BH3-only proteins: integrated control point of apoptosis

Bcl-2 family proteins play central roles in the regulation of most, if not all, apoptotic pathways, and hence this family plays a critical role in oncogenesis. The Bcl-2 homology 3 (BH3)-only members of this family are "proapoptotic," promoting apoptosis by sensing cellular stresses: that is, they are activated or induced in response to stress stimuli. These BH3-only proteins then interfere with the function of "prosurvival" Bcl-2 family members, thereby promoting the progression of apoptosis. It has long been recognized that the induction and activation of each of the BH3-only proteins are subject to the type of stress stimulus. Although it was originally assumed that all the BH3-only proteins exert similar effects on the downstream apoptotic machinery, recent studies have uncovered their distinct functional properties, indicating the operation of more intricate, versatile control mechanisms of apoptosis. In this review, we will summarize recent findings on the diversity in the activation and function of BH3-only proteins. In particular, we focus on the overlapping and individual roles of 2 BH3-only proteins, Puma and Noxa, in the context of the apoptotic response induced by the tumor suppressor p53.     

The BH3 mimetic S1 induces autophagy through ER stress and disruption of Bcl-2/Beclin 1 interaction in human glioma U251 cells

Previous results showed that a novel BH3 mimetic S1 could induce cell death in a wide range of cancer types in vitro through Bax/Bak-dependent apoptosis. We demonstrated that in addition to mitochondrial pathway apoptosis, endoplasmic reticulum (ER) stress-associated apoptosis was also induced by S1. Moreover, S1 can induce autophagy in U251 cells, which may occur through ER stress and disruption of the association of Bcl-2 and Beclin 1. Inhibition of autophagy by the autophagic inhibitors 3-methyladenine (3-MA) or chloroquine (CQ) increased S1-induced apoptosis. In conclusion, autophagy plays an important role in S1-induced U251 cell death.     

Downregulations of B‐cell lymphoma 2 and myeloid cell leukemia sequence 1 by microRNA 153 induce apoptosis in a glioblastoma cell line DBTRG‐05MG

MicroRNA‐153 (miR‐153) is a brain‐specific miRNA that is expressed at a significantly lower level in glioblastoma (GBM) relative to non‐neoplastic brain tissue. Although the expression pattern of miR‐153 has been extensively established, its target genes and cellular mechanism remain undefined. To investigate into the potential function of miR‐153 in glioblastmas, we transfected a GBM cell line DBTRG‐05MG with synthetic miR‐153 oligos and observed decreased cell proliferation and increased apoptosis. Bioinformatics analysis revealed that anti‐apoptosis family member B‐cell lymphoma 2 (Bcl‐2) and myeloid cell leukemia sequence 1 (Mcl‐1) are potential targets of miR‐153. Indeed, Western blot analysis indicated that miR‐153 downregulated both Bcl‐2 and Mcl‐1 at the protein levels. Single strand miR‐153 inhibitor, which forms complementary base‐pair with endogenous miR‐153, efficiently blocked the apoptosis and target protein degradation induced by overexpression of miR‐153. By luciferase reporter assays, we further showed that miR‐153 inhibited Bcl‐2 and Mcl‐1 expressions by directly targeting the 3′UTR regions of their respective mRNAs.     

Bcl‐XL and MCL‐1 constitute pertinent targets in ovarian carcinoma and their concomitant inhibition is sufficient to induce apoptosis

In ovarian carcinomas, recurrence and acquired chemoresistance are the first leading causes of therapeutic failure and are responsible for the poor overall survival rate. Cisplatin exposure of sensitive cells has been previously associated with a down‐regulation of Bcl‐X_L expression and apoptosis, whereas recurrence was systematically observed when Bcl‐X_L expression was maintained. Bcl‐X_L down‐regulation could thus constitute an interesting chemosensitizing strategy. We showed that a Bcl‐X_L targeted RNA interference strategy efficiently sensitized chemoresistant ovarian carcinoma cells to cisplatin, but some of them were still able to re‐proliferate. Considering the possible cooperation between Bcl‐X_L and MCL‐1, we investigated the possibility to avoid recurrence in vitro using a multi‐targeted RNAi strategy directed against these two anti‐apoptotic proteins. We showed that their concomitant inhibition lead to massive apoptosis in absence of cisplatin, this multi‐targeted RNAi approach being much more efficient than conventional chemotherapy. We thus demonstrated that Bcl‐X_L and MCL‐1 cooperate to constitute together a strong molecular “bolt”, which elimination could be sufficient to allow chemoresistant ovarian carcinoma cells apoptosis. Moreover, we demonstrated that in presence of a low concentration of cisplatin, the concomitant down‐regulation of Bcl‐X_L and MCL‐1 allowed a complete annihilation of tumour cells population thus avoiding subsequent recurrence in vitro in cell lines highly refractory to any type of conventional chemotherapy. Therefore, Bcl‐X_L and MCL‐1 targeted strategies could constitute an efficient therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in association with conventional chemotherapy.     

Functional screening identifies a microRNA,miR‐491 that induces apoptosis by targeting Bcl‐XL in colorectal cancer cells

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain‐of‐function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD‐1). We discovered a number of miRNAs that increased or decreased cell viability in DLD‐1. They included known oncogenic miRNAs such as miR‐372 and miR‐373, and tumor suppressive miRNAs such as miR‐124a, but also some for which this information was novel. Among them, miR‐491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl‐X_L was a direct target of miR‐491, and its silencing contributed to miR‐491‐induced apoptosis. Moreover, treatment of miR‐491 suppressed in vivo tumor growth of DLD‐1 in nude mice. Our study provides a new regulation of Bcl‐X_L by miR‐491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl‐X_L.     

Inhibition of cyclin E1 overcomes temozolomide resistance in glioblastoma by Mcl‐1 degradation

Glioblastoma (GBM) is one of the major causes of brain cancer‐related mortality worldwide. Temozolomide (TMZ) is an important agent against GBM. Acquired TMZ‐resistance severely limits the chemotherapeutic effect and leads to poor GBM patient survival. To study the underlying mechanism of drug resistance, two TMZ resistant GBM cell lines, A172 and U87, were generated. In this study, the TMZ resistant cells have less apoptosis and cell‐cycle change in response to the TMZ treatment. Western blot results revealed that cyclin E1 was upregulation in TMZ resistant cells. Inhibition or depletion of cyclin E1 re‐sensitized the resistant cells to the TMZ treatment, which indicated the induction of cyclin E1 is the cause of TMZ resistance in GBM cells. Furthermore, we also found the expression of cyclin E1 stabilized the expression of Mcl‐1, which contributes to the TMZ resistance in GBM cells. Finally, our in vivo xenograft data showed that the combination of flavopiridol, a cyclin E1/CDK2 inhibitor, overcomes the TMZ resistant by inducing higher apoptosis. Overall, our data provided a rationale to overcome the TMZ resistant in GBM treatment by inhibiting the cyclin E1 activity.     

Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch

Cancer cells have developed novel mechanisms for evading chemotherapy-induced apoptosis and autophagy-associated cell death pathways. Upon the discovery that chemotherapeutics could target these cell death pathways in a manner that was not mutually exclusive, new discoveries about the interrelationship between these two pathways are emerging. Key proteins originally thought to be "autophagy-related proteins" are now found to be involved in either inducing or inhibiting apoptosis. Similarly, apoptosis inhibiting proteins can also block autophagy-associated cell death. One example is the complex formed by the autophagy protein, Beclin 1, and anti-apoptotic protein Bcl-2, which leads to inhibition of autophagy-associated cell death. Researchers have been investigating additional mechanisms that form/disrupt this complex in order to better design chemotherapeutics. This review will highlight the role Bcl-2 and Beclin 1 play in cancer development and drug resistance, as well as the role the Bcl-2:Beclin 1 complex in the switch between autophagy and apoptosis.     

Induction of apoptosis in prostate carcinoma cells by BH3 peptides which inhibit Bak/Bcl-2 interactions.

Interactions between proteins of the Bcl-2 family play an important role in the regulation of apoptosis. Anti-apoptotic family members can heterodimerize with pro-apoptotic family members and antagonize their function, thus protecting against death. In cells protected from death by overexpression of Bcl-2 much of the Bax is present in Bax/Bcl-2 hetero-multimers and its death signal is blocked as it cannot homodimerize. This led us to use the Bcl-2/Bax heterodimer as a target for new compounds which may provide a therapy particularly suited to tumour cells for which resistance to conventional therapy is associated with elevated expression of Bcl-2. We assessed whether apoptosis could be induced in prostate tumour cells by blocking this heterodimerization with synthetic peptide sequences derived from the BH3 domain of pro-apoptotic Bcl-2 family members. Prostate cells were found to undergo up to 40% apoptosis 48 h following the introduction of synthetic peptides from the BH3 domains of Bax and Bak. The caspase inhibitor z-VAD.fmk provided protection against apoptosis mediated by these peptides. Immunoprecipitation studies revealed that introduction of peptides derived from the BH3 regions of Bak and Bax into cells blocked Bak/Bcl-2 heterodimerization. These data suggest that by blocking the dimerization through which Bcl-2 would normally inhibit apoptosis the apoptotic pathway driven by Bak was re-opened.     

Identification of predictive factors of response to the BH3‐mimetic molecule ABT‐737: An ex vivo experiment in human serous ovarian carcinoma

Ovarian cancers are addicted to Bcl‐x_L and Mcl‐1, antiapoptotic members of the Bcl‐2 family. Bcl‐x_L can be inhibited by the BH3‐mimetic ABT‐737. In vitro, ABT‐737 can induce apoptosis of cancer cells, and its activity is potentiated by Mcl‐1 inactivation. Herein, we assessed the sensitivity of human ovarian tumor nodes to ABT‐737 when combined with carboplatin, which can indirectly inhibit Mcl‐1. Fresh samples from 25 patients with high‐grade serous ovarian cancer (HGSOC) who were chemo‐naïve and had undergone surgery were prospectively exposed ex vivo to ABT‐737 ± carboplatin. The treatment effect was studied on sliced tumor nodes by assessment of cleaved‐caspase 3 immunostaining. We also studied the association between baseline Bcl‐2 family protein expression (via immunohistochemistry) and the response of nodes to treatment. ABT‐737 induced apoptosis as a single agent but its efficacy was not improved by the addition of carboplatin. Bim was frequently expressed (20/25) and its absence or low expression was associated with the absence of response to ABT‐737, p value = 0.019 by Fisher's test and sensitivity = 93%, (95% confidence interval, 66–100). Moreover, we observed that in tumors in which Bim was expressed, a low expression of phospho‐Erk1/2 or Mcl‐1 improved the proportion of responses. This pilot study showed that ABT‐737 has promise as monotherapy for HGSOC in a specific subgroup of tumors. Bim, Mcl‐1, and phospho‐Erk1/2 appeared to be relevant biomarkers that could be used for the selection of patients in the design of clinical trials using Navitoclax (an orally available compound related to ABT‐737).     

Targeting CDK9 by wogonin and related natural flavones potentiates the anti‐cancer efficacy of the Bcl‐2 family inhibitor ABT‐263

Tumor initiation, progression and resistance to therapies are tightly associated with over‐expression of anti‐apoptotic proteins Bcl‐2, Bcl‐x_L, Bcl‐w and Mcl‐1. ABT‐263 (Navitoclax), an orally bio‐available small‐molecule mimetic of the Bcl‐2 homology domain 3, inhibits Bcl‐2, Bcl‐x_L, and Bcl‐w and has shown anti‐cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT‐263 in patients is dose‐limited due to causing thrombocytopenia via inhibition of Bcl‐x_L in platelets. ABT‐199 specifically inhibits Bcl‐2; however, its use is limited to tumors over‐expressing only Bcl‐2. Besides, many tumors resist treatment due to high levels of Mcl‐1 expression or develop resistance via up‐regulation of Mcl‐1 during long‐term exposure. These obstacles highlight the demand to improve the ABT‐263‐based therapy. In this study, we show that anti‐cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT‐263‐induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down‐regulation of Mcl‐1 expression. Importantly, wogonin does not enhance the toxicity of ABT‐263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT‐263 in cancer cells which have acquired resistance to ABT‐263. Furthermore, we show that combination of wogonin with ABT‐263 promotes in vivo tumor regression in a human T‐cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT‐263 thereby possibly decreasing the risk of adverse side effects.     

The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.     

Linoleic acid and butyrate synergize to increase Bcl‐2 levels in colonocytes

The biological properties of polyunsaturated fatty acid (PUFA) classes have been the source of much contention. For example, n‐3 PUFA are chemoprotective, whereas n‐6 PUFA may promote tumor development. Since dietary components can have combinatorial effects, we further examined the apoptotic properties of n‐3 or n‐6 fatty acids when combined with different fiber sources. Mice were fed diets supplemented with either fish oil (FO; enriched in n‐3 PUFA) or corn oil (CO; enriched in n‐6 PUFA) and nonfermentable (cellulose) or fermentable (pectin) fiber sources. In complementary experiments, immortalized young adult mouse colonic (YAMC) cells were treated with docosahexaenoic acid (DHA; 22:6n‐3) or linoleic acid (LA; 18:2n‐6) with or without butyrate. Mice fed a FO and pectin diet had significantly (p < 0.05) increased levels of apoptosis in colonocytes compared to all other diets. Similarly, apoptosis was highly induced in DHA and butyrate cotreated YAMC cells. In contrast, in both YAMC and mouse models, LA/CO with butyrate/pectin treatment reduced apoptosis and enhanced expression of bcl‐2. The LA and butyrate induced antiapoptotic phenotype was reversed by knocking down bcl‐2 using targeted siRNA. In comparison, overexpression of bcl‐2 blocked the proapoptotic effect of DHA and butyrate. These data provide new mechanistic insights into the regulation of apoptosis by dietary PUFA and fiber.