoptosis. The mechanisms of action appear to be partly via cell cycle arrest, ROS generation and inhibition of ATX. The present study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer.
... Apo2L/TRAIL has emerged as a promising anticancer agent due to its selectivity in inducing apoptosis in many types of malignant cells while paring normal cells [10][11][12]. However, HCC cells are intrinsically resistant to Apo2L/TRAIL-induced apoptosis, which could be due to several distinct mechanisms, including compensatory activation of Akt [13,14] and NF-jB [15][16][17][18][19], increased expression of antiapoptotic Mcl-1 and cIAP2 [20][21][22], and decreased caspase-8 and caspase-3 activities [19]. Our data indicate that cotreatment of HCC cells with Apo2L/ TRAIL and OSU-A9 led to a dose-dependent reduction in the expression and nuclear localization of NF-jB/p65 by suppressing Akt activation and RelA expression, accompanied by parallel decreases in the expression of the NF-jB target gene products, including Bcl-xL, Mcl-1, cIAP1, cIAP2 and survivin. ...
Hepatocellular carcinoma (HCC) cells are intrinsically resistant to tumour necrosis factor-related apoptosis ligand (Apo2L/TRAIL), in part, due to the compensatory activation of nuclear factor-kappaB (NF-κB). To broaden the clinical utility of Apo2L/TRAIL in HCC, OSU-A9, a potent indole-3-carbinol-derived Akt/NF-κB signalling inhibitor, was used to overcome the intrinsic resistance. The antitumour effects of OSU-A9, Apo2L/TRAIL, and the therapeutic combination were assessed by MTT assays, caspase activation and PARP cleavage, and the synergistic interactions were determined by Calcusyn analysis. NF-κB reporter gene and RT-PCR were tested for the activation of NF-κB and the expression of death receptor (DR)4 and 5. OSU-A9 could sensitize HCC cells to Apo2L/TRAIL with high potency through down-regulation of Akt/NF-κB signalling. OSU-A9 dose-dependently reduced Akt phosphorylation and the expression and nuclear localization of RelA/p65, accompanied by parallel decreases in the expression of NF-κB target products, including Bcl-xL, Mcl-1, cIAP1, cIAP2, and survivin. Moreover, OSU-A9 increased DR5 expression through a ROS-dependent mechanism. Concertedly, these mechanisms underlie the synergistic interaction between OSU-A9 and Apo2L/TRAIL in mediating apoptotic death in HCC cells. The ability of OSU-A9 to accentuate Apo2L/TRAIL-induced apoptosis by inactivating Akt/NF-κB signalling might foster a promising therapeutic strategy for HCC. This article is protected by copyright. All rights reserved.
... NF-κB is constitutively activated in myeloma, with a number of NF-κB-activating gene mutations reported in myeloma cell lines and primary specimens [5,6]. Additionally, NF-κB is overexpressed in drug resistant myeloma cell lines and elevated in patients at time of relapse [7][8][9]. ...
Multiple myeloma cells are highly sensitive to the oncolytic effects of vesicular stomatitis virus (VSV), which specifically targets and kills cancer cells. Myeloma cells are also exquisitely sensitive to the cytotoxic effects of the clinically-approved proteasome inhibitor bortezomib. Therefore, we sought to determine if the combination of VSV and bortezomib would enhance tumor cell killing. However, as shown here, combining these two agents in vitro results in antagonism. We show that bortezomib inhibits VSV replication and spread. We found that bortezomib inhibits VSV-induced NF-κB activation and, using the NF-κB-specific inhibitor BMS-345541, that VSV requires NF-κB activity in order to efficiently spread in myeloma cells. In contrast to other cancer cell lines, viral titer is not recovered by BMS-345541 when myeloma cells are pre-treated with interferon (IFN)-β. Thus, inhibiting NF-κB activity, either with bortezomib or BMS-345541, results in reduced VSV titers in myeloma cells in vitro. However, when VSV and bortezomib are combined in vivo, in two syngeneic, immunocompetent myeloma models, the combination reduces tumor burden to a greater degree than VSV as a single agent. Intra-tumoral VSV viral load is unchanged when mice are concomitantly treated with bortezomib as compared to VSV treatment alone. To our knowledge, this is the first report analyzing the combination of VSV and bortezomib in vivo. Although antagonism between VSV and bortezomib is seen in vitro, analyzing these cells in the context of their host environment shows that bortezomib enhances VSV response, suggesting that this combination will also enhance response in myeloma patients.
... Also highlighting the important role of the NF-B pathway in drug resistance, microarray analysis of the FN-adhered cells revealed overexpression of 11 NF-B-regulated genes (58). Finally, NF-B has been found to be overexpressed in MM cell lines that have been selected for resistance to various chemotherapeutic agents (59), and NF-B levels have been found to be elevated in response to chemotherapy as well as in patient samples collected at the time of relapse (37,(60)(61)(62). Thus, inhibition of NF-B, using agents such as the proteasome inhibitor bortezomib, is likely vital for the successful treatment of multiple myeloma. ...
The Fanconi Anemia (FA)/BRCA DNA damage repair pathway plays a critical role in the cellular response to stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA DNA damage repair pathway genes are overexpressed and causative for resistance in multiple myeloma (MM) cell lines selected for resistance to melphalan. We hypothesized that the FA/BRCA DNA damage repair pathway mediates response and resistance to chemotherapeutic agents used to treat multiple myeloma and other cancers, and targeting this pathway is vital to overcoming drug resistance. In this dissertation, we show that FA/BRCA pathway genes are collectively overexpressed in MM, prostate, and ovarian cancer cell lines selected for resistance to melphalan and cisplatin, respectively. Interestingly, cells selected for resistance to topoisomerase II inhibitors selectively overexpress only FANCF.
We also show that FA/BRCA pathway expression can be inhibited by the proteasome inhibitor bortezomib. FA/BRCA pathway mRNA expression was inhibited by bortezomib in myeloma cell lines and patient samples. FANCD2 gene and protein expression are downregulated by bortezomib, and remain attenuated in the face of melphalan treatment. Melphalan-induced FANCD2 foci formation was also inhibited by bortezomib, and this drug enhanced melphalan-induced DNA damage, likely via inhibition of FA-mediated DNA damage repair. Next, we analyzed regulation of the FA/BRCA pathway. We demonstrate that NF-kappaB, specifically the Re1B/p50 subunits, transcriptionally regulates members of the FA/BRCA pathway, and inhibition of these subunits by siRNA, BMS-345541, and bortezomib reduces FA/BRCA pathway expression. Furthermore, knocking down Re1B and p50 simultaneously attenuates FANCD2 protein expression and results in diminished DNA repair and enhanced sensitivity to melphalan.
Importantly, melphalan resistance was restored when FANCD2 was re-expressed in these cells. We also show that bortezomib regulates FANCD2 protein expression directly, by inhibiting FANCD2 synthesis. Finally, we demonstrate that low-dose bortezomib arrests cells in G0/G1 and also overcomes the S-phase arrest induced by melphalan, likely via inhibition of ATR. Overall, our findings provide evidence for targeting the FA/BRCA pathway, either directly or indirectly, via inhibition of NF-kappaB or ATR, to enhance chemotherapeutic response and reverse drug resistance in multiple myeloma and other cancers.
... Deregulated activity of the transcription factor NF-B has frequently been observed in several cancers (22,33). The NF-B pathway impinges on multiple aspects of cell growth and apoptosis and has been shown to protect against various apoptotic stimuli (1), including chemotherapeutic agents (7,8). Hence, understanding the regulatory mechanisms that control NF-B in tumors is of paramount interest. ...
Antiapoptotic activity of NF-κB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IκB is
a seminal step in activation of NF-κB. The IκB kinases, IKK1 and IKK2, have been implicated in both IκB degradation and subsequent
modifications of NFκB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2−/−), we document a novel IκB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin
(DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IκBα. Degradation of IκBα is
partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-κB generated
by DoxR-induced IκB degradation in IKK1/2−/− cells is able to activate chromatin based NF-κB reporter gene and expression of the endogenous target gene, IκBα. These results
also imply that modification of NF-κB by IKK1 or IKK2 either prior or subsequent to its release from IκB is not essential
for NF-κB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2−/− MEFs is enhanced by simultaneous inhibition of NF-κB activation by blocking the proteasome activity. These results reveal
an additional pathway of activating NF-κB during the course of anticancer therapy and provide a mechanistic basis for the
observation that proteasome inhibitors could be used as adjuvants in chemotherapy.
... Activated NF-κB binds to specific DNA sequences in target genes, designated as kB elements and regulates transcription of over 400 genes involved in immunoregulation, growth, inflammation, carcinogenesis and apoptosis. Several chemotherapeutic agents including 5-fluorouracil (5-FU) have been reported to induce NF-κB activation in various cell lines (Cusack, 1999). Cytotoxic drugs induce NF-κB with delayed kinetics. ...
... It is considered that several factors can play a role. Among these mechanisms, the transcriptional nuclear factor-κB (NF-κB) is a key regulator of genes involved in cellular proliferation, secretion of soluble factors such as TNFα and up-regulation of antiapoptotic genes [15][16][17][18][19]. ...
Chemotherapeutic drugs like Adriamycin (ADR) induces apoptosis or senescence in cancer cells but these cells often develop resistance and generate responses of short duration or complete failure. The methylxantine drug Pentoxifylline (PTX) used routinely in the clinics setting for circulatory diseases has been recently described to have antitumor properties. We evaluated whether pretreatment with PTX modifies apoptosis and senescence induced by ADR in cervix cancer cells.
HeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, ADR or PTX + ADR. The cellular toxicity of PTX and survival fraction were determinated by WST-1 and clonogenic assay respectively. Apoptosis, caspase activation and ADR efflux rate were measured by flow cytometry, senescence by microscopy. IkappaBalpha and DNA fragmentation were determinated by ELISA. Proapoptotic, antiapoptotic and senescence genes, as well as HPV-E6/E7 mRNA expression, were detected by time real RT-PCR. p53 protein levels were assayed by Western blot.
PTX is toxic (WST-1), affects survival (clonogenic assay) and induces apoptosis in cervix cancer cells. Additionally, the combination of this drug with ADR diminished the survival fraction and significantly increased apoptosis of HeLa and SiHa cervix cancer cells. Treatments were less effective in HaCaT cells. We found caspase participation in the induction of apoptosis by PTX, ADR or its combination. Surprisingly, in spite of the antitumor activity displayed by PTX, our results indicate that methylxantine, per se does not induce senescence; however it inhibits senescence induced by ADR and at the same time increases apoptosis. PTX elevates IkappaBalpha levels. Such sensitization is achieved through the up-regulation of proapoptotic factors such as caspase and bcl family gene expression. PTX and PTX + ADR also decrease E6 and E7 expression in SiHa cells, but not in HeLa cells. p53 was detected only in SiHa cells treated with ADR.
PTX is a good inducer of apoptosis but does not induce senescence. Furthermore, PTX reduced the ADR-induced senescence and increased apoptosis in cervix cancer cells.
Multiple myeloma (MM, plasma cell myeloma) is a malignant hematologic disease characterized by the clonal proliferation of malignant plasma cells. The treatment of MM has changed dramatically in recent years, with the introduction of new drugs into therapeutic strategies, both in the front line setting and in relapsed refractory disease. However, most patients eventually relapse and often demonstrate multiple drug resistance. Therefore there is still an urgent and unmet need to define the molecular mechanisms of resistance for available drugs in order to enhance the use of existing treatments and design more effective therapies. Genetic abnormalities are well known to play a central role in MM resistance to available drugs, and epigenetic aberrations mainly affecting the patterns of DNA methylation and histone modifications of genes, especially tumor suppressors, can be involved in the resistance mechanism. Moreover, defects in the mechanisms of apoptosis, senescence and DNA repair could also contribute to drug resistance. In addition, mutations or alterations in the expression of the drug target can influence response to therapy. Achieving a better understanding of the pathways and protein expression involved in MM drug resistance and the development of novel therapeutic strategies are important goals for further progress in the treatment of MM. This review gives a critical overview of the role of cellular, microenvironmental and molecular mechanisms of drug resistance in MM.
Melanoma is notorious for its resistance to cytotoxic and radiation therapy, and patients with advanced melanoma has a poor prognosis. Despite development of new cytotoxic drugs, immunotherapies, and treatment strategies combining the two, overall survival of patients with metastatic melanoma has not improved significantly. Recent studies provide a modicum of optimism regarding our understanding of the biology of melanoma and potentially regarding targeted therapy for this disease. Genomic mutations in N-Ras, BRaf, and PTEN have demonstrated intimate involvement in the progression, invasion,and survival of neoplastic cells in most cutaneous melanomas. An increasing number ofdrugs inhibit the signal transduction pathways activated by these aberrations, and recent clinical studies have shown cautiously promising responses to these drugs. Many of these studies are also elucidating the role of angiogenesis and immunoregulation in melanoma, opening the door to a wide variety of new strategies for tumor environment modulation and immunostimulation for more effective melanoma therapy. Because of the complicated signal transduction pathways in melanoma cells and the complex interactions of these tumors with the surrounding environment and immune system, combining these drugs with other targeted therapies, cytotoxic agents, or immunotherapies will likely be required for successful treatment of melanoma.
The transcription factor nuclear factor kappa B (NF-κB) is a member of the Rel family of proteins that plays an important role in a variety of cellular response mechanisms including immunity, inflammation, cell growth, and apoptosis. Recent evidence suggests that this transcription factor is induced following exposure of cancer cells to apoptotic stimuli such as tumor necrosis factor (TNF), chemotherapy, and irradiation. The activation of NF-κ involves the proteasome-dependent degradation of an inhibitor of NF-κB and results in a cascade of events leading to the suppression of apoptosis. This mechanism of inducible cancer therapy resistance may be overcome by inhibitors of NF-κ activation. The preliminary studies that led to the development of novel cancer therapies that combine chemotherapy and proteasome inhibition are discussed.
It has been reported that (-)Menthol can inhibit the growth of rat liver epithelial tumor cells and is a potent chemopreventive agent. The purpose of the present experiment was to examine and identify cellular processes leading to cell death which are affected by (-)Menthol in human gastric SNU-5 cancer cells.. Cell death (cytotoxicity) was examined and analyzed by trypan blue stain and flow cytometric methods. It was shown that (-)Menthol inhibited the proliferation of the cells in a dose- and time-dependent manner, inhibited topoisomerase I, II alpha and II beta, but promoted the levels of NF-kappa B gene expression based on the Western blot and polymerase chain reaction (PCR) and cDNA microarray methods. These data suggest that (-)Menthol may induce cytotoxicity through inhibiting gene expression of topoisomerase I, II alpha a and II beta and promoting the gene expression of NF-kappa B in SNU-5 cells.
The first three clinical studies of bortezomib tested regimens of differing dose-intensities: once weekly for 4 wk on a 6 wk cycle (least intensive), twice weekly for 2 wk of a 3 wk cycle, and twice weekly for 4 wk of a 6 wk cycle (most intensive). From these studies, the intermediate-intensity regimen has been advanced, because it is the best tolerated but still achieves a high level of proteasome activity. Patients on this regimen treated with 1.0–1.50 mg/m2bortezomib had a reduction in proteasome activity to about 40% of baseline but recovered most activity within the 72 h period between doses. Doselimiting toxicities for this regimen were peripheral sensory neuropathy (PSN) and diarrhea. Patients with preexisting damage from prior neurotoxic chemotherapy may be more likely to develop PSN, and this possibility is being investigated in ongoing trials. Diarrhea is also adequately managed with loperamide. Notably, hematologic events in the early phase I trials were uncommon—thrombocytopenia was not dose-limiting; febrile neutropenia was rare; and hepatic, renal, and cardiotoxicity have not been noted. Given this favorable side effect profile, bortezomib may be particularly effective in combination-treatment regimens. In preclinical studies, bortezomib has shown at least an additive effect with CPT-11, gemcitabine, and docetaxel, and trials are in progress to determine the optimum dosing schedules for these combinations. In these ongoing trials, no unexpected or additive toxicities have been observed yet.
Suppression of apoptosis by survival signals is considered a hallmark of malignant transformation and resistance to anti-cancer therapy. The phosphoinositide-3 kinase (PI3k)/Akt pathway and NF-κB transcription factors are potent mediators of tumour cell survival. The carbocyclic lactone-lactam antibiotic rapamycin, a widely used immunosuppressant, inhibits the oncogenic transformation of human cells induced by PI3k or Akt by blocking the downstream mTOR kinase. However, inhibition of the PI3k/Akt/mTOR cascade may not be the only mechanism whereby rapamycin exerts anticancer effects. We previously demonstrated that rapamycin inhibits NF-κB by acting on FKBP51, a large immunophilin whose isomerase activity is essential for the functioning of the IKK kinase complex. This suggested that rapamycin may be effective also against neoplasias that express the tumour suppressor PTEN, which, by reducing cellular levels of phosphatidyl-inositol triphosphate, antagonizes the action of PI3k. To address this issue, we over-expressed PTEN in a human melanoma cell line characterized by high phospho-Akt and phospho-mTOR levels, and examined the effect of rapamycin on the apoptotic response to the NF-κB inducer doxorubicin versus cisplatin, which does not activate NF-κB. Rapamycin increased both cisplatin-and doxorubicin-induced apoptosis. Transient transfection of PTEN remarkably decreased phospho-mTOR levels and increased sensitivity to cisplatin's cytotoxic effect. Under these conditions, rapamycin failed to enhance cisplatin-induced apoptosis. This finding supports the notion that inhibition of a survival pathway increases the efficacy of cytotoxic drugs, and suggests that the pro-apoptotic effect of the rapamycin-cisplatin association requires activated mTOR. Rapamycin retained the capacity to enhance doxorubicin-induced apoptosis in cells over-expressing PTEN, which confirms our earlier observation that inhibition of the PI3k/Akt/mTOR pathway is not involved in the effect exerted by the rapamycin-doxorubicin association. These findings indicate that constitutive activation of mTOR is sufficient but not necessary for rapamycin's anti-cancer effect. Finally, we show that a decrease in FKBP51 expression levels, obtained with the small interfering RNA technique in the leukemic cell line Jurkat, increased doxorubicin-induced apoptosis, suggesting that this rapamycin ligand is involved in resistance to chemotherapy-induced apoptosis. In conclusion, rapamycin affects more than one signalling survival pathway and more than one target. Our data may impact on the synthesis of rapamycin derivatives. Thus far, rapamycin derivatives used in clinical trials have been tested for their mTOR-inhibiting effect. Our study opens the door to a novel class of anti-cancer drugs that specifically target immunophilins.
Ether á go-go 1 (Eag1) is frequently highly expressed in various malignant cancers and its excessive expression is correlated with poor prognosis in various cancers. However, the relationship of Eag1 expression with the clinical outcome of patients having ovarian cancer treated with cisplatin-based adjuvant chemotherapy is still unknown. In this study, we measured the expression of Eag1 in ovarian cancer and investigated the association between cisplatin chemosensitivity of ovarian cancer cells and Eag1 expression level. We demonstrate that decreased expression of Eag1 correlates with favorable prognosis in patients treated with cisplatin-based adjuvant chemotherapy and predicts higher cisplatin sensitivity in ovarian cancer cells. In vitro, knockdown of Eag1 by small interfering RNA facilitated the sensitivity of ovarian cancer cells (SKOV3 and TYK) to cisplatin-induced apoptosis via nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) pathway. Furthermore, knockdown of Eag1 expression was associated with decreased expression of the P-glycoprotein without affecting multidrug resistance-associated protein 1 expression. Taken together, Eag1 may serve as a potential indicator to predict Eag1 chemosensitivity, and silencing Eag1 may represent a potential therapeutic strategy for ovarian cancer.
© The Author(s) 2015.
Chemotherapeutic agents simultaneously induce transcription factors p53 and NFκB. p53 induction can activate an apoptotic program, and resistance to chemotherapy correlates with the loss of a functional p53 pathway. By contrast, NFκB prevents apoptosis in response to chemotherapeutic agents. We have analyzed the p53 response in IKK1/2−/− MEFs, which lack detectable NFκB activity. Compared to WT fibroblasts, IKK1/2−/− fibroblasts showed increased cell death and p53 induction in response to the chemotherapeutic agent, doxorubicin. Reconstitution of IKK2, but not IKK1, increased Mdm2 levels and decreased doxorubicin-induced p53 stabilization and cell death. IKK2-mediated effects required its kinase function and were abrogated by coexpression of the dominant negative IκBαM, implying a role for NFκB in blocking chemotherapy-induced p53 and cell death.
Le facteur de transcription NF-κB apparaît comme étant un acteur important du processus de tumorigenèse et a été décrit pour la première fois en 1986. En particulier, NF-κB est un facteur important de survie des cellules cancéreuses. Son activité est induite par des stimuli de nature variée dont des conditions de stress comme celles produites par les radiations ionisantes ou les substances de chimiothérapie utilisées en cancérologie. En retour, NF-κB induit l’expression d’un vaste répertoire de gènes qui rendent compte de ses actions pléiotropiques. De nombreuses études ont montré que l’inhibition de l’activation de NF-κB par divers procédés, augmente la sensibilité à l’effet apoptotique de nombreux stimuli comme le TNF ou de façon intéressante aux radiations ionisantes et à la chimiothérapie. De ces études a émergé le concept de l’utilisation thérapeutique du blocage de l’activation de NF-κB comme thérapie adjuvante du cancer. Cette revue a pour but de présenter les propriétés et les fonctions de NF-κB et de discuter de son intérêt en radiothérapie.
Chemotherapy causes unwanted side effects and chemoresistance limiting its effectiveness. Therefore, phytochemicals are now used as alternative treatments. Thymoquinone is used to treat different cancers, including colon cancer. Irinotecan resistant (CPT-11-R) LoVo colon cancer cell line was previously constructed by step-wise CPT-11 challenges to un-treated parental LoVo cells. Thymoquinone dose-dependently increased total cell death index and activated apoptosis at 2 μM, which then diminished at increasing doses. The possibility of autophagic cell death was then investigated. Thymoquinone caused mitochondrial outer membrane permeability (MOMP) and activated autophagic cell death. JNK and p38 inhibitors (SP600125 and SB203580, respectively) reversed TQ-autophagic cell death. Thymoquinone was also found to activate apoptosis before autophagy and direction of cell death was switched towards autophagic cell death at initiation of autophagosome formation. Therefore, thymoquinone resulted in caspase-independent, autophagic cell death via MOMP and activation of JNK and p38 in CPT-11-R LoVo colon cancer cells.
Rugulactone and its analogues were synthesized following Horners-Wadsworth-Emmons and ring-closing metathesis as the key reactions. A library of new Rugulactone analogues were designed, synthesized and evaluated for their anticancer activity in breast cancer cells. All analogues have shown anti-proliferative activity, while some of them exhibited significant cytotoxicity. In assays related to cell-cycle distribution, these conjugates induced G1 cell-cycle arrest in MDA-MB-231 cells. The cell cycle arrest nature was further confirmed by examining the effect on Cyclin E and Cdk2 proteins that acts at G1-S phase transition. Immunocytochemistry assay revealed that these compounds inhibited nuclear translocation of NF-kB protein, thereby activation of NF-kB was inhibited. The expression of NF-kB target genes such as Cyclin D1 and Bcl-xL were severely affected. Apart from acting on NF-kB, these compounds also regulate class I Histone deacetylase proteins such as (HDAC-3 and 8) that have a crucial and regulatory role in cell-proliferation. Simultaneously, the apoptotic inducing nature of these compounds was confirmed by activation of PARP protein, a protein that plays a key role in DNA damage and repair pathways. Among all compounds of this series 3gis the most potent compound and can be used for further studies.
Most NF-κB inhibitors target the IKK complex, IκB proteins, or NF-κB transcription factors. The most promising classes of inhibitors include antioxidants, antiinflammatory compounds, natural compounds, statins, proteasome inhibitors, IKKβ inhibitors, biologics, gene therapy, and RNA interference. Targeting NF-κB is limited by intrinsic pathway complexity, cross-talk with other pathways, a lack of biomarkers, poor drug specificity, drug resistance, and difficulty with drug delivery. Future NF-κB targeting will be improved through better understanding of the pathway, more specific inhibitors, and multimodality therapies.
Several reports within the last 5 years have suggested that nuclear factor (NF)-κB activation suppresses apoptosis through expression of anti-apoptotic genes. In the present report, we provide evidence from four independent lines that NF-κB activation is required for the cytotoxic effects of doxorubicin. We used doxorubicin and its structural analogues WP631 and WP744, to demonstrate that anthracyclines activate NF-κB, and this activation is essential for apoptosis in myeloid (KBM-5) and lymphoid (Jurkat) cells. All three anthracyclines had cytotoxic effects against KBM-5 cells; analogue WP744, was most potent, with an ic50 of 0.5μM, and doxorubicin was least active, with an ic50 of 2μM. We observed maximum NF-κB activation at 1μM with WP744 and at 50μM with doxorubicin and WP631, and this activation correlated with the IκBα degradation. Because the anthracycline analogue (WP744), most active as a cytotoxic agent, was also most active in inducing NF-κB activation and the latter preceded the cytotoxic effects, suggests that NF-κB activation may mediate cytotoxicity. Second, receptor-interacting protein-deficient cells, which did not respond to doxorubicin-induced NF-κB activation, were also protected from the cytotoxic effects of all the three anthracyclines. Third, suppression of NF-κB activation by pyrrolidine dithiocarbamate, also suppressed the cytotoxic effects of anthracyclines. Fourth, suppression of NF-κB activation by NEMO-binding domain peptide, also suppressed the cytotoxic effects of the drug. Overall our results clearly demonstrate that NF-κB activation and IκBα degradation are early events activated by doxorubicin and its analogues and that they play a critical pro-apoptotic role.
Purpose of review: To examine the links of nuclear factor-κB (NF-κB) to treatment-induced signaling in breast cancer and to propose further studies to elucidate the role of NF-κB in breast cancer response to chemotherapy and radiation. Recent findings: The authors' group and others have investigated the clinical relevance of ubiquitously expressed NF-κB in breast cancer. Possibly through its effects on apoptosis, NF-κB has been implicated in tumor resistance to chemotherapy and radiation in many types of tumors. Furthermore, both in vitro and in vivo studies have shown that targeted inhibition of NF-κB can sensitize tumor cells to chemotherapy and radiation. Summary: The molecular mechanisms involved in chemotherapy-induced and radiation-induced cell death in breast cancer are not fully known, nor are the mechanisms of treatment resistance. NF-κB is a transcription factor for a number of genes involved in tumor progression and resistance to systemic therapies and is a major regulator of the apoptotic pathway. Gaining further insights into molecular factors such as NF-κB as biomarkers for treatment response may help clinicians predict treatment outcome and lead to the development of targeted therapeutics.
Activation of the basic helix-loop-helix (bHLH) gene TAL1 is the most common genetic event seen in both childhood and adult T cell acute lymphoblastic leukemia (T-ALL). Despite recent success in treating T-ALL patients, TAL1 patients do not respond well to current therapies. In hopes of leading the way to better therapies for these patients, we have sought to determine the mechanism(s) of Tal1 induced leukemia in mice. By generating a DNA-binding mutant Tal1 transgenic mouse we have determined that the DNA binding activity of Tal1 is not required to induce leukemia. We have also shown that Tal1 expression in the thymus affects thymocyte development and survival. We demonstrate that Tal1 heterodimerizes with the class I bHLH proteins E47 and HEB in our mouse models of TAL1 induced leukemia. Severe thymocyte differentiation arrest and disease acceleration in Tal1/E2A+/- and Tal1/HEB+/- mice provides genetic evidence that Tal1 causes leukemia by inhibiting the function of the transcriptional activators E47 and HEB which have been previously shown to be important in T cell development. In pre-leukemic Tal1 thymocytes, we find the co-repressor mSin3A/HDAC1 bound to the CD4 enhancer, whereas an E47/HEB/p300 complex is detected in wild type thymocytes. Furthermore, mouse Tal1 tumors are sensitive to pharmacologic inhibition of HDAC and undergo apoptosis. These data demonstrate that Tal1 induces T cell leukemia by repressing the transcriptional activity of E47/HEB and suggests that HDAC inhibitors may prove efficacious in T-ALL patients that express TAL1.
The proteasome is a multiprotease complex that degrades the majority of cellular proteins in a highly regulated manner. The elimination of many key proteins by the proteasome is required for essential cellular processes, including cell-cycle progression, cell survival and cellular homeostasis. Conversely, inhibition of the proteasome results in cell-cycle arrest or programmed cell death. The observation that malignant cells were more susceptible to the effects of proteasome inhibition than normal cells raised the notion of proteasome inhibition as a novel approach in cancer therapy. The present review will focus on the potential for proteasome inhibitor therapy in cancer with bortezomib (Velcade(TM); formerly known as PS-341; Millennium Pharmaceuticals, Inc., Cambridge, MA, USA). Bortezomib is an extremely potent and selective proteasome inhibitor. In cell culture and animal models of cancer, it has :potent tumoricidal effects and sensitizes cancer cells to conventional anticancer agents. Bortezomib is the only proteasome inhibitor that has entered clinical trials in patients with cancer. With approximately 200 patients treated in phase I trials to date, bortezonnib has been generally well tolerated at doses that achieve a desired degree of proteasome inhibition. Encouraging antitumor activity has been observed. These data served as the basis for phase 11 clinical trials of bortezomib in patients with a broad range of tumors and also for clinical studies of bortezomib plus chemotherapy. The early results of combination trials show that bortezomib was generally well tolerated at doses that resulted in a good level of proteasome inhibition when combined with chemotherapy in patients. in these trials. No major overlapping toxicities have been observed to date and there was evidence of antitumor activity by many of the combinations tested in chemorefractory patients. Phase 11 or definitive phase III studies of bortezomib and chemotherapy will be considered after the completion of these initial trials and should serve to contribute to a further understanding of the potential role of bortezomib in the treatment of human malignancies in the near future.
Many cellular processes are regulated by the reversible reaction of protein phosphorylation on serine, threonine and tyrosine residues. Deregulation of the signal transduction cascade upsets this well-balanced system and has been implicated in many diseases, including cancer. Identification of the proteins and post-translational modifications (PTMs) involved in the breakdown of certain cell signaling pathways provide unique insight into the disease's pathology. The importance of phosphorylation on a molecular level has been implicated specifically within signaling pathways involved in the pathogenesis of cancer. Emerging phosphoproteomic technologies have proven to be valuable tools in identifying new biomarkers, which presents an opportunity to probe phosphoproteins in specific cancer related pathways for both diagnosis and therapeutic exploitation. This review will discuss the development and impact of current phosphoproteomic identification and quantification methodologies and the utility of these technologies to assist in the determination of phosphoproteins as potential biomarkers and/or drug targets in cancer.
New chemotherapeutic agents are still required to further optimise treatment of leukemia patients. Proteasome inhibition by bortezomib, PR-171 (carfilzomib) and NPI-0052 (salinosporamide A) has been successfully used for the treatment of multiple myeloma and mantle cell lymphoma and is considered also as novel treatment strategy in leukemia. Combination of proteasome inhibitors bortezomib and NPI-0052 induces synergistic anti-multiple myeloma activity both in vitro using multiple myeloma cells and in vivo in a human plasmacytoma xenograft mouse model. Cell death resulting from proteasome inhibition requires caspase activation and increased levels of reactive oxygen species. While bortezomib induces several caspases, NPI-0052 activates predominantly caspase-8-dependent pathway. We studied the effect of bortezomib (10 nM) on DNA synthesis and apoptosis in human acute myeloid cell lines KASUMI-1, ML-1, ML-2 and CTV-1 cells. Bortezomib was potent inhibitor of DNA synthesis in all four types of leukemia cells and induced apoptosis in KASUMI-1, ML-2 and CTV-1 cells but not in ML-1 cells. Other research groups showed that histone deacetylase in-hibitors (valproic acid or benzamide derivative MS-275) in combination with NPI-0052 or PR-171 induced greater levels of acute leukemia cell death than in combination with bortezomib. Proteasome inhibition as monotherapy and its combi-nation with many conventional therapies as novel treatment strategies in leukemia are promising. Malignant cells are more sensitive to this treatment than normal hematopoietic cells.
Resistance toward chemotherapy remains one of the principle obstacles to the effective treatment of malignancies. As our knowledge
of mechanisms involved in cancer biology expands, new molecular targets emerge. This chapter aims to overview the major resistance
mechanisms, in order to identify potential targets appropriate for developmental therapeutics. An emphasis on the role of
transcription factor NF-κB in colorectal cancer is presented as an example of how targeted therapies may advance from the
bench to the bedside.
Much has been written regarding the use of pro-inflammatory cytokines in the treatment of cancer. This stems from their potential
in the acute situation to induce death of diseased cells, as well as to destroy tumor blood vessels and activation of proteins
involved in apoptosis. However, as discussed earlier, it is important to consider that several chronic inflammatory conditions
lead to the development of the malignant phenotype. Examples include ulcerative colitis/Crohn disease, associated with colorectal
carcinoma; Barrett’s esophagus with esophageal cancer; schistosomiasis with bladder cancer; and Helicobacter pylori with gastric carcinoma/lymphoma. Stimulation of fibroblastic and tumor stroma growth, as well as induction of angiogenic
and anti apoptotic factors have all been reported in the presence of chronic inflammation (1,2). In addition, secretion of a pro-inflammatory chemokine, macrophage migration inhibitory factor (MIF) has been reported to
result in suppression of p53 function (3). This chapter will discuss the rationale for inhibiting pro-inflammatory cytokines and their potential use to treat cancer
and its sequelae. Given the availability of agents to block TNF and IL-1, these proteins will be discussed in detail.
Curcumin, a yellow pigment present in turmeric, possess potential anti-proliferative and anti-inflammatory activities but poor aqueous solubility limits its applications. In this study we report a novel comparative study of the formulation and characterization of curcumin nanoparticles (nanocurcumin) using two poly (lactide-co-glycolide) (PLGA) combinations, 50:50 and 75:25 having different lactide to glycolide ratios. Nanocurcumin 50:50 showed smaller size with higher encapsulation efficiency. Thermal evaluation suggested the presence of curcumin in molecular dispersion form which supported its sustained release up to a week where nanocurcumin 50:50 showed faster release. Cellular uptake studies in human epithelial cervical cancer cells (HeLa) exhibited enhanced intracellular fluorescence with nanocurcumin when compared to free curcumin, when both given in purely aqueous media. Antiproliferative studies using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Annexin V/propidium iodide staining, poly (ADP-ribose) polymerase (PARP) cleavage and downregulation of clonogenic potential of HeLa cells proved the better antitumor activity of nanocurcumin 50:50 administered in aqueous media. Superior efficacy of nanocurcumin 50:50 in comparison to free curcumin was further demonstrated by electrophoretic mobility shift assay and immunocytochemical analysis. In conclusion, the enhanced aqueous solubility and higher anticancer efficacy of nanocurcumin administered in aqueous media clearly demonstrates its potential against cancer chemotherapy, with dependence on the combination of PLGA.
Chemotherapy agents have been shown to induce the transcription factor nuclear factor-kappaB (NF-kappaB) and subsequent chemoresistance in fibrosarcomas and other cancers. The mechanism of NF-kappaB-mediated chemoresistance remains unclear, with a previous report suggesting that doxorubicin induces this response independent of the inhibitory kappaB kinases (IKK). Other studies have indicated that IKKbeta, but not IKKalpha, is required. Mouse embryo fibroblasts devoid of IKKalpha, IKKbeta, or both subunits (double knockout) were treated with doxorubicin. The absence of either IKKalpha or IKKbeta or both kinases resulted in impaired induction of NF-kappaB DNA-binding activity in response to doxorubicin. To provide a valid clinical correlate, HT1080 human fibrosarcoma cells were transfected with small interference RNA specific for IKKalpha or IKKbeta and then subsequently treated with doxorubicin. Knockdown of IKKalpha severely impaired the ability of doxorubicin to initiate NF-kappaB DNA-binding activity. However, a decrease in either IKKalpha or IKKbeta resulted in decreased phosphorylation of p65 in response to doxorubicin. The inhibition of doxorubicin-induced NF-kappaB activation by the knockdown of either catalytic subunit resulted in increased cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase and increased apoptosis when compared with doxorubicin alone. The results of this study validate current approaches aimed at NF-kappaB inhibition to improve clinical therapies. Moreover, we show that IKKalpha plays a critical role in NF-kappaB-mediated chemoresistance in response to doxorubicin and may serve as a potential target in combinational strategies to improve chemotherapeutic response.
Colorectal cancer is the third most common malignancy in the United States. Modest advances with therapeutic approaches that include oxaliplatin (L-OHP) have brought the median survival rate to 22 months, with drug resistance remaining a significant barrier. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is undergoing clinical evaluation. Although human colon carcinomas express TRAIL receptors, they can also demonstrate TRAIL resistance. Constitutive NF-kappaB activation has been implicated in resistance to TRAIL and to cytotoxic agents. We have demonstrated constitutive NF-kappaB activation in five of six human colon carcinoma cell lines; this activation is inhibited by quinacrine. Quinacrine induced apoptosis in colon carcinomas and potentiated the cytotoxic activity of TRAIL in RKO and HT29 cells and that of L-OHP in HT29 cells. Similarly, overexpression of IkappaBalpha mutant (IkappaBalphaM) or treatment with the IKK inhibitor, BMS-345541, also sensitized these cells to TRAIL and L-OHP. Importantly, 2 h of quinacrine pretreatment resulted in decreased expression of c-FLIP and Mcl-1, which were determined to be transcriptional targets of NF-kappaB. Extended exposure for 24 h to quinacrine did not further sensitize these cells to TRAIL- or L-OHP-induced cell death; however, exposure caused the down-regulation of additional NF-kappaB-dependent survival factors. Short hairpin RNA-mediated knockdown of c-FLIP or Mcl-1 significantly sensitized these cells to TRAIL and L-OHP. Taken together, data demonstrate that NF-kappaB is constitutively active in colon cancer cell lines and NF-kappaB, and its downstream targets may constitute an important target for the development of therapeutic approaches against this disease.
