Proteins of the bcl-2 family in apoptosis signalling: from mechanistic insights to therapeutic opportunities

1. Proteins of the Bcl-2 family are central regulators of apoptosis and are thought to act primarily on the mitochondria. 2. Members of the Bcl-2 family possess either anti-apoptotic or pro-apoptotic function. They are characterized by the presence of conserved sequence motifs, known as Bcl-2 homology (BH) domains. Anti-apoptotic members share all four BH domains, designated as BH1-4; the multidomain pro-apoptotic members contain BH1-3 domains, whereas another subgroup of pro-apoptotic members only have a BH3 domain. 3. The BH3-only proteins act as sensors for distinct apoptosis pathways, whereas multidomain pro-apoptotic Bax and Bak are executioners of death orders relayed by the BH3-only proteins. 4. Anti-apoptotic Bcl-2 family members appear to function, at least in part, by interacting with and antagonizing pro-apoptotic family members. The BH1-3 domains of BclXL form an elongated hydrophobic groove, which is the docking site for the BH3 domains of pro-apoptotic binding partners. 5. The deregulation of the various Bcl-2 proteins has been implicated in many pathological conditions. 6. Knowledge derived from the understanding of the function and regulation of the Bcl-2 family of proteins has allowed us to contemplate new therapeutic strategies for diseases where apoptosis signalling mechanisms can potentially be manipulated. 7. The anti-apoptotic Bcl-2 members have been targeted successfully using an antisense approach, BH3-peptides and small molecular weight chemicals that are inhibitors of their anti-apoptotic function.     

Inhibitors of the anti-apoptotic Bcl-2 proteins: a patent review

INTRODUCTION: The B-cell lymphoma-2 (Bcl-2) family of proteins is central to the regulation of apoptosis, which is vital for proper tissue development and cellular homeostasis. Anti-apoptotic proteins, members of the Bcl-2 family, are an important survival factor for many cancers and their overexpression has been associated with tumor initiation, progression, and resistance to current anticancer therapies. Therefore, strategies seeking to antagonize the function of Bcl-2 anti-apoptotic proteins have been extensively studied for developing a novel cancer therapy. AREAS COVERED: This review covers research and patent literature of the last 15 years dealing with the discovery and development of inhibitors of the Bcl-2 protein family. EXPERT OPINION: The feasibility of disrupting protein-protein interactions between anti-apoptotic and pro-apoptotic proteins, members of the Bcl-2 family, using peptidomimetics and small-molecule inhibitors has been successfully established. Three small-molecule inhibitors have entered human clinical trials, which will allow the evaluation of this potential therapeutic approach in cancer patients. It will be important to gain a better understanding of pan and selective Bcl-2 inhibitors in order to facilitate future drug design efforts.     

Studying the effect of LPS on cytotoxicity and apoptosis in PC12 neuronal cells: Role of Bax,Bcl-2, and Caspase-3 protein expression

Gram-negative bacteria and their endotoxins may be a causal or complicating factor in many serious diseases. Bacterial lipopolysaccharides (LPS) could potentially be human pathogens. Among the many disorders induced by LPS, neurodegenerative diseases such as Parkinson’s are reported and are of great interest. Despite the evidence on LPS-induced neurodegeneration, the exact mechanism is unknown. The purpose of this study was to investigate the cytotoxic effect of LPS and also to examine the involvement of Bax, pro-apoptotic, Bcl-2, anti-apoptotic, and caspase-3, the executioner of apoptosis, protein expression, during LPS-induced apoptosis in neuronal PC12 cells. The cell viability was evaluated by MTT assy. The expression of pro-apoptotic Bax and anti-apoptotic Bcl2 and caspase -3 protein expressions were measured by immunoblotting. The results showed that LPS could reduce cell viability after 72?h in a dose-dependent manner which was statistically significant in concentration of 200 µg/ml. In western blotting analysis, LPS (200 µg/ml) also enhanced expression of pro-apoptotic Bax and pro-caspase-3 proteins compared to controls, while the expression of Bcl-2 protein was not changed significantly. The Bax/Bcl-2 ratio was significantly increased in LPS-treated cells compared to controls. From the present results, it might be concluded that LPS can cause PC12 cell death, in which apoptosis plays an important role, possibly by the mitochondrial pathway through higher expression of the Bax as well as caspase 3 protein.     

作用于Bcl-2家族抗凋亡亚族蛋白的小分子抑制剂的研究进展

细胞的凋亡是维持机体平衡的重要因素。细胞凋亡由一系列细胞因子调控。Bcl-2蛋白家族是细胞凋亡的关键性调节因子。Bcl-2家族分为抗凋亡和促凋亡两个亚族，他们的相互作用对细胞凋亡信号传导起调控作用。很多肿瘤细胞高表达Bcl-2抗凋亡亚族成员Bcl-2/Bcl-xL。近年来，随着Bcl-2家族各成员的晶体结构相继阐明，人们开始寻找作用于Bcl-2家族抗凋亡亚族蛋白的小分子抑制剂。本文从药物设计角度对该方面的进展作一综述。     

HPMA copolymer-bound doxorubicin induces apoptosis in ovarian carcinoma cells by the disruption of mitochondrial function

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin has showed greater potency than free doxorubicin in the treatment of ovarian cancer in vivo and in vitro. The promising activity of the conjugate demonstrated in clinical trials has generated considerable interest in understanding the mechanism of action of this macromolecular therapeutic. In this study, the involvement of the mitochondrial pathway in HPMA copolymer-bound doxorubicin-induced apoptosis in the human ovarian cancer cell line A2780 was investigated. Through a series of in vitro assays, including confocal microscopy, flow cytometry, and spectrofluorimetry, a significant decrease in mitochondrial membrane potential in A2780 cells treated with HPMA copolymer-bound doxorubicin was found. The most dramatic changes in mitochondrial membrane potential were observed between 2 and 12 h of continuous drug exposure. The potential of the mitochondrial membrane remained collapsed when drug treatment continued up to 24 h. For the first time, it was shown that HPMA copolymer-bound doxorubicin induces apoptosis in ovarian cancer cells by simultaneous activation of both caspase-dependent and caspase-independent pathways of DNA damage. This was determined by monitoring the translocation of the mitochondrial proteins cytochrome c and apoptosis-inducing factor to cytosol. The altered balance between anti-apoptotic and pro-apoptotic members of the Bcl-2 family of proteins was responsible for the mitochondrial function distraction. HPMA copolymer-bound doxorubicin induced a time-dependent decrease in the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins, which control cell survival. At the same time, the expression level of pro-apoptotic members (Bax, Bad) of the Bcl-2 family was increased under the chosen experimental conditions. Altogether, these results indicate that HPMA copolymer-bound doxorubicin induced apoptosis in ovarian cancer cells through the mitochondrial pathway.     

Bcl-2-targeted cancer therapeutics

The antiapoptotic members of the Bcl-2 family of proteins play multiple roles in cancer. These membrane-integrated proteins inhibit the pro-apoptotic activity of oncogenes during oncogenesis, support the survival of established cancer cells, and increase resistance to chemotherapy. Hence, strategies aimed at inhibiting the expression or activity of Bcl-2 proteins are predicted to have therapeutic value. Several antisense oligonucleotides (AO), capable of reducing expression of either Bcl-2 or Bcl-X_L, were shown to induce apoptosis in cancer cells, to inhibit tumour growth in certain mouse tumour models, and to sensitise cancer cells to chemotherapy. One such AO, oblimersen, is presently being evaluated in combination with standard therapy in patients with advanced cancers, including chronic lymphocytic leukaemia and multiple myeloma. Bcl-2 proteins are thought to inhibit apoptosis by interacting with the pro-apoptotic proteins Bax and Bak, and preventing their activation. Small molecules capable of inhibiting this interaction have been discovered and shown to induce apoptosis in cancer cells. Gossypol and chelerythrine are two such molecules that inhibit tumour growth in mouse tumour models. This review summarises the evidence supporting the role of Bcl-2 proteins in cancer and then examines patented therapeutic strategies that target Bcl-2 protein expression or activities.     

Bcl-xL and Mcl-1 are involved in prevention of in vitro apoptosis in rat late-stage erythroblasts derived from bone marrow

Apoptosis controls erythroid homeostasis by balancing survival and death of erythroid cells. The mitochondrial pathway of apoptosis involves regulation of apoptotic events caused by the Bcl-2 family proteins, including the anti-apoptotic and pro-apoptotic members. However, little has been reported on the role of the anti-apoptotic Bcl-2 family members in rat late-stage erythroblasts that are no longer erythropoietin (EPO)-dependent. In the present study, to investigate this we analyzed changes in apoptosis-related factors that occurred in vitro. EPO stimulation resulted in reduced apoptotic cell death of the late-stage erythroblasts accompanied by decreased caspase-3 and caspase-9 activities, which is indicative of the induction of apoptosis through the mitochondrial pathway. Analysis of mRNA expression of the Bcl-2 family proteins demonstrated that EPO stimulation up-regulated the Bcl-xL mRNA, resulting in decreases in the mRNA ratios of Bak, Bax, and Bad to Bcl-xL. Also, the mRNA ratios of Bak and Noxa to Mcl-1 were decreased, mainly due to up-regulation of Mcl-1 mRNA. These results showed a close association between reduced apoptotic cell death and increased mRNA levels of Bcl-xL and Mcl-1 in the presence of EPO. Thus, the present study suggests that Bcl-xL may be an important anti-apoptotic factor of rat late-stage erythroblasts as has been reported in murine erythroblasts. Moreover, the results also indicate the possibility that Mcl-1 may act on the rat late-stage erythroblasts as an anti-apoptotic factor.     

Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain.

1. This study was designed to assess the influence of activation and blockade of the endogenous opioid system in the brain on two key proteins involved in the regulation of programmed cell death: the pro-apoptotic Fas receptor and the anti-apoptotic Bcl-2 oncoprotein. 2. The acute treatment of rats with the mu-opioid receptor agonist morphine (3-30 mg x kg(-1), i.p., 2 h) did not modify the immunodensity of Fas or Bcl-2 proteins in the cerebral cortex. Similarly, the acute treatment with low and high doses of the antagonist naloxone (1 and 100 mg x kg(-1), i.p., 2 h) did not alter Fas or Bcl-2 protein expression in brain cortex. These results discounted a tonic regulation through opioid receptors on Fas and Bcl-2 proteins in rat brain. 3. Chronic morphine (10-100 mg x kg(-1), 5 days, and 10 mg x kg(-1), 13 days) induced marked increases (47-123%) in the immunodensity of Fas receptor in the cerebral cortex. In contrast, chronic morphine (5 and 13 days) decreased the immunodensity of Bcl-2 protein (15-30%) in brain cortex. Chronic naloxone (10 mg x kg(-1), 13 days) did not alter the immunodensities of Fas and Bcl-2 proteins in the cerebral cortex. 4. The concurrent chronic treatment (13 days) of naloxone (10 mg x kg(-1)) and morphine (10 mg x kg(-1)) completely prevented the morphine-induced increase in Fas receptor and decrease in Bcl-2 protein immunoreactivities in the cerebral cortex. 5. The results indicate that morphine, through the sustained activation of opioid receptors, can promote abnormal programmed cell death by enhancing the expression of pro-apoptotic Fas receptor protein and damping the expression of anti-apoptotic Bcl-2 oncoprotein.     

