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BCL-2: Found bound and drugged!
Abstract
The BCL-2 gene was first cloned in 1985 from the t(14;18) chromosomal translocation found in nearly all follicular lymphomas. BCL-2 is understood to have a central role in inhibiting apoptosis. Now, Oltersdorf et al. report the development of a high-affinity, mechanistically validated small-molecule antagonist of BCL-2 that kills cancer in mouse xenograft models and primary human cancer cells in vitro. The two decades spanning these two achievements provide an interesting case study of rational drug development. The investigation of antiapoptotic protein antagonists holds new promise for selectively inducing programmed cell death in cancer cells.
... BH3 mimetics bind into the BH3 groove of pro-survival BCL-2 proteins thereby displacing them from their inhibitory binding to BAX or BAK. [19][20][21][22] We utilized the BH3 mimetics ABT-737 and the BCL-2-specific compound . ABT-737 binds with high affinity (K i ⩽ 1 nM) to BCL-2, BCL-X L and BCL-W, but not to MCL-1 or A1. ...
... ABT-737 binds with high affinity (K i ⩽ 1 nM) to BCL-2, BCL-X L and BCL-W, but not to MCL-1 or A1. [19][20][21][22] The BCL-2-selective compound termed ABT-199 has a sub-nanomolar affinity for BCL-2 (K i o 0.010 nM) and binds 1 less avidly to BCL-X L (K i = 48 nM) or BCL-W (K i = 245 nM). 18 It has no measurable binding to MCL-1 (K i 4444 nM). ...
Deregulated apoptosis is an identifying feature of Myelodysplastic Syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-XL/-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/sAML patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34(+) cells and the decreased colony forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by ABT-737 or ABT-199 treatment and implies that BH3-mimetics might delay disease progression in higher-risk MDS or sAML patients.Leukemia accepted article preview online, 08 July 2015. doi:10.1038/leu.2015.179.
... 19,20 ABT-737, a BH3-only mimetic that binds Bcl-2, Bcl-X L and Bcl-w, acts by increasing the amount of free BH3-only proteins. [21][22][23][24][25][26] The death receptor pathway is stimulated by ligands from the tumor necrosis factor (TNF) family, including TNF-related apoptosis-inducing ligand (TRAIL), binding to death receptors DR-4 (TRAIL-R1) or DR-5 (TRAIL-R2)) on human cells, or DR-5 on murine cells. 27,28 Indeed, we have demonstrated that combining vorinostat with an agonistic anti-TRAIL receptor (TRAILR) antibody is more effective than single-agent treatment of breast cancer cell lines, 29,30 whereas ABT-737 resensitizes Bcl-2-and Bcl-X L -overexpressing lymphoma cells to vorinostat. ...
... Given that all four MM cell lines expressed high levels of Bcl-2 and/or Bcl-X L , we assessed their sensitivity to ABT-737. 23,24 All four cell lines were sensitive to ABT-737, with the U266 line being slightly more resistant (Figure 2b). ...
Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.
... Bax and Bak), which induce a death effect by promoting their insertion into mitochondrial membranes and oligomerization [6-8]. Bcl2 and related antiapoptotic proteins block the progression of a death signal by preventing Bax/Bak oligomerization [9]. ...
... Importantly, Mcl-1 is overexpressed in about 50% of HCC patients [1], suggesting that Mcl-1 is a potential therapeutic target for some patients with HCC. In contrast to Bcl2 and Bcl-XL, Mcl-1 is rapidly inducible with a shorter half-life and seems to be more widely expressed in HCC [1,9,14]. Mcl-1 is mainly localized to the outer mitochondrial membrane via its C-terminal TM domain [15,16]. ...
Glycochenodeoxycholate (GCDA) is one of the major human bile salts. Bile salts stimulate cell survival and proliferation through the mitogen-activated protein kinase, but the downstream signaling mechanism(s) remains enigmatic. Mcl-1 is an antiapoptotic molecule of the Bcl2 family that is extensively overexpressed in tumor tissues of patients with hepatocellular carcinoma (HCC).
Here we found that exposure of HepG2 cells to GCDA results in activation of ERK1 and ERK2 and phosphorylation of Mcl-1 in a PD98059 (MEK inhibitor)-sensitive manner. GCDA stimulates Mcl-1 phosphorylation in cells expressing WT but not T163A Mcl-1 mutant, indicating that GCDA-induced Mcl-1 phosphorylation occurs exclusively at the T163 site in its PEST region. GCDA-induced Mcl-1 phosphorylation at T163 enhances the half-life of Mcl-1. Treatment of HepG2 cells with GCDA facilitates Mcl-1 dissociation from Mule (a physiological Mcl-1 ubiquitin E3 ligase). Specific depletion of Mcl-1 from HepG2 cells by RNA interference increases sensitivity of HepG2 cells to chemotherapeutic drugs (i.e. cisplatin and irinotecan). In addition to activation of the ERK/Mcl-1 survival pathway, GCDA can also induce dose-dependent apurinic/apyrimidinic (AP) sites of DNA lesions, which may partially neutralize its survival activity.
Our findings suggest that bile salt may function as a survival agonist and/or potential carcinogen in the development of HCC. Molecular approaches that inactivate Mcl-1 by blocking its T163 phosphorylation may represent new strategies for treatment of HCC.
... Given that the development of an anti-apoptotic phenotype is one of the hallmark characteristic that is required for cells to become cancerous, our understanding of the processes that can lead to anti-apoptosis is probably best characterized in cancer cells [38,43,158,192,193]. In effect, the prototypical Bcl-2 family member, Bcl-2 was originally identified as an oncogene gene whose expression was increased in most follicular lymphomas due to a reciprocal chromosomal translocation (t14; 18) [194,195]. Overexpression studies revealed that Bcl-2 was an atypical oncogene since it did not serve to promote growth instead it was found to prevent cell death in response to different stresses. These studies served as a prelude to the development of our understanding of the central importance of the different pro-and anti-apoptotic members of the Bcl-2 family in the regulation of cell death [74]. ...
... The anti-apoptotic Bcl-2 is likely the most studied and potent antiapoptotic gene and it displays many of the characteristics of the ideal anti-apoptotic gene [44,195]. Bcl-2 is ubiquitously and constitutively expressed so that its inactivation results in enhanced cell death in response to stimuli. ...
Type I programmed cell death (PCD) or apoptosis is critical for cellular self-destruction for a variety of processes such as development or the prevention of oncogenic transformation. Alternative forms, including type II (autophagy) and type III (necrotic) represent the other major types of PCD that also serve to trigger cell death. PCD must be tightly controlled since disregulated cell death is involved in the development of a large number of different pathologies. To counter the multitude of processes that are capable of triggering death, cells have devised a large number of cellular processes that serve to prevent inappropriate or premature PCD. These cell survival strategies involve a myriad of coordinated and systematic physiological and genetic changes that serve to ward off death. Here we will discuss the different strategies that are used to prevent cell death and focus on illustrating that although anti-apoptosis and cellular survival serve to counteract PCD, they are nevertheless mechanistically distinct from the processes that regulate cell death.
... Instead of BAX/BAK activation, this subclass counterbalances antiapoptotic proteins. 69,70 Meanwhile, anti-apoptotic proteins block the death signaling pathway through the direct inhibition of activator BH3-only proteins or BAX/BAK activation. 71 Anti-apoptotic proteins (Bcl-2 and Bcl-xl) cause cancer progression 72 and therefore increase the resistance of tumor cells to different types of cell death stimuli, including anticancer drugs. ...
Cancer is the second leading cause of mortality worldwide. Conventional therapies , including surgery, radiation, and chemotherapy, have limited success because of secondary resistance. Therefore, safe, non-resistant, less toxic, and convenient drugs are urgently required. Natural products (NPs), primarily sourced from medicinal plants, are ideal for cancer treatment because of their low toxicity and high success. NPs cure cancer by regulating different pathways, such as PI3K/AKT/mTOR, ER stress, JNK, Wnt, STAT3, MAPKs, NF-kB, MEK-ERK, inflammation, oxidative stress, apoptosis, autophagy, mito-phagy, and necroptosis. Among the NPs, steroid saponins, including polyphyllins (I, II, D, VI, and VII), have potent pharmacological, analgesic, and anticancer activities for the induction of cytotoxicity. Recent research has demonstrated that polyphyllins (PPs) possess potent effects against different cancers through apoptosis, autophagy, inflammation, and necroptosis. This review summarizes the available studies on PPs against cancer to provide a basis for future research.
... This subclass neutralizes antiapoptotic proteins instead of directly activating BAX/BAK. 48,49 Meanwhile, antiapoptotic proteins block death signaling by directly inhibiting the activation of BAX/BAK or activator BH3-only proteins. 50 Antiapoptotic proteins, including Bcl-2 and Bclxl, are involved in cancer progression 51 and thereby induce the resistance of tumor cells to many types of apoptotic stimuli, including cytotoxic anticancer drugs. ...
The second foremost cause of mortality around the word is cancer. Conventional therapies, such as radiation, surgery, and chemotherapy have limited accessibility owing to secondary resistance. Therefore, convenient, safe, and nonresistant drugs are urgently needed. Plant-derived natural products have attracted considerable interest owing to their high efficacy, low toxicity, and convenience. Gypenosides (Gyp) inhibit invasion, migration, metastasis, and proliferation and induce apoptosis in different cancers, including oral, lung, colorectal, hepatocellular, and leukemic cancers through different molecular pathways. This review summarizes Gyp studies on cancer to serve as a reference for further research and clinical trials.
... This subclass neutralizes antiapoptotic proteins instead of directly activating BAX/BAK. 48,49 Meanwhile, antiapoptotic proteins block death signaling by directly inhibiting the activation of BAX/BAK or activator BH3-only proteins. 50 Antiapoptotic proteins, including Bcl-2 and Bclxl, are involved in cancer progression 51 and thereby induce the resistance of tumor cells to many types of apoptotic stimuli, including cytotoxic anticancer drugs. ...
The second foremost cause of mortality around the word is cancer. Conventional therapies, such as radiation, surgery, and chemotherapy have limited accessibility owing to secondary resistance. Therefore, convenient, safe, and nonresistant drugs are urgently needed. Plant-derived natural products have attracted considerable interest owing to their high efficacy, low toxicity, and convenience. Gypenosides (Gyp) inhibit invasion, migration, metastasis, and proliferation and induce apoptosis in different cancers, including oral, lung, colorectal, hepatocellular, and leukemic cancers through different molecular pathways. This review summarizes Gyp studies on cancer to serve as a reference for further research and clinical trials.
... The concept of selective shutting of the cell survival pathways in cancer cells has persisted in academia and drug industry (2). The idea received attention from the time when Korsmeyer first proposed a very simplistic rheostat model that a balance between pro-survival and pro-death family proteins can predict the life and death of cell (3). ...
Despite several decades of extensive research focus in the development of small molecule inhibitors that target the pro-survival BH-3 family members especially Bcl-2, a clinically successful agent is yet to emerge. The majority of agents either show poor target engagement in humans or their use is limited by acute toxicity, especially thrombocytopenia. In the recent paper in Science Translational Medicine , Leverson and colleagues investigate a novel combination of Bcl-2 family inhibitors that is predicted to present a less toxic yet highly selective way of targeting the Bcl-2 pathway (1).
... Agents that target Nrf2 fit into the second category [6]; aberrant Nrf2 activation appears to be a strategy whereby cancer cells are protected from elevated oxidative stress [173,174]. The third category includes drugs being developed to antagonize BCL-2's antiapoptotic function (e.g., ABT-737) [175]. ...
In the absence of oxygen human life is measured in minutes. In the presence of oxygen, normal metabolism generates reactive species (ROS) that have the potential to cause cell injury contributing to human aging and disease. Between these extremes, organisms have developed means for sensing oxygen and ROS and regulating their cellular processes in response. Redox signaling contributes to the control of cell proliferation and death. Aberrant redox signaling underlies many human diseases. The attributes acquired by altered redox homeostasis in cancer cells illustrate this particularly well. This teaching review and the accompanying illustrations provide an introduction to redox biology and signaling aimed at instructors of graduate and medical students.
Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
... To further test the possibility that an apoptotic-like program could be activated by coumarin A/AA, Bax and Bcl-2 protein expression was studied. These two proteins belong to the Bcl-2 family and are partially responsible for regulating the status of the mitochondrial permeability transition pore (mtPTP), thus allowing the release and translocation of diverse proapoptotic proteins from the mitochondria to the cytoplasm and nucleus [33][34][35]. Bax and Bcl-2 can determine the proapoptotic balance of the cell. In the present work, we have shown that 12 h of treatment with coumarin A/AA induces the overexpression of Bax protein and, 3 h later the inhibition of Bcl-2 protein expression ( Figure 6A) was observed. ...
... The former consists of Bax and Bak, which are essential for apoptosis [28]. The BH3-only proteins are further divided into two subclasses: "activators" (e.g., Bim and tBid), which directly activate Bax/Bak to induce mitochondrial outer membrane permeabilization (MOMP), and "sensitizers/derepressors" (e.g., Bad, Bik, Bmf, Hrk, Noxa, and Puma), which do not activate Bax/Bak directly but instead neutralize antiapoptotic proteins [29,30]. The central role that Bax/Bak play in apoptosis is supported by evidence that BH3-only proteins fail to trigger apoptosis in Bax/Bak-deficient cells [31,32]. ...
Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction.
... Other BH3-only proteins (i.e., Bad, Bik, Noxa, and PUMA) lack the ability to directly activate Bax but can oppose the action of antiapoptotic family members. Bcl2 and related antiapoptotic proteins block the progression of a death signal by preventing Bax/Bak oligomerization [22]. Importantly, we have recently discovered that the cigarette smoke components, nicotine and NNK, can induce phosphorylation of Bcl2, Mcl-1, Bax, and Bad, which leads to activation of Bcl2 and Mcl-1 [5,23] and inactivation of Bax and Bad in association with increased chemoresistance in human lung cancer cells [24,25]. ...
Lung cancer is the leading cause of cancer death and has a strong etiological association with cigarette smoking. Nicotine and nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are two major components in cigarette smoke that significantly contribute to the development of human lung cancer. Nicotine is able to stimulate survival of both normal human lung epithelial and lung cancer cells. In contrast to nicotine, NNK is a more potent carcinogen that not only induces single-strand DNA breaks and oxidative DNA damage but also stimulates survival and proliferation of normal lung epithelial and lung cancer cells. However, the molecular mechanism(s) by which nicotine and NNK promote cell survival, proliferation, and lung tumor development remains elusive. The fate of cells (i.e., survival or death) is largely decided by the Bcl2 family members. In the past several years, multiple signaling links between nicotine/NNK and Bcl2 family members have been identified that regulate survival and proliferation. This review provides a concise, systematic overview of the current understanding of the role of the pro- or antiapoptotic proteins in cigarette smoking, lung cancer development, and treatment resistance.
... Synergistic drug combinations can be rationalized using knowledge of single-drug action mechanisms. Since promoting apoptosis in tumor cells is a promising strategy for cancer therapy, we reasoned that directly enhancing proapoptotic Bax activities and simultaneously inhibiting antiapoptotic Bcl-2 protein function would achieve superior therapeutic effects (17,52). Indeed, the combination of our Bax activator with the Bcl-2 and Bcl-x L inhibitor ABT-737 functioned synergistically to induce apoptosis in human lung and pancreatic tumor cells, allowing decreased doses of each drug to achieve effects comparable to the effect of either drug alone at the usual dose. ...
The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus,
compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics.
In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen
approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly,
this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer
membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis
in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the
development of new therapeutic agents for cancer.
... 13 Meanwhile, antiapoptotic proteins such as Bcl-2 and Bcl-xl prevent apoptosis by inhibiting the action of pro-apoptotic proteins. 14,15 Accordingly, when the balance of activity between pro-and antiapoptotic members is upset, the permeability of mitochondrial membrane is lost and mitochondrial reactive oxygen species (ROS) is induced. 16,17 Apoptogenic proteins like cytochrome c or apoptotic inducing factors are then released to the cytosol, which activate pro-caspases to induce apoptosis. ...
Mitochondrial dysfunction and synaptic damage are critical early features of Alzheimer’s disease (AD) associated with amyloid β (Aβ) and τ. We previously reported that the scaffolding protein RanBP9, which is overall increased in AD, simultaneously promotes Aβ generation and focal adhesion disruption by accelerating the endocytosis of APP and β1-integrin, respectively. Moreover, RanBP9 induces neurodegeneration in vitro and in vivo and mediates Aβ-induced neurotoxicity. However, little is known regarding the mechanisms underlying such neurotoxic processes. Here, we show that RanBP9 induces the loss of mitochondrial membrane potential and increase in mitochondrial superoxides associated with decrease in Bcl-2, increase in Bax protein and oligomerization, fragmentation of mitochondria, and cytochrome c release. RanBP9-induced neurotoxic changes are significantly prevented by the mitochondrial fission inhibitor Mdivi-1 and by classical inhibitors of the mitochondrial apoptosis, XIAP, Bcl-2, and Bcl-xl. RanBP9 physically interacts with the tumor suppressor p73 and increases endogenous p73α levels at both transcriptional and post-translational levels;moreover, the knockdown of endogenous p73 by siRNA effectively blocks RanBP9 and Aβ1-42-induced mitochondria-mediated cell death. Conversely, siRNA knockdown of endogenous RanBP9 also suppresses p73-induced apoptosis, suggesting that RanBP9 and p73 have cooperative roles in inducing cell death. Taken together, these finding implicate the RanBP9/p73 complex in mitochondria-mediated apoptosis in addition to its role in enhancing Aβ generation.
... One promising BH3 mimetic is ABT-737 (developed by Abbott). ABT-737 is a mimetic of the BH3-only pro-apoptotic protein Bad, and is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X L , and Bcl-w with an affinity 2–3 orders of magnitude higher than any previously reported compounds (Letai, 2005; Oltersdorf et al., 2005 ). It acts like a BH3-only protein to antagonize antiapoptotic Bcl-2 family members, thereby diminishing their ability to inhibit apoptosis (Oltersdorf et al., 2005). ...
The BH3 mimetic ABT-737 is a potent inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L), and Bcl-w. The Bcl-2 family modulates sensitivity to anticancer drugs in many cancers, including melanomas. In this study, we examined whether ABT-737 is effective in killing melanoma cells either alone or in combination with a proteasome inhibitor already in clinical use (Bortezomib) in vitro and in vivo, and further evaluated the mechanisms of action. Results showed that ABT-737 alone induced modest cytotoxicity in melanoma cells, but only at higher doses. Knock-down of the anti-apoptotic proteins Bcl-2, Bcl-X(L), or Mcl-1 with siRNAs demonstrated that Mcl-1 is the critical mediator of melanoma's resistance to ABT-737 treatment. However, ABT-737 displayed strong synergistic lethality when combined with Bortezomib. Immunoblot analyses demonstrated that Bortezomib increased expression of Noxa, a pro-apoptotic Bcl-2 member that antagonizes Mcl-1. Additionally, siRNA-mediated inhibition of Noxa expression protected melanoma cells from cytotoxicity induced by the combination treatment. These results demonstrate that Bortezomib synergizes with ABT-737 by neutralizing Mcl-1's function via increased levels of Noxa. In a xenograft mouse model, although drug doses were limited due to toxicity, ABT-737 or Bortezomib slowed melanoma tumor growth compared to the control, and the drug combination significantly decreased growth compared to either drug alone. These data imply that less toxic drugs fulfilling a function similar to Bortezomib to neutralize Mcl-1 are promising candidates for combination with ABT-737 for treating melanomas.
... The mitochondrial apoptotic pathway is controlled by a balance between the proapoptotic protein members (i.e., the multidomain proapoptotic Bax, Bak, and BH3only proapoptotic Bid, Bim, Bad, Bik, Noxa, Puma, Bmf, Hrk) and antiapoptotic protein members (i.e., the multidomain antiapoptotic Bcl-2, Bcl-X L , Bcl-w, Mcl-1, Bfl/A1) of the Bcl-2 family. Those multidomain Bcl-2 proteins are functionally regulated by the BH3-only proteins (42,43). Bid is localized in the cytosolic fraction of cells as an inactive precursor (44) and truncated Bid (t-Bid), the active form of Bid, is generated upon proteolytic cleavage by caspase-8 (45,46). ...
Fenretinide (4-HPR) is a cytotoxic retinoid with minimal systemic toxicity that has shown clinical activity against recurrent high-risk neuroblastoma. To identify possible synergistic drug combinations for future clinical trials, we determined whether ABT-737, a small-molecule BH3-mimetic that inhibits most proteins of the antiapoptotic Bcl-2 family, could enhance 4-HPR activity in neuroblastoma.
Eleven neuroblastoma cell lines were tested for the cytotoxic activity of 4-HPR and ABT-737 as single agents and in combination using the DIMSCAN fluorescence digital imaging cytotoxicity assay. The effect of these agents alone and in combination on mitochondrial membrane depolarization and apoptosis (by flow cytometry), cytochrome c release, caspases, Bax-α, t-Bid, and Bak activation, and subcutaneous xenografts in nu/nu mice was also determined.
Multilog synergistic cytotoxicity was observed for the drug combination in all of the 11 neuroblastoma cell lines tested, including MDR lines and those insensitive to either drug as single agents. 4-HPR + ABT-737 induced greater mitochondrial membrane depolarization and mitochondrial cytochrome c release, greater activation of caspases, Bax-α, t-Bid, and Bak, and a higher level of apoptosis than either drug alone. In vivo, 4-HPR + ABT-737 increased the event-free survival of the MDR human neuroblastoma line CHLA-119 implanted subcutaneously in nu/nu mice (194.5 days for the combination vs. 68 days for ABT-737 and 99 days for 4-HPR).
Thus, the combination of 4-HPR with a BH3-mimetic drug warrants clinical trials in recurrent neuroblastoma.
... This could, at least partly, be mediated by Bim, a BH3-only pro-apoptotic protein. Bim was classified as an ''activator'' in view of its purported ability to act directly and to activate Bax and Bak [39]. It has been well documented that Bim is critical for HDACI-induced apoptosis of both solid tumor and leukemia cells [40,41]. ...
Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.
Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.
Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.
Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.
... Bim has been classified as an "activator" in view of its purported ability to engage directly and activate Bax and Bak. 39 It has been well documented that Bim is critical for HDACIinduced apoptosis of both solid tumor and leukemia cells. 33,34 In this study, we demonstrated that Bim also plays critical roles in cytarabine and cytarabine plus VPA induced apoptosis in pediatric AML cells. ...
To determine the possibility of synergistic antileukemic activity and the underlying molecular mechanisms associated with cytarabine combined with valproic acid (VPA; a histone deacetylase inhibitor and a Food and Drug Administration-licensed drug for treating both children and adults with epilepsy) in pediatric acute myeloid leukemia (AML).
The type and extent of antileukemic interactions between cytarabine and VPA in clinically relevant pediatric AML cell lines and diagnostic blasts from children with AML were determined by MTT assays and standard isobologram analyses. The effects of cytarabine and VPA on apoptosis and cell cycle distributions were determined by flow cytometry analysis and caspase enzymatic assays. The effects of the two agents on DNA damage and Bcl-2 family proteins were determined by Western blotting.
We showed synergistic antileukemic activities between cytarabine and VPA in four pediatric AML cell lines and nine diagnostic AML blast samples. t(8;21) AML blasts were significantly more sensitive to VPA and showed far greater sensitivities to combined cytarabine and VPA than non-t(8;21) AML cases. Cytarabine and VPA cooperatively induced DNA double-strand breaks, reflected in induction of γH2AX and apoptosis, accompanied by activation of caspase-9 and caspase-3. Further, VPA induced Bim expression and short hairpin RNA knockdown of Bim resulted in significantly decreased apoptosis induced by cytarabine and by cytarabine plus VPA.
Our results establish global synergistic antileukemic activity of combined VPA and cytarabine in pediatric AML and provide compelling evidence to support the use of VPA in the treatment of children with this deadly disease.
... A recently described inhibitor of Bcl-2, ABT-737 [17], has attracted much attention. This molecule provides a remarkable example of drug discovery [17,18] which has been reviewed previously [19,20]. Apoptosis induced by ABT-737 is dependent on Bax and Bak [11] suggesting that its primary mechanism of action is through regulating the intrinsic apoptosis pathway. ...
Conventional chemotherapy for cancer utilizes cytotoxic agents which elicit their therapeutic effect in part through the induction of apoptosis. In contrast, drugs which have been developed more recently and which are referred to as "targeted therapy" may exhibit less unwanted toxicity but in some cases these drugs are cytostatic. The recent development of drugs which target the apoptotic machinery offers a means to combine these two approaches. The intrinsic apoptotic pathway is controlled by the balance between anti-apoptotic proteins belonging to the Bcl-2 family and pro-apoptotic proteins bearing a single BH3 domain. Anti-apoptotic Bcl-2 family members are able to sequester the pro-apoptotic proteins by binding their BH3 domain. Compounds which inhibit this interaction are expected to promote apoptosis by preventing sequestration of the pro-apoptotic protein. Recently, a number of drugs have been developed which accomplish this, eg ABT-737, and some of these are progressing to clinical trials in oncology. These drugs may induce apoptosis on their own or synergize with existing chemotherapy. For example, ABT-737 is able to induce apoptosis when used as a single agent to treat leukemic and lung cancer cells and has also been shown to synergize with conventional chemotherapeutic agents in several cancer types. The spectrum of Bcl-2 family members expressed in a tumor cell, and the specificity of the inhibitor for these different anti-apoptotic proteins, helps determine whether Bcl-2 antagonists induce apoptosis when used as single agents. The ability of cytotoxic drugs to alter the expression of pro- and anti-apoptotic proteins is likely to help determine whether Bcl-2 antagonists synergize with cytotoxic therapy. Finally, as we begin to understand the pathways that regulate the expression of pro- and anti-apoptotic pathways, several new therapeutic strategies can be envisioned.
... To further test the possibility that an apoptotic-like program could be activated by coumarin A/AA, Bax and Bcl-2 protein expression was studied. These two proteins belong to the Bcl-2 family and are partially responsible for regulating the status of the mitochondrial permeability transition pore (mtPTP), thus allowing the release and translocation of diverse proapoptotic proteins from the mitochondria to the cytoplasm and nucleus [33][34][35]. Bax and Bcl-2 can determine the proapoptotic balance of the cell. In the present work, we have shown that 12 h of treatment with coumarin A/AA induces the overexpression of Bax protein and, 3 h later the inhibition of Bcl-2 protein expression ( Figure 6A) was observed. ...
It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral-blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis-inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase-3 was not detected. The overall results indicate that coumarin A/AA induces a caspase-independent apoptotic-like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology.
... Currently, there are no known drugs designed to activate Bcl-2. Indeed, the majority of drugs targeting Bcl-2 are inhibitors, which are being tested for their utility to selectively induce programmed cell death in cancer cells [46]. It seems logical that using the same approach to synthesize small molecule inhibitors to Bcl-2 should be able to generate activators of this anti-apoptotic molecule. ...
Mounting evidence suggests the involvement of caspases in the disease process associated with Alzheimer's disease (AD). The activation of caspases may be responsible for the neurodegeneration associated with AD and several recent studies have suggested that caspases may also play a role in promoting pathogenic mechanisms underlying this disease. Thus, caspase activation and cleavage of the amyloid precursor protein (APP) and tau may facilitate both the production of beta-amyloid (Abeta) as well as the formation of neurofibrillary tangles (NFTs). Because the activation of caspases in AD may be a proximal event that is not just associated with neurodegeneration, caspases are potential therapeutic targets for the treatment of this disorder. In this review, studies documenting the role of caspases in the AD brain will be discussed. In this context, a discussion of the therapeutic value of targeting caspase inhibition in the treatment of AD will be evaluated including drug targets, delivery and selectivity.
... ABT-737 (developed by Abbott Laboratories) is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X L , and Bcl-w with an affinity 2-3 orders of magnitude more potent than any previously reported compounds (Letai, 2005;Oltersdorf et al., 2005). It acts like a BH3-only protein to antagonize anti-apoptotic Bcl-2 family members, thereby diminishing their ability to inhibit apoptosis (Oltersdorf et al., 2005). ...
The Bcl-2 family is important in modulating sensitivity to anticancer drugs in many cancers, including melanomas. The BH3 mimetic ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2/Bcl-X(L)/Bcl-w. In this report, we examined whether ABT-737 is effective in killing melanoma cells in combination with the proteasome inhibitor MG-132, and further evaluated the mechanisms of action. Viability, morphological, and Annexin V apoptosis assays showed that ABT-737 alone exhibited little cytotoxicity, yet it displayed strong synergistic lethality when combined with MG-132. In addition, the detection of Bax/Bak activation indicated that the combination treatment synergistically induced mitochondria-mediated apoptosis. Furthermore, mechanistic analysis revealed that this combination treatment induced expression of the pro-apoptotic protein Noxa- and caspase-dependent degradation of the anti-apoptotic protein, Mcl-1. Finally, siRNA-mediated inhibition of Mcl-1 expression significantly increased sensitivity to ABT-737 in these cells, and knocking down Noxa expression protected the cells from cytotoxicity induced by the combination treatment. These findings demonstrate that ABT-737 combined with MG-132 synergistically induced Noxa-dependent mitochondrial-mediated apoptosis. In summary, this study indicates promising therapeutic potential of targeting anti-apoptotic Bcl-2 family members in treating melanoma, and it validates rational molecular approaches that target anti-apoptotic defenses when developing cancer treatments.
... Bcl-2 is an anti-apoptotic protein primarily expressed in mitochondria and the outer nuclear membrane, which prevents caspase-9 activation through an interaction with Apaf-1 ( LeBlanc, 2003). The rationale for using Bcl-2 is that Bcl-2 represents a critical convergence point that prevents the activation of the intrinsic receptor pathway of apoptosis implicated in AD (Su et al., 1996;Letai, 2005;Bredesen et al., 2006). Moreover, overexpression of Bcl-2 has been shown to prevent neuronal apoptosis (Martinou et al., 1994) and delay symptom onset in a transgenic mouse model of amyotrophic lateral sclerosis (Kostic et al., 1997). ...
Alzheimer's disease (AD) is characterized by the accumulation of plaques containing beta-amyloid (Abeta) and neurofibrillary tangles (NFTs) consisting of modified tau. Although Abeta deposition is thought to precede the formation of NFTs in AD, the molecular steps connecting these two pathologies is not known. Previous studies have suggested that caspase activation plays an important role in promoting the pathology associated with AD. To further understand the contribution of caspases in disease progression, a triple transgenic Alzheimer's mouse model overexpressing the anti-apoptotic protein Bcl-2 was generated. Here we show that overexpression of Bcl-2 limited caspase-9 activation and reduced the caspase cleavage of tau. Moreover, overexpression of Bcl-2 attenuated the processing of APP (amyloid precursor protein) and tau and reduced the number of NFTs and extracellular deposits of Abeta associated with these animals. In addition, overexpression of Bcl-2 in 3xTg-AD mice improved place recognition memory. These findings suggest that the activation of apoptotic pathways may be an early event in AD and contributes to the pathological processes that promote the disease mechanisms underlying AD.
Atrazine (ATR), a bio accumulative herbicide is frequently used in agriculture to control unwanted weeds. Due to continuous application, atrazine persists in the environment and causes deleterious impacts including neurotoxicity, hepatotoxicity, and gut microbiota disorders. Therefore, this study for the first time reports the variation in the gut microbiota, induction of process of apoptosis and autophagy in mice induced by ATR. Results indicated that TUNEL-positive hepatocytes suggestive of apoptosis were increased in livers of different experimental mice. Results on metabolic analysis in liver tissues indicated an overall change in seventy-six metabolites particularly Uridine 5'-diphosphate, Propenoylcarnitine and Chinenoside V resulting in generation of energy-related metabolic disorders and imbalance of oxidation/autoxidation status. Results on gut microbiome inquisition showed that ATR changed the richness and diversity of gut microbiota of mice and number of Firmicutes. Moreover, results also revealed that ATR induced apoptosis via disruption of apoptotic (Bax, Bcl2, and Casp3) and autophagy (LC3/Map1lc3a, Beclin1/Becn1 and P62/Sqstm1) genes. Results of our experimental study confirmed that changes in gut microbiota play a significant role in process of gut immune regulation and inflammation via different metabolites. In conclusion, the findings of our study provide a new idea for the involvement of mechanisms of detoxification in liver and inquisition of gut microbiota plays crucial role in regulation of physiological activities through liver-gut axis to mitigate toxic effects in animals.
Boswellia sacra Flueck essential oil is obtained from frankincense obtained from Boswellia sacra trees. The aim of this study was to investigate the in vitro anti-cancer activity of Boswellia sacra (BS) essential oil in CD133+ and CD133- Colo-320 cells. Essential oil was analyzed by GC-MS. CD133+ cells, cancer stem cells, were obtained from the Colo-320 by the MiniMACS system. Cytotoxicity were evaluated with MTT assay. Anti-cancer activity of BS essential oil was investigated by immunocytochemistry. 41 compounds were identified in the essential oil. 1:2 dilution BS essential oil was found to be active against all the cells for 24 h incubation. β-catenin immunoreactivity was significantly decreased in both BS essential oil-treated CD133+ and CD133- Colo-320 cells. Caspase-3 immunoreactivity significantly increased in BS essential oil-treated CD133- Colo-320 cells. We conclude that BS essential oil decreased β-catenin signaling molecules which play a crucial role in cancer cell proliferation. BS essential oil promoted apoptosis in CD133- Colo-320 cells.
Acute myeloid leukemia (AML) is a malignant disease of the bone marrow, comprising various subtypes. We have investigated seven different AML cell lines that showed different sensitivities toward the inducer of apoptosis ABT-737, with IC50 concentrations ranging from 9.9 nM to 1.8 µM. Besides, the AML cell lines revealed distinct differences in ¹⁸F-FDG uptake ranging from 4.1 to 11.0%. Moreover, the Pearson coefficient (0.363) suggests a moderate correlation between ¹⁸F-FDG uptake and the IC50 values of ABT-737. Differentiation of the AML cell lines NB-4 and AML-193 with all-trans-retinoic-acid (ATRA) induced a significant increase in sensitivity towards ABT-737 along with a reduced uptake of ¹⁸F-FDG. Therefore, ¹⁸F-FDG uptake could be predictive on sensitivity to treatment with ABT-737. Furthermore, because differentiation treatment of AML cells using ATRA reduced ¹⁸F-FDG uptake and increased sensitivity towards ABT-737, a combined treatment regimen with ATRA and ABT-737 might be a promising therapeutic option in the future.
A multitude of maleimides is grafted onto the backbone of a phosphorothioate antisense oligonucleotide (ASO) to generate the construct of maleimide-grafted ASO (Mal-g-ASO). Through click conjugation with cell membrane thiols...
Objective: To explore the effect of VEGF-C gene transfection on the expression of VEGF-C in human cervical carcinoma HeLa cells and the mechanisms of its anti-apoptosis effect. Methods: The constructed pcDNA3.1(+)A/EGF-C vector was transformed into human cervical cancer HeLa cells and was selected by G418. The changes in the expression level of VEGF-C mRNA and protein were determined by semi-quantitive RT-PCR and ELISA. HeLa cells with overexpression of VEGF-C were named as HeLa/S1. The expression level of NF-κB and bcl-2 mRNA was determined by RT-PCR in transfected cells. Results: After transfection by liposome, the VEGF-C mRNA level and the expression of VEGF-C protein in transfected cells were higher than those in the control groups. HeLa/S1 cell line was successfully established. In HeLa/S1 cells, the expression of NF-κB (2.06±0.09 vs 1.35±0.02 vs 1.38±0.02 P<0.05) and bcl-2 gene mRNA (2.02± 0.67 vs 0.41 ±0.06 vs 0.37±0.06, P<0.05) level were higher than those in the control groups. Conclusion: VEGF-C gene transfection by liposome can increase the expression of VEGF-C in human cervical cancer HeLa cells. NF-κB is stimulated and induces the overexpression of bcl-2 gene in HeLa/S1 cells.
The effect of combining MYB suppression with the histone deacetylase inhibitor LBH589 was studied in human myeloid leukemia cell lines. MYB knockdown inhibited proliferation and induced apoptosis in U937 and K562 cells in vitro, and also sensitized both to the pro-apoptotic effect of LBH589. This was accompanied by enhanced expression of the pro-apoptotic BCL2 family members BOK and BIM. U937 cells carrying inducible MYB shRNA were also transplanted into NOD/SCID mice. The combination of MYB knockdown and LBH589 prolonged survival compared to either treatment alone, suggesting that further development of such combinations might lead to effective and safe leukemia therapies.
A new and versatile strategy has been developed towards HA 14-1 analogues, selectively modified on position 4 and/or on the primary amine function. An important aspect was the appropriate selection of the phenol protective group in the 5-bromosalicylaldehyde, allowing the isolation of the key intermediate the 2H-benzopyrane-2-imine 2′.
Bu-Shen-Ning-Xin Decoction (BSNXD) administration has alleviated the early pathologic damage of atherosclerosis by inhibiting the adhesion molecule expression and upregulating the estrogen receptor (ER) β expression in endothelial cells, and increasing the serum nitric oxide (NO) level without any effect on serum lipid status, endometrium and fat deposition in liver in ovariectomized rabbits. The BSNXD-derived serum increases ER β expression in the human umbilical vein endothelial cells (HUVECs), and decreases malondialdehyde (MDA) production, and upregulates eNOS expression then increases NO synthesis through ERβ-dependent pathway. NO not only suppresses the LPS-induced NF-κB transcription in HUVECs, but also decreases apoptosis of endothelial cells. The BSNXD-derived serum decreases monocyte chemoattractant protein-1 production, and suppresses cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) expression in HUVECs injured by oxidized low-density lipoproteins (ox-LDL), and these effects can be abolished by ERβ antagonist (R,RTHC) and NO synthase inhibitor (L-NAME). The BSNXD-derived serum-treated HUVECs supernatant reduces CCR2, LFA-1 and VLA-4 expression in monocytes cell line U937 cells, which in turn inhibits adherence of U937 to injured endothelial cells. NO synthesis increases, and MDA production decreases through ERβ-mediated pathway that suppresses apoptosis and NF-κB activity in endothelial cells that downregulates adhesion molecules expression on endothelial cells via ERβ/NO/NF-κB pathway, and in turn leukocyte adhesion, which suggests BSNXD potential value in prophylaxis atherosclerosis.
The process of regulated or programmed cell death (PCD) executed through genetically encoded intrinsic cellular machinery is widely accepted to represent one of the key cellular responses to extrinsic and intrinsic stimulation. Extensive analysis of PCD carried out during the last decade clearly established that proper execution of PCD is important for normal mammalian development and also for homeostasis of the adult organism. Deregulation of PCD has been linked to development of many severe human diseases like cancer, autoimmunity, stroke, and some neurodegenerative diseases. Although apoptosis, which is the first discovered form of PCD, has been and remains the mainstay of PCD research, better understanding of the process of apoptosis led to the surprising discovery that other forms of PCD also exist and play multiple important roles in health and disease. Small-molecule inhibitors of apoptotic and nonapoptotic cell death have been successfully developed and proved very useful in defining the mechanisms and functional role of various PCD processes. Furthermore, some molecules have been developed extensively and represent emerging new therapies for human pathologies. In this article, we will discuss the major PCD-related protein targets of chemical inhibitors and describe the major classes of small molecule inhibitors developed to this point.
The proteasome inhibitors have emerged as a new and promising class of cancer therapeutics for hematological malignanices.
In multiple myeloma and mantle cell lymphoma, bortezomib is the first-in-class proteasome inhibitor to be approved by the
US Food and Drug Administration for treatment of these malignancies. In vitro and in vivo data are suggestive of the utility
of proteasome inhibitors for acute leukemias and importantly, a Phase-I clinical trial in adult leukemia patients demonstrated
biological activity (Cortes et al., Clin Cancer Res. 10:3371-3376, 2004). Here, we describe the rationale for targeting the
proteasome, the molecular pharmacology of the proteasome inhibitors and summarize results from clinical trials using proteasome
inhibitors as single agents and as a component of multidrug combination therapies. In acute lymphoblastic leukemia (ALL) patients
with refractory disease and poor outcomes, the proteasome inhibitors represent an untapped therapeutic resource. Since low
doses of proteasome inhibitors display striking synergy with low doses of other agents such as epigenetically targeted drugs
(Miller et al. (Blood. 110:267-277, 2007) (Blood. 113(18):4289-4299, 2009)), these investigations hold promise for pediatric
and young adult patients, where long-term toxicities and late effects could be minimized by optimizing the use of this class
of drugs.
Mutations and loss of the von Hippel-Lindau (VHL) tumor suppressor gene are associated with most renal cancers as well as several other types of human tumors, but the potential role of the VHL protein (pVHL) in patients with chondrosarcoma has not been characterized. The purpose of the present study was to investigate the expression profiles of pVHL in chondrosarcoma and its association with clinicopathologic parameters, Bax expression, the apoptosis index, and overall survival of patients with chondrosarcoma.
The messenger RNA (mRNA) and protein levels of VHL in fresh specimens from eight chondrosarcomas were studied with use of real-time polymerase chain reaction and Western blot, respectively. The protein expression of VHL and Bax was investigated by means of immunohistochemical analysis of paraffin-embedded clinical specimens from seventeen benign cartilage tumors and thirty-four chondrosarcomas. The apoptosis index in chondrosarcoma was examined by means of the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. Curves for overall survival were drawn according to the Kaplan-Meier method, and differences were analyzed with the log-rank test. The association of pVHL expression with the clinicopathologic parameters, Bax expression, apoptosis index, and overall survival for patients with chondrosarcoma was also analyzed.
Levels of VHL protein (p = 0.005) and mRNA (p = 0.008) were significantly reduced in chondrosarcoma tissues as compared with the paired adjacent normal tissues. Immunohistochemical analysis showed decreased pVHL in a significantly higher proportion of chondrosarcomas (64.7%) than benign cartilage tumors (29.4%). pVHL expression was positively correlated with Bax expression and the apoptosis index in chondrosarcoma. Longitudinal studies of a cohort of patients with chondrosarcomas showed that decreased pVHL expression significantly correlated with increased tumor grade (p = 0.026) but was not independently predictive of overall survival.
Reduced pVHL expression was associated with decreased apoptosis and increasing chondrosarcoma grade, but the relationship between these findings and chondrosarcoma pathogenesis requires further study.
To investigate the in vivo antitumor efficacy of quercetin in U937 xenografts and the functional roles of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia.
Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercetin as well as Mcl-1 expression and Bax activation were investigated in xenografts of U937 cells.
Administration of quercetin caused pronounced apoptosis in both transformed and primary leukemia cells but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was accompanied by Mcl-1 downregulation and Bax conformational change and mitochondrial translocation that triggered cytochrome c release. Knockdown of Bax by siRNA reversed quercetin-induced apoptosis and abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-mediated Bax activation, translocation, and cell death. Conversely, interruption of Mcl-1 by siRNA enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence of Bax had no effect on quercetin-mediated Mcl-1 downregulation. Furthermore, in vivo administration of quercetin attenuated tumor growth in U937 xenografts. The TUNEL-positive apoptotic cells in tumor sections increased in quercetin-treated mice as compared with controls. Mcl-1 downregulation and Bax activation were also observed in xenografts.
These data suggest that quercetin may be useful for the treatment of leukemia by preferentially inducing apoptosis in leukemia versus normal hematopoietic cells through a process involving Mcl-1 downregulation, which, in turn, potentiates Bax activation and mitochondrial translocation, culminating in apoptosis.
The Bcl-2 antagonist ABT-737 kills transformed cells in association with displacement of Bim from Bcl-2. The histone deactetylase
(HDAC) inhibitor suberoyl bis-hydroxamic acid (SBHA) was employed to determine whether and by what mechanism ABT-737 might
interact with agents that upregulate Bim. Expression profiling of BH3-only proteins indicated that SBHA increased Bim, Puma,
and Noxa expression, while SBHA concentrations that upregulated Bim significantly potentiated ABT-737 lethality. Concordance
between SBHA-mediated Bim upregulation and interactions with ABT-737 was observed in various human leukemia and myeloma cells.
SBHA-induced Bim was largely sequestered by Bcl-2 and Bcl-xL, rather than Mcl-1; ABT-737 attenuated these interactions, thereby triggering Bak/Bax activation and mitochondrial outer
membrane permeabilization. Knockdown of Bim (but not Puma or Noxa) by shRNA or ectopic overexpression of Bcl-2, Bcl-xL, or Mcl-1 diminished Bax/Bak activation and apoptosis. Notably, ectopic expression of these antiapoptotic proteins disabled
death signaling by sequestering different proapoptotic proteins, i.e., Bim by Bcl-2, both Bim and Bak by Bcl-xL, and Bak by Mcl-1. Together, these findings indicate that HDAC inhibitor-inducible Bim is primarily neutralized by Bcl-2
and Bcl-xL, thus providing a mechanistic framework by which Bcl-2 antagonists potentiate the lethality of agents, such as HDAC inhibitors,
which upregulate Bim.
Drug resistance remains a major clinical challenge for cancer treatment. One mechanism by which tumor cells develop resistance to cytotoxic agents and radiation is related to resistance to apoptosis. Apoptosis is a well-organised process of cell death pre-programmed inside the cell. Apoptosis can be initiated either by activation of death receptors on the cell surface membranes (extrinsic pathway) or through a series of cellular events primarily processed at mitochondria (intrinsic pathway). Apoptosis has been shown to be important for tumorigenesis and cancer treatment. Defects in apoptosis can result in the expansion of a population of neoplastic cells. However, because the death of tumor cells induced by chemotherapy and radiotherapy is largely mediated by activation of apoptosis, inhibition of apoptosis will make tumor cells resistant to anti-tumor treatment. Herein, we will review the molecular changes that have the potential to cause apoptotic dysregulation, including activation of antiapoptotic factors (Bcl-2, BCLX(L), Bfl1/A1 etc.), inactivation of pro-apoptotic effectors (p53, p53 pathway), and /or reinforcement of survival signals (Survivin, FLIP, NF-kappaB etc). Furthermore, we will discuss therapeutic intervention and/or strategies that can lower the threshold for apoptosis of tumor cells that could became useful approaches to treat cancer with special emphasis placed on the important priority to develop new cancer therapeutics toward tumor stem cells.
This study assessed the role of oxidative stress and loss of glutathione in ABT-737-induced apoptosis.
Jurkat human acute lymphocytic leukemia cells and HeLa cells transfected with a tet-regulated Bcl-2 expression system were treated with ABT-737 or its less active stereoisomer. GSH concentrations, intracellular reactive oxygen species (ROS), caspase activation and apoptotic DNA fragmentation were measured.
ABT-737 induced oxidative stress through decreased GSH and increased intracellular hydrogen peroxide and superoxide levels. Apoptotic DNA fragmentation and caspase activation were the consequences of this oxidative stress. Combining ABT-737 with ROS-inducing agents such as adaphostin or etoposide enhanced cell death.
These results demonstrate that inhibition of Bcl-2 causes a loss of GSH, an increase in ROS, caspase activation and subsequent apoptosis. Clinically, redox alterations as a consequence of Bcl-2 inhibition by ABT-737 should be considered in devising combination therapies with this novel agent or its derivatives.
Neoadjuvant chemotherapy may improve the outcome of esophageal cancer after esophagectomy, but is accompanied by considerable toxicity by collateral destruction of normal cells. Such side effects may be avoided by developing therapies that specifically target molecular characteristics of tumors. The aim of the present study was to determine the proportion of esophageal squamous cell carcinoma (ESCC) patients that could possibly benefit from (a combination of) currently available targeted therapies, by assessing the frequency of immunohistochemical expression of their target molecular markers in ESCC tissues. Sections from a validated tissue microarray comprising 108 ESCCs were immunohistochemically stained for Bcl-2, c-KIT, cyclo-oxygenase-2 (COX-2), cyclin D1, estrogen receptor (ER), epidermal growth factor receptor (EGFR), Her-2/neu, progesterone receptor (PR), and vascular endothelial growth factor (VEGF). VEGF, cyclin D1, EGFR, and COX-2 could be detected in 55, 42, 40, and 40%, respectively. Her-2/neu, Bcl-2, and c-KIT were detected in 12, 11, and 10% of the tumors, respectively. No nuclear expression of ER or PR was noticed. Concurrent expression of two markers was noticed in 28% of ESCCs, whereas 25% of ESCCs showed concurrent expression of three markers. The concurrent expression of two of the most frequently expressed markers (VEGF, cyclin D1, EGFR, and COX-2) ranged from 11 (COX-2 and EGFR) to 26% (cyclin D1 and VEGF). The expression of all of these four markers was seen in 5% of ESCCs. Promising targets for molecular therapy in ESCC appear to be COX-2, VEGF, EGFR, and cyclin D1, as they are frequently overexpressed. Phase II clinical studies on these molecular markers may therefore be warranted. The role for targeted therapy against ER, PR, Her-2/neu, c-KIT, or Bcl-2 in ESCC seems limited.
Nonsteroidal anti-inflammatory drugs (NSAIDs) exert anti-tumor action in a variety of cancer cells. However, several treatment side effects such as gastrointestinal injury, cardiovascular toxicity, and acute renal failure limit their clinical use. We found that indomethacin caused renal epithelial cell injury independently of cyclooxygenase inhibition. Indomethacin treatment was associated with the disruption of mitochondrial transmembrane potential, release of cytochrome c, down-regulation of Bcl-2 and Mcl-1, upregulation of Bax, and elevation of caspases activity. Enhanced Mcl-1 but not Bcl-2 expression alleviated indomethacin-increased caspase-3 activity. Down-regulation of Akt-related and signal transducer and activator of transcription (STAT-3)-related pathways was found in indomethacin-treated cells. Pharmacological and genetic studies revealed a potential mechanistic link between Akt/Mcl-1 and STAT-3/Mcl-1 signaling pathways and indomethacin-induced cytotoxicity. Mcl-1 is a determinant molecule for the induction of epithelial cell injury caused by indomethacin. Therefore, the maintenance of Mcl-1 levels is important for prevention of renal epithelial cell injury and apoptosis.
The Bcl-2 antagonist ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, which may confer resistance to this novel agent. Here, we show that Mcl-1 down-regulation by the cyclin-dependent kinase (CDK) inhibitor roscovitine or Mcl-1-shRNA dramatically increases ABT-737 lethality in human leukemia cells. ABT-737 induces Bax conformational change but fails to activate Bak or trigger Bax translocation. Coadministration of roscovitine and ABT-737 untethers Bak from Mcl-1 and Bcl-xL, respectively, triggering Bak activation and Bax translocation. Studies employing Bax and/or Bak knockout mouse embryonic fibroblasts (MEFs) confirm that Bax is required for ABT-737+/-roscovitine lethality, whereas Bak is primarily involved in potentiation of ABT-737-induced apoptosis by Mcl-1 down-regulation. Ectopic Mcl-1 expression attenuates Bak activation and apoptosis by ABT-737+roscovitine, whereas cells overexpressing Bcl-2 or Bcl-xL remain fully sensitive. Finally, Mcl-1 knockout MEFs are extremely sensitive to Bak conformational change and apoptosis induced by ABT-737, effects that are not potentiated by roscovitine. Collectively, these findings suggest down-regulation of Mcl-1 by either CDK inhibitors or genetic approaches dramatically potentiate ABT-737 lethality through cooperative interactions at two distinct levels: unleashing of Bak from both Bcl-xL and Mcl-1 and simultaneous induction of Bak activation and Bax translocation. These findings provide a mechanistic basis for simultaneously targeting Mcl-1 and Bcl-2/Bcl-xL in leukemia.
ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, which confers resistance to this novel agent. Here, we summarize recent findings indicating that Mcl-1 represents a critical determinant of ABT-737 sensitivity and resistance, and that Mcl-1 down-regulation by various pharmacologic agents or genetic approaches dramatically increases ABT-737 lethality in diverse malignant cell types. These findings also show that the multidomain proapoptotic proteins Bax and Bak play important functional roles in ABT-737-mediated apoptosis, and that Bak activation is essential in potentiation of ABT-737 lethality by agents that down-regulate Mcl-1. Collectively, these findings suggest a novel therapeutic strategy targeting multiple arms of the apoptotic machinery.
Defects in apoptosis signaling contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), and overexpression of antiapoptotic Bcl-2 (Bcl-2 and Bcl-X(L)) family proteins has been observed in ALL. ABT-737 is a small-molecule BH3-mimetic that inhibits the antiapoptotic Bcl-2 family proteins. We evaluated the cytotoxicity of ABT-737 in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in 7 ALL cell lines. Multilog synergistic cytotoxicity was observed in all 7 cell lines with ABT-737 plus L-asparaginase or vincristine, and in 5 of 7 cell lines with ABT-737 plus dexamethasone or VXL. In leukemia cells, but not in normal lymphocytes, ABT-737 plus L-asparaginase induced greater mitochondrial depolarization (JC-1 staining); mitochondrial cytochrome c release; activation of Bax, Bid, and caspases (immunoblotting); and eventually apoptosis (annexin V staining) than did either drug alone. In mouse xenografts derived from patients with ALL at diagnosis (ALL-7) or at relapse (ALL-19), event-free survival (EFS) was significantly enhanced with ABT-737 plus VXL relative to VXL or ABT-737 alone (P </= .02). Thus, ABT-737 synergistically enhanced VXL cytotoxicity in ALL cell lines via a mitochondrial death pathway and enhanced EFS in VXL-treated mice bearing ALL xenografts. Combining VXL with a BH3-mimetic warrants clinical investigation in ALL at relapse and potentially in chemotherapy-resistant ALL subgroups.
Induction of apoptosis in tumor cells by direct activation of the Bcl-2-regulated apoptosis pathway by small molecule drugs carries high hopes to overcome the shortcomings of current anticancer therapies. This novel therapy concept builds on emerging insights into how Bcl-2-like molecules maintain mitochondrial integrity and how pro-apoptotic BH3-only proteins lead to its disruption. Means to unleash the pro-apoptotic potential of BH3-only proteins in tumor cells, or to bypass the need for BH3-only proteins by directly blocking possible interactions of Bcl-2-like pro-survival molecules with Bax and/or Bak, constitute interesting options for the design of novel anticancer therapies. For the optimization and clinical implementation of these novel anticancer strategies, a detailed understanding of the role of individual BH3-only proteins in cell death signaling in healthy cells and during tumor suppression is required. In this review, we will touch on the latest findings on BH3-only protein function and attempts to define the molecular properties of the so-called 'BH3 mimetics,' a novel class of anticancer agents, able to prompt apoptosis in tumor cells, regardless of their p53 or Bcl-2 status.
A tetrahydroaminoquinoline-based library was generated with the goals of finding small molecule modulators of protein-protein interactions. Several library members as well as other related intermediates were tested for their ability to bind to Bcl-X(L) and Mcl-1 by in silico and (15)N NMR studies. The NMR study led to the identification of the tetrahydroaminoquinoline-based nude scaffold, 7 as a weak binder (K(d)=200 microM for Bcl-X(L) and K(d)=300 microM for Mcl-1) to both proteins. Using this scaffold as the starting material, we then synthesized a focused library of only 9 derivatives by applying the principles of a fragment-based approach. All these derivatives were then tested by NMR and this led to the discovery of a novel, small molecule (MIPRALDEN, 17) as a binder to Mcl-1 and Bcl-X(L) (K(D)=25 and 70 microM). This finding is novel because to our knowledge there are not many small molecules known in the literature that bind to Mcl-1.
TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.
Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.
Critical issues in apoptosis include the importance of caspases versus organelle dysfunction, dominance of anti- versus proapoptotic BCL-2 members, and whether commitment occurs upstream or downstream of mitochondria. Here, we show cells deficient for the downstream effectors Apaf-1, Caspase-9, or Caspase-3 display only transient protection from “BH3 domain-only” molecules and die a caspase-independent death by mitochondrial dysfunction. Cells with an upstream defect, lacking “multidomain” BAX, BAK demonstrate long-term resistance to all BH3 domain-only members, including BAD, BIM, and NOXA. Comparison of wild-type versus mutant BCL-2, BCL-XL indicates these antiapoptotics sequester BH3 domain-only molecules in stable mitochondrial complexes, preventing the activation of BAX, BAK. Thus, in mammals, BH3 domain-only molecules activate multidomain proapoptotic members to trigger a mitochondrial pathway, which both releases cytochrome c to activate caspases and initiates caspase-independent mitochondrial dysfunction.
Follicular lymphoma, the most common human lymphoma, characteristically has a t(14; 18) interchromosomal translocation. It is typically an indolent disease comprised of small resting B cells, but frequently develops into a high-grade lymphoma. The t(14; 18) translocates the Bcl-2 gene, generating a deregulated Bcl-2-immunoglobulin fusion gene. Bcl-2 is a novel inner mitochondrial membrane protein that extends the survival of certain cells by blocking programmed cell death. To determine the oncogenic potential of the t(14; 18) translocation, we produced transgenic mice bearing a Bcl-2-immunoglobulin minigene that structurally mimicked the t(14; 18). An indolent follicular hyperplasia in these transgenic mice progressed to a malignant diffuse large-cell lymphoma. The long latency, progression from polyclonal to monoclonal disease, and histological conversion, are all suggestive of secondary changes. Half of the immunoblastic high-grade lymphomas had a rearranged c-myc gene. Our transgenic mice provide an animal model for tumour progression in t(14; 18) lymphoma and show that prolonged B-cell life increases tumour incidence.
High levels of human bcl-2 protein(s) result in (i) the tumorigenic conversion of mouse NIH3T3 cells, (ii) the better survival of mouse myeloid cells in the absence of the required growth factor and (iii) give a growth advantage to human EBV-lymphoblastoid B cells both in low serum medium and limiting dilutions. The effect of the high levels of bcl-2 protein in EBV-B cells was further investigated. This revealed that high levels of bcl-2 alpha protein made EBV-B cells more resistant to a variety of stresses including the application of heat shock, ethanol, methotrexate and the absence of serum. Stress resistance was not observed in EBV-B cells with elevated level of c-myc protein. The mechanism of stress resistance conferred by the bcl-2 alpha protein is yet to be determined although the resistance does not seem to be the result of an increase in major heat shock proteins, hsp70 and hsp90, nor the arrest of cells in G1/G0 phase. The increased viability was observed in control transfectants but not in bcl-2 transfectants when cells are seeded at higher density in the absence of serum. Thus the improved survival of cells as a result of high levels of the bcl-2 alpha protein is not specific to the absence of growth factor but is found to occur with a variety of stresses.
Human follicular B cell lymphomas possess a t(14;18) interchromosomal translocation that juxtaposes the putative proto-oncogene bcl-2 with the immunoglobulin (Ig) heavy chain locus. We generated minigene constructs representing the bcl-2-Ig fusion gene found at this chromosomal breakpoint. These constructs were placed into the germ line of mice to assess the effects of the t(14;18) during development. The transgene demonstrates a lymphoid pattern of expression and uniformly results in an expanded follicular center cell population. Hyperplastic splenic follicles coalesce to form massive regions of splenic white pulp. Mice over 15 weeks of age demonstrate regional lymphadenopathy with abnormal cellular infiltrates. The expanded lymphoid compartment is composed predominantly of polyclonal B220-positive, IgM/IgD-positive B cells. Provocatively, the bcl-2-Ig transgene confers a survival advantage to a population of mature B cells assessed in vitro. bcl-2-Ig transgenic mice document a prospective role for the t(14;18) in B cell growth and the pathogenesis of follicular lymphoma.
The t(14;18)(q32;21) chromosomal translocation characteristic of follicular lymphomas is the most common cytogenetic abnormality known to be associated with any specific type of hematolymphoid malignancy. A fragment of DNA containing the crossover point between chromosomes 14 and 18 was cloned from the tumor cells of a patient with a follicular lymphoma carrying this translocation. Nucleotide sequence analysis of the breakpoint DNA revealed that the break in chromosome 14 occurred in joining region 4(J4) of the nonfunctional immunoglobulin heavy chain allele. This finding and other structural similarities of the breakpoint with the functional diversity region-joining region (D-J) joint in this lymphoma suggest that D-J recombination enzymes played a role in the mechanism of the t(14;18) translocation. Hybridization analysis of DNA from 40 follicular lymphomas showed that the majority of t(14;18) translocations occur on chromosome 18 DNA within 4.2 kilobases of the cloned breakpoint. A DNA probe from this breakpoint-cluster region detects transcription products in the tumor cells from which it was cloned and in a B-lymphoma cell line containing a t(14;18) translocation.
A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.
Recombinant DNA probes were cloned for the areas flanking the breakpoint on chromosome 18 in cells from a patient with acute
lymphocytic leukemia of the B-cell type; cells of this line carry the t(14;18) chromosomal translocation. Two of the probes
detected DNA rearrangements in approximately 60 percent of the cases of follicular lymphoma screened. In follicular lymphoma,
most of the breakpoints in band q21 of chromosome 18 were clustered within a short stretch of DNA, approximately 2.1 kilobases
in length. Chromosome 18-specific DNA probes for the areas flanking the breakpoints also detected RNA transcripts 6 kilobases
in length in various cell types. The gene coding for these transcript (the bcl-2 gene) seems to be interrupted in most cases
of follicular lymphomas carrying the t(14;18) chromosomal translocation.
Specific chromosomal translocations found in distinct neoplasms suggest that genes that flank such breakpoints play a critical role in transformation. We have characterized the t(14;18)(q32;q21) chromosomal translocation present in over 60% of human follicular lymphomas. We exploited an unexpected rearrangement of an Ig heavy-chain gene to clone the chromosomal breakpoint. An element isolated from 18q21 mediated translocations in all four t(14;18) bearing cell lines and in six of 11 follicular lymphomas, but did not normally rearrange in other B or non-B cells. The breakpoints clustered within a small 4.3 kb region on chromosome 18. The breakpoints on chromosome 14 were focused within or immediately 5' to JH. These breakpoints retained the Ig enhancer region close to a new transcriptional unit identified on chromosome segment 18q21. Since none of the cellular oncogenes are known to map to 18q21, cloning this element provides an opportunity to characterize a potentially new transforming gene.
Excerpt
The genetic control of tumorigenesis has been greatly advanced by the discovery of oncogenes. A central dogma holds that oncogenes induce an overt proliferation providing the driving force for oncogenesis. Cell death has long been recognized as a physiological event during embryonic development. Moreover, a distinct morphological form of cell destruction, apoptosis, had also been described even within cancer tissue (Kerr et al. 1972). In many respects, cancer can be viewed as a violation of normal tissue homeostasis. The maintenance of a relatively constant number of cells in normal tissues reflects a balanced equation between cell proliferation and cell death (Fig. 1). Aberrations of homeostasis that manifest as tumorigenesis would include events that promote proliferation or repress cell death. The history of cancer genetics is replete with examples of oncogenes that promote proliferation. However, bcl-2 provided the first certain example of an oncogene that regulated cell demise. The overexpression of...
A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites
of a protein are identified, optimized, and linked together to produce high-affinity ligands. The approach is called “SAR
by NMR” because structure-activity relationships (SAR) are obtained from NMR. With this technique, compounds with nanomolar
affinities for the FK506 binding protein were rapidly discovered by tethering two ligands with micromolar affinities. The
method reduces the amount of chemical synthesis and time required for the discovery of high-affinity ligands and appears particularly
useful in target-directed drug research.
We propose that deregulation of proliferation, together with a reduction in apoptosis, creates a platform that is both necessary and can be sufficient for cancer. The secondary traits of diverse neoplasms are a consequence of cell proliferation, tissue expansion, and other outcomes of this platform.
The "BH3-only" proteins of the BCL-2 family require "multidomain" proapoptotic members BAX and BAK to release cytochrome c from mitochondria and kill cells. We find short peptides representing the alpha-helical BH3 domains of BID or BIM are capable of inducing oligomerization of BAK and BAX to release cytochrome c. Another subset characterized by the BH3 peptides from BAD and BIK cannot directly activate BAX, BAK but instead binds antiapoptotic BCL-2, resulting in the displacement of BID-like BH3 domains that initiate mitochondrial dysfunction. Transduced BAD-like and BID-like BH3 peptides also displayed synergy in killing leukemic cells. These data support a two-class model for BH3 domains: BID-like domains that "activate" BAX, BAK and BAD-like domains that "sensitize" by occupying the pocket of antiapoptotic members.
Apoptosis, the cell-suicide programme executed by caspases, is critical for maintaining tissue homeostasis, and impaired apoptosis is now recognized to be a key step in tumorigenesis. Whether a cell should live or die is largely determined by the Bcl-2 family of anti- and proapoptotic regulators. These proteins respond to cues from various forms of intracellular stress, such as DNA damage or cytokine deprivation, and interact with opposing family members to determine whether or not the caspase proteolytic cascade should be unleashed. This review summarizes current views of how these proteins sense stress, interact with their relatives, perturb organelles such as the mitochondrion and endoplasmic reticulum and govern pathways to caspase activation. It briefly explores how family members influence cell-cycle entry and outlines the evidence for their involvement in tumour development, both as oncoproteins and tumour suppressors. Finally, it discusses the promise of novel anticancer therapeutics that target these vital regulators.
Programmed cell death is a distinct genetic and biochemical pathway essential to metazoans. An intact death pathway is required for successful embryonic development and the maintenance of normal tissue homeostasis. Apoptosis has proven to be tightly interwoven with other essential cell pathways. The identification of critical control points in the cell death pathway has yielded fundamental insights for basic biology, as well as provided rational targets for new therapeutics.
Resistance to apoptosis, often achieved by the overexpression of antiapoptotic proteins, is common and perhaps required in the genesis of cancer. However, it remains uncertain whether apoptotic defects are essential for tumor maintenance. To test this, we generated mice expressing a conditional BCL-2 gene and constitutive c-myc that develop lymphoblastic leukemia. Eliminating BCL-2 yielded rapid loss of leukemic cells and significantly prolonged survival, formally validating BCL-2 as a rational target for cancer therapy. Loss of this single molecule resulted in cell death, despite or perhaps attributable to the presence of other oncogenic events. This suggests a generalizable model in which aberrations inherent to cancer generate tonic death signals that would otherwise kill the cell if not opposed by a requisite apoptotic defect(s).
The expanding role of mitochondria in apoptosis
- Wang