TGFβ1 treatment of B cell lymphomas decreases c-myc gene expression and induces apoptosis. Since we have demonstrated NF-κB/Rel factors play a key role in transcriptional control of c-myc, we explored the effects of TGFβ1 on WEHI 231 immature B cells. A reduction in NF-κB/Rel activity followed TGFβ1 treatment. In WEHI 231 and CH33 cells, we observed an increase in IκBα, a specific NF-κB/Rel inhibitor, due to transcriptional induction. Engagement of surface CD40 or ectopic c-Rel led to maintenance of NF-κB/Rel and c-Myc expression and protection of WEHI 231 cells from TGFβ1-mediated apoptosis. Ectopic c-Myc expression overrode apoptosis induced by TGFβ1. Thus, downmodulation of NF-κB/Rel reduces c-Myc expression, which leads to apoptosis in these immature B cell models of clonal deletion. The inhibition of NF-κB/Rel activity represents a novel TGFβ signaling mechanism.
The pathogenesis and etiology of Hodgkin's disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkin's disease and for developing new therapeutic strategies against it.
By differential screening of tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS)-
activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a
member of the inhibitor of apoptosis (iap) gene family. iap genes function to protect cells from
undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap1, hiap2,
and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory
cytokines TNF-α, interleukin 1β, and LPS, reagents that lead to activation of the nuclear transcription factor κB (NF-κB). Indeed, overexpression of IκBα, an inhibitor of NF-κB, suppresses the induced expression of iap genes and sensitizes ECs to TNF-α–induced apoptosis.
Ectopic expression of one member of the human iap genes, human X-chromosome–linked iap
(xiap), using recombinant adenovirus overrules the IκBα effect and protects ECs from TNF-α–
induced apoptosis. We conclude that xiap represents one of the NF-κB–regulated genes that
counteracts the apoptotic signals caused by TNF-α and thereby prevents ECs from undergoing
apoptosis during inflammation.
T cell receptor engagement activates transcription factors important for cytokine gene regulation. Additionally, this signaling
pathway also leads to activation-induced apoptosis in T lymphocytes that is dependent on FasL transcription and expression.
Here we demonstrate that nuclear factor κB (NF-κB), which is involved in the transcriptional regulation of many cytokine genes
expressed in activated lymphocytes, also plays a role in T cell activation-induced FasL expression. Inhibition of NF-κB activity
in a T cell hybridoma leads to decreased FasL expression and apoptosis upon T cell receptor stimulation. We identified the
NF-κB site in the FasL promoter that contributes to such regulation. Co-expression of p65 (Rel A) with the FasL promoter enhanced
its activity, and co-expression of IκB dramatically inhibited the inducible promoter activity. In contrast, the transcription
factor AP-1 is not required for activation-induced FasL promoter activity. These results define a role for NF-κB in mediating
FasL expression during T cell activation.
Bcl-2-family proteins are key regulators of the apoptotic response. Here, we demonstrate that the pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB. We show that bfl-1 gene expression is dependent on NF-kappaB activity and that it can substitute for NF-kappaB to suppress TNFalpha-induced apoptosis. bfl-1 promoter analysis identified an NF-kappaB site responsible for its Rel/NF-kappaB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kappaB in the immune system. The activation of Bfl-1 may be the means by which NF-kappaB functions in oncogenesis and promotes cell resistance to anti-cancer therapy.
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.
NF-κB, which consists of two polypeptides, p50 (M_r 50K) and p65/RelA (M_r 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses. Here we describe the generation of mice deficient in the RelA subunit of NF-κB. Disruption of the relA locus leads to embryonic lethality at 15–16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for IκBα and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-κB-regulated pathways.
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.
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.
NF-kappa B, which consists of two polypeptides, p50 (M(r) 50K) and p65/RelA (M(r) 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses. Here we describe the generation of mice deficient in the RelA subunit of NF-kappa B. Disruption of the relA locus leads to embryonic lethality at 15-16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for I kappa B alpha and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-kappa B-regulated pathways.
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.
TGF beta 1 treatment of B cell lymphomas decreases c-myc gene expression and induces apoptosis. Since we have demonstrated NF-kappa/Rel factors play a key role in transcriptional control of c-myc, we explored the effects of TGF beta1 on WEHI 231 immature B cells. A reduction in NF-kappa B/Rel activity followed TGF beta 1 treatment. In WEHI 231 and CH33 cells, we observed an increase in I kappa B alpha, a specific NF-kappa B/Rel inhibitor, due to transcriptional induction. Engagement of surface CD40 or ectopic c-Rel led to maintenance of NF-kappa B/Rel and c-Myc expression and protection of WEHI 231 cells from TGF beta 1-mediated apoptosis. Ectopic c-Myc expression overrode apoptosis induced by TGF beta 1. Thus, downmodulation of NF-kappa B/Rel reduces c-Myc expression, which leads to apoptosis in these immature B cell models of clonal deletion. The inhibition of NF-kappa B/Rel activity represents a novel TGF beta signaling mechanism.
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.
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.
Expression of nuclear factor-kappaB (NF-kappaB)/Rel transcription factors has recently been found to promote cell survival, inhibiting the induction of apoptosis. In most cells other than B lymphocytes, NF-kappaB/Rel is inactive, sequestered in the cytoplasm. For example, nuclear extracts from two human untransformed breast epithelial cell lines expressed only very low levels of NF-kappaB. Unexpectedly, nuclear extracts from two human breast tumor cell lines displayed significant levels of NF-kappaB/Rel. Direct inhibition of this NF-kappaB/ Rel activity in breast cancer cells induced apoptosis. High levels of NF-kappaB/Rel binding were also observed in carcinogen-induced primary rat mammary tumors, whereas only expectedly low levels were seen in normal rat mammary glands. Furthermore, multiple human breast cancer specimens contained significant levels of nuclear NF-kappaB/Rel subunits. Thus, aberrant nuclear expression of NF-kappaB/Rel is associated with breast cancer. Given the role of NF-kappaB/Rel factors in cell survival, this aberrant activity may play a role in tumor progression, and represents a possible therapeutic target in the treatment of these tumors.
The transcription factor NF-kappa B, more than a decade after its discovery, remains an exciting and active area of study. The involvement of NF-kappa B in the expression of numerous cytokines and adhesion molecules has supported its role as an evolutionarily conserved coordinating element in the organism's response to situations of infection, stress, and injury. Recently, significant advances have been made in elucidating the details of the pathways through which signals are transmitted to the NF-kappa B:I kappa B complex in the cytosol. The field now awaits the discovery and characterization of the kinase responsible for the inducible phosphorylation of I kappa B proteins. Another exciting development has been the demonstration that in certain situations NF-kappa B acts as an anti-apoptotic protein; therefore, elucidation of the mechanism by which NF-kappa B protects against cell death is an important goal. Finally, the generation of knockouts of members of the NF-kappa B/I kappa B family has allowed the study of the roles of these proteins in normal development and physiology. In this review, we discuss some of these recent findings and their implications for the study of NF-kappa B.
Transcription factors of the nuclear factor–κB/rel (NF-κB) family may be important in cell survival by regulating unidentified,
anti-apoptotic genes. One such gene that protects cells from apoptosis induced by Fas or tumor necrosis factor type α (TNF),IEX-1L, is described here. Its transcription induced by TNF was decreased in cells with defective NF-κB activation, rendering
them sensitive to TNF-induced apoptosis, which was abolished by transfection with IEX-1L. In support, overexpression of antisense IEX-1L partially blocked TNF-induced expression ofIEX-1L and sensitized normal cells to killing. This study demonstrates a key role of IEX-1L in cellular resistance to TNF-induced apoptosis.
Tumor necrosis factor α (TNF-α) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription
factor nuclear factor kappa B (NF-κB), which suppresses apoptosis by an unknown mechanism. The activation of NF-κB was found
to block the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins
c-IAP1 and c-IAP2 were identified as gene targets of NF-κB transcriptional activity. In cells in which NF-κB was inactive,
all of these proteins were required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 were sufficient to
suppress etoposide-induced apoptosis. Thus, NF-κB activates a group of gene products that function cooperatively at the earliest
checkpoint to suppress TNF-α–mediated apoptosis and that function more distally to suppress genotoxic agent–mediated apoptosis.
We investigated whether the cell growth and apoptosis of multiple cytokine-producing bladder cancer cells can be regulated by nuclear factor kappaB (NF-kappaB). The bladder cancer cell line KU-19-19, obtained from a 76-year-old man who demonstrated marked leukocytosis, produces multiple cytokines and demonstrates autocrine growth by granulocyte colony-stimulating factor (G-CSF). Electrophoretic mobility shift assay (EMSA) revealed that NF-kappaB was activated in KU-19-19 but not in other bladder cancer cell lines (KU-1, KU-7, or T-24, respectively). The inhibition of NF-kappaB DNA-binding activity with adenovirus vectors expressing the stable form of the NF-kappaB inhibitor IkappaBalpha (multiplicity of infection [MOI] of 10) inhibited growth and induced apoptosis of KU-19-19, but not KU-1, KU-7, or T-24. The production of several cytokines was suppressed significantly in KU-19-19 by this gene delivery. Although dexamethasone (10 microM) could also suppress cytokine production, it did not induce dramatic cell death in KU-19-19 because it could not inhibit NF-kappaB activation stably and strongly. These results suggest that NF-kappaB activation maintains the cell viability as well as regulates cytokine production in cytokine-producing cancer cells and therefore these in vitro experiments support a rationale for preclinical in vivo studies to demonstrate growth inhibition in established tumors.
Several reports indicated that nuclear factor kappaB (NF-kappaB) activation by cytokines, cytotoxic drugs, or ionizing radiation protects cells against apoptosis. Therefore, we investigated the consequence of NF-kappaB inhibition on the efficiency of antineoplastic agents. HPB, HCT116, MCF7, and OVCAR-3 cells stably expressing a dominant negative IkappaBalpha inhibitor showed a decreased NF-kappaB activation following treatment with tumor necrosis factor a and various chemotherapeutic agents. However, there was no difference in survival between parental cells and cells expressing mutated IkappaBalpha. These studies suggest that, at least in these cell lines, stable NF-kappaB inhibition did not modify the response to cytotoxic drugs.
In response to different extracellular signals, Rel/NF-kappaB transcription factors are critical regulators of apoptosis in a variety of cell types. Here we show that in normal B and T cells, expression of the Bcl-2 prosurvival homolog, A1, is rapidly induced in a Rel-dependent manner by mitogens. In B-cell lines derived from c-rel-/- mice, which like primary cells lacking Rel undergo apoptosis in response to antigen receptor ligation, constitutive expression of an A1 transgene inhibits this pathway to cell death. These findings are the first to show that Rel/NF-kappaB regulates physiologically the expression of a Bcl-2-like protein that is critical for the control of cell survival during lymphocyte activation.
Programmed cell death (apoptosis) seems to be the principal mechanism whereby anti-oncogenic therapies such as chemotherapy and radiation effect their responses. Resistance to apoptosis, therefore, is probably a principal mechanism whereby tumors are able to overcome these cancer therapies. The transcription factor NF-kappaB is activated by chemotherapy and by irradiation in some cancer cell lines. Furthermore, inhibition of NF-kappaB in vitro leads to enhanced apoptosis in response to a variety of different stimuli. We show here that inhibition of NF-kappaB through the adenoviral delivery of a modified form of IkappaBalpha, the inhibitor of NF-kappaB, sensitizes chemoresistant tumors to the apoptotic potential of TNFalpha and of the chemotherapeutic compound CPT-11, resulting in tumor regression. These results demonstrate that the activation of NF-kappaB in response to chemotherapy is a principal mechanism of inducible tumor chemoresistance, and establish the inhibition of NF-kappaB as a new approach to adjuvant therapy in cancer treatment.
NF-κB-regulated XIAP gene expression protects endothelial cells from TNFα-induced apoptosis
- C Stehlik
- R Martin
- I Kumabashiri
- J Schmid
- B Binder
Stehlik C, de Martin R, Kumabashiri I, Schmid J, Binder B, and Lipp J. NF-κB-regulated XIAP gene expression protects endothelial cells from TNFα-induced apoptosis. J Exp Med 1998; 188: 211–216.
NF-κB-regulated XIAP gene expression protects endothelial cells from TNFα-induced apoptosis
- Stehlik