Pro-apoptotic Activity of BH3-only Proteins and BH3 Mimetics: from Theory to Potential Cancer Therapy

The evasion of cancer cells from the induction of cell death pathways results in the resistance of tumor to current treatment modalities. Therefore, the resistance to cell death, one of the hallmarks of cancer, is a major target in the development of new approaches to selectively affect cancer cells. The complex interplay between individual members of Bcl-2 family regulates both cell survival and the mitochondrial pathway of apoptosis by maintaining mitochondrial membrane integrity (anti-apoptotic Bcl-2 subfamily) and by triggering its disruption in response to stress stimuli (Bax-like subfamily). BH3-only proteins, another Bcl-2 subfamily, act either by direct stimulation of pro-apoptotic proteins of the Bax subfamily or by interfering with anti-apoptotic proteins of the Bcl-2 subfamily. Thus, pro-apoptotic BH3 mimetics, thought to function as BH3-only proteins, are expected to improve the effectiveness of cancer treatment. BH3 mimetics could be either natural or synthetic, peptidic or only based on a helical peptide-like scaffold. Experimental and clinical evidence indicates that BH3 mimetics may not be sufficient to cure cancer patients when used as a single agent. BH3 profiling of cancer cells was introduced to better predict the in vivo responsiveness of tumor to BH3 mimetics combined with conventional therapies. In summary, targeting the Bcl-2 proteins is a promising tool with potential to generate new treatment modalities and to complement existing anti-cancer therapies. This review presents the current knowledge on BH3-only proteins and the spectrum of strategies employing BH3 mimetics in preclinical and clinical studies that aim at tumor targeting.     

Obatoclax mesylate : pharmacology and potential for therapy of hematological neoplasms

INTRODUCTION: Augmentation and acceleration of apoptosis for cancer therapy are logical therapeutic strategies given the increasing body of data suggesting the dysregulation of control of cell death in many neoplasms. Apoptosis is particularly well studied in hematological neoplasms, thus these varied diseases present opportunities for pro-apoptotic drug development both as single agents and in combination with established therapies. Accordingly, several agents targeting function of anti-apoptotic Bcl-2 family members have entered clinical trials in the last decade and are discussed. AREAS COVERED: The pan Bcl-2 family member BH3 domain mimetic obatoclax (GX15-070) is currently under clinical evaluation in solid tumors and hematological neoplasms. This agent offers the attractive property of uniformly inhibiting all of the anti-apoptotic members of the Bcl-2 protein family. Its chemistry and preclinical development and activity are reviewed. Pharmacology, pharmacodynamics, drug resistance and clinical use of this agent in leukemias and lymphomas are discussed. The prospects for obatoclax in changing clinical practice are addressed. EXPERT OPINION: Obatoclax may not prove to have dramatic single agent activity for hematological neoplasms. It seems more likely that its activity will be manifest in combination therapy with other agents, particularly cytotoxic chemotherapies. Results of ongoing studies are awaited.     

Small-molecule inhibitors of Bcl-2 family proteins as therapeutic agents in cancer

This review focuses on the recent patents and use of small-molecule inhibitors (SMIs) of Bcl-2 family proteins as therapeutic agents against cancer. Bcl-2 members are crucial regulators of apoptotic cell death. Apoptosis is an evolutionarily conserved process of programmed cell death that plays an essential role in organism development and tissue homeostasis. Several mechanisms exist allowing cells to escape programmed cell death among them is the overexpression of the antiapoptotic proteins. Cancer cells are often found to overexpress many of these members such as Bcl-2, Bcl-X(L), Mcl-1, Bcl-w and A1/Bfl1 and are usually resistant to a wide range of anti-cancer drugs and treatments. Many groups have been working to develop anti-cancer drugs that block the function of anti-apoptotic Bcl-2 members, thus favoring cell death. Methods include the downregulation of Bcl-2 expression or the use of peptides or small organic molecules to the Bcl-2 binding pocket, preventing its sequestration of proapoptotic proteins such as Bid and Bim. One of the most promising aspects of SMIs in treating cancer is that their targets and mechanisms of action are different from those of cytotoxic drugs and radiation. This makes it feasible to combine SMIs with other treatments, creating a synergistic therapy, without likely development of cross-resistance or increased toxicity. A broad-spectrum or "pan" SMI which targets multiple Bcl-2 family proteins is the goal.     

Dexamethasone differentially regulates Bcl-2 family proteins in human proliferative chondrocytes: Role of pro-apoptotic Bid

Glucocorticoids (GCs) are widely used to treat inflammatory diseases and cancers. A multitude of undesired side effects have been reported in GC-treated patients including decreased linear bone growth. We have previously reported that GCs activate the caspase cascade and trigger Bax-mediated mitochondrial apoptosis in growth plate chondrocytes causing growth retardation in young mice. To further explore the role of mitochondrial apoptosis in GC-induced bone growth retardation, a number of pro- and anti-apoptotic proteins were studied in ex vivo cultures of human growth plate cartilage and human HCS-2/8 proliferative chondrocytes exposed to dexamethasone. Dexamethasone was found to increase the pro-apoptotic proteins Bcl-xS, Bad, and Bak as well as the proteolysis of Bid. Anti-Bid small interfering RNA partially rescued the chondrocytes from dexamethasone-induced apoptosis. Taken together, our data suggest that GC treatment differentially regulates Bcl-2 family member proteins to facilitate mitochondrial apoptosis in proliferative chondrocytes thereby contributing to GC-induced bone growth impairment. Prevention of this imbalance between pro- and anti-apoptotic Bcl-2 family proteins may provide a new strategy to protect from adverse effects of GCs on bone growth.     

水飞蓟宾对异丙肾上腺素引起的大鼠乳鼠心肌细胞损伤的保护作用及其机制

水飞蓟宾(silibinin)为从菊科植物水飞蓟(Silybum marianum)的种子和果实中提取得到的黄酮类单体成分。本研究观察水飞蓟宾对β-肾上腺素受体激动剂异丙肾上腺素对培养乳鼠心肌细胞损伤的影响，并对其机制进行研究。采用MTT法测定细胞存活率，酶联法测定细胞丙二醛(maleic dialdehyde，MDA)和细胞培养液中乳酸脱氢酶(lactate dehydrogenase，LDH)的含量以及超氧化物歧化酶(superoxide dismutase，SOD)的活性，流式细胞仪测定线粒体膜电位(ΔΨ)，以及Western blotting检测与线粒体相关蛋白的表达。与模型组相比，水飞蓟宾使细胞受损伤的程度降低，并且增加SOD的活性，抑制了细胞膜电位的降低，并且改善Bcl-2家族蛋白中Bax/Bcl-2的表达比率，上调Bax上游去乙酰化酶SIRT1蛋白的表达。水飞蓟宾通过上调线粒体上游Bax/Bcl-2的表达比率与SIRT1蛋白的表达，改善了线粒体的功能，从而对由异丙肾上腺素引起的培养乳鼠心肌细胞损伤有明显的保护作用。     

Peptido-targeting of the mitochondrial transition pore complex for therapeutic apoptosis induction

The permeability transition pore (PTPC), a polyprotein complex, participates in the mitochondrial homeostasis as well as in the mitochondrial phase of the intrinsic pathway of apoptosis. It integrates multiple death signals including alterations of the intracellular milieu, translocation of pro-apoptotic members of the Bax/Bcl-2 family, p53, and viral proteins. As a consequence, PTPC can act as a coordinator of the pro-apoptotic mitochondrial membrane permeabilization process and the release of pro-apoptotic intermembrane space proteins into the cytosol. Moreover, the deregulation of PTPC has been involved in several major human pathologies such as cancer, neurodegeneration, ischemia/reperfusion, aging, as well as hepatotoxicity. Therefore, PTPC has emerged as a promising potential therapeutic target. Here, we will review the current knowledge concerning the two opposite functions of the PTPC and its implication in various pathologies. We will discuss the possibility to target this complex with peptides to modulate apoptosis in an innovative therapeutic perspective.     

Bcl-2 proteins: targets and tools for chemosensitisation of tumor cells

Proteins of the Bcl-2 family share one or several Bcl-2 homology (BH) regions and behave as pro- or anti-apoptotic proteins. Prosurvival members such as Bcl-2 and Bcl-X(L) are supposed to preserve mitochondrial outer membrane integrity, thus preventing the release of soluble apoptogenic molecules. Pro-apoptotic members include BH3-only proteins that act as sensors of cellular damage and initiate the death process and Bax-like proteins that act downstream of BH3-only proteins to permeabilise the mitochondrial outer membrane. Whether BH3-only proteins directly activate Bax-like proteins or prevent prosurvival members of the family from inhibiting Bax-like proteins or both remains a matter of controversy. Expression of these proteins is altered in various human tumours and this abnormal expression may contribute to oncogenesis and tumour cell resistance to anticancer drug-induced cell death. Based on these observations, prosurvival proteins are attractive intracellular targets for inducing tumour cell death or sensitising tumour cells to death induced by chemotherapeutic drugs. The use of 18-mer antisense oligonucleotides (G3139 or Genasense) targeting the first six codons of bcl-2 mRNA is currently developed in clinics with phase I studies demonstrating that thrombocytopenia may be the main dose-limiting side effect. This strategy, that efficiently decreases Bcl-2 protein expression in some tumour cells, is currently tested in phase II and phase III trials. Alternative approaches to achieve the functional knock-out of Bcl-2 include the use of either peptides mimicking the BH3 domain of Bcl-2-related proteins or more stable, non peptidic BH3 mimetics and the pharmacological modulation of the post-translational modifications of the protein.     

Differential effects of anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-xL on Celecoxib-induced apoptosis

The cyclooxygenase-2 inhibitor Celecoxib is a potent inducer of apoptosis in tumor cells. In most cellular systems Celecoxib induces apoptosis via an intrinsic, mitochondrial apoptosis pathway. We recently showed that in Bax-negative Jurkat cells expression of pro-apoptotic Bak is essential for Celecoxib-induced mitochondrial damage and apoptosis induction. Aim of the present study was to identify specific pro- and anti-apoptotic members of the Bcl-2 family involved in the regulation of Bak activation, and subsequent apoptosis upon treatment with Celecoxib in the Jurkat cell model.Our results show that apoptosis in response to Celecoxib required the presence of Noxa and downregulation of the anti-apoptotic protein Mcl-1. Celecoxib-induced Bak activation and subsequent apoptosis could be inhibited by overexpression of Bcl-xL but not by the very similar Bcl-2. In Bcl-xL-overexpressing cells neutralization of both, Mcl-1 and Bcl-xL, was prerequisite for an efficient induction of apoptosis. Our data reveal an important role of the Mcl-1/Noxa axis for Celecoxib-induced apoptosis and suggest that Celecoxib may be of value for treatment of tumors addicted to Mcl-1 and for combined treatment approaches targeting anti-apoptotic Bcl-2 family members.     

The role of major apoptotic proteins in Ca<Superscript>2+</Superscript> dependent cell homeostasis

The key role of Bcl-2 (B-cell lymphoma) family proteins in programmed cell death (apoptosis) is reliably established, but particular molecular mechanisms of downregulation involving these anti-and pro-apoptotic proteins are still insufficiently clear. There are several hypotheses explaining the cell-protective (Bcl-2, Bcl-X_L) and pro-apoptotic (Bcl-x, Bak) functions of Bcl-2 family proteins. This paper briefly summarizes the available information concerning the molecular mechanisms of action of the pro-and anti-apoptotic proteins of Bcl-2 family. In particular, an original hypothesis is put forward according to which homo-and heterodimerization of these proteins are the key events in the mechanism of Ca²⁺ ion dependent homeostasis regulation. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 12, pp. 3–8, December, 2005.     

High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (～195,000 compounds) using high-throughput flow cytometry.