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Objectives: Autophagy is a complex cellular process that maintains homeostasis in systemic lupus erythematosus. Abnormally high expression of Bcl-2 was observed in B and T lymphocytes in the peripheral blood in SLE patients. These may be responsible for the survival of self-reactive lymphocytes and the development of lupus, and the study aims at evaluating interaction between apoptosis and autophagy in Egyptian lupus patients. Methods: Sixty patients with SLE were diagnosed by fulfilling the Systemic Lupus International Collaborating Clinics (SLICC) classification criteria for SLE and sixty healthy age and sex matched control. All patients were subjected to full medical history and clinical examination. Activity was assessed using SLEDAI-2K score. Gene expression of Beclin-1, Bcl-2-L2, and Bcl-2 was measured. Results: The study revealed that the expression of anti-apoptotic Bcl-2 and Bcl-2-L2 was significantly higher in SLE patients than control subjects, as well as the major apoptotic agent (Beclin-1) mRNA, p = 0.03, < 0.001 and 0.02, respectively. The apoptotic Beclin-1 mRNA was positively correlated with SLE disease severity index, r = 0.25; p = 0.0.4; therefore, our results showed that expression of the Beclin-1 was significantly higher in SLE patients than control (p < 0.02). Conclusion: Our results showed that expression of the Beclin 1 were significantly higher in SLE patients than control (p < 0.02). [ABSTRACT FROM AUTHOR]

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells’ viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.

A series of 5-(1H-indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines 8a–j has been designed, synthesized and tested in vitro as potential pro-apoptotic Bcl-2-inhibitory anticancer agents based on our previous lead compound 8a . Synthesis of the target compounds was readily accomplished through a cyclisation reaction between indole-3-carboxylic acid hydrazide ( 5 ) and substituted isothiocyanates 6a–j , followed by oxidative cyclodesulfurization of the corresponding thiosemicarbazide 7a–j using 1,3-dibromo-5,5-dimethylhydantoin. Active compounds of the series 8a–j were found to have sub-micromolar IC 50 values selectively in Bcl-2 expressing human cancer cell lines; notably the 2-nitrophenyl analogue 8a was found to exhibit potent activity, and compounds 8a and 8e possessed comparable Bcl-2 binding affinity (ELISA assay) to the established natural product-based Bcl-2 inhibitor, gossypol. Molecular modeling studies helped to further rationalise anti-apoptotic Bcl-2 binding, and identified compounds 8a and 8e as candidates for further development as Bcl-2 inhibitory anticancer agents. [ABSTRACT FROM AUTHOR]

Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to cancer. Found at the outer mitochondrial membrane, VDAC1 assumes a crucial position in the cell, controlling the metabolic cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC1 to interact with proteins that mediate and regulate the integration of mitochondrial functions with other cellular activities. As a metabolite transporter, VDAC1 contributes to the metabolic phenotype of cancer cells. This is reflected by VDAC1 over-expression in many cancer types, and by inhibition of tumor development upon silencing VDAC1 expression. Along with regulating cellular energy production and metabolism, VDAC1 is also a key protein in mitochondria-mediated apoptosis, participating in the release of apoptotic proteins and interacting with anti-apoptotic proteins. The involvement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space is discussed, as is VDAC1 oligomerization as an important step in apoptosis induction. VDAC also serves as an anchor point for mitochondria-interacting proteins, some of which are also highly expressed in many cancers, such as hexokinase (HK), Bcl2, and Bcl-xL. By binding to VDAC, HK provides both metabolic benefit and apoptosis-suppressive capacity that offers the cell a proliferative advantage and increases its resistance to chemotherapy. VDAC1-based peptides that bind specifically to HK, Bcl2 or Bcl-xL abolished the cell’s abilities to bypass the apoptotic pathway. Moreover, these peptides promote cell death in a panel of genetically characterized cell lines derived from different human cancers. These and other functions point to VDAC1 as a rational target for the development of a new generation of therapeutics.

Our focus in the past several years has been on the identification of novel and effective pan-Bcl-2 antagonists. We have recently reported a series of Apogossypolone (ApoG2) derivatives, resulting in the chiral compound (+/-) BI97D6. We report here the synthesis and evaluation on its optically pure (-) and (+) atropisomers. Compound (-) BI97D6 potently inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl-1 with IC50 values of 76 ± 5, 31 ± 2, 25 ± 8 and 122 ± 28 nM, respectively. In a cellular assay, compound (-) BI97D6 effectively inhibits cell growth in the PC-3 human prostate cancer and H23 human lung cancer cell lines with EC50 values of 0.22 ± 0.08 and 0.14 ± 0.02 µM, respectively. Similarly, compound (-) BI97D6 effectively induces apoptosis in the BP3 human lymphoma cell line in a dose-dependent manner. The compound also shows little cytotoxicity against bax-/-/bak-/- cells, suggesting that it kills cancers cells predominantly via a Bcl-2 pathway. Moreover, compound (-) BI97D6 displays in vivo efficacy in both a Bcl-2 transgenic mouse model and in a prostate cancer xenograft model in mice. Therefore, compound (-) BI97D6 represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.

Abstract: A series of substituted 3-(benzylthio)-5-(1H-indol-3-yl)-4H-1,2,4-triazol-4-amines has been synthesised and tested in vitro as potential pro-apoptotic Bcl-2-inhibitory anticancer agents. Synthesis of the target compounds was readily accomplished in good yields through a cyclisation reaction between indole-3-carboxylic acid hydrazide and carbon disulfide under basic conditions, followed by S-benzylation. Active compounds, such as the nitrobenzyl analogue 6c, were found to exhibit sub-micromolar IC50 values in Bcl-2 expressing human cancer cell lines. Molecular modelling and ELISA studies further implicated anti-apoptotic Bcl-2 as a candidate molecular target underpinning anticancer activity. [Copyright &y& Elsevier]

Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to cancer. Found at the outer mitochondrial membrane, VDAC1 assumes a crucial position in the cell, controlling the metabolic cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC1 to interact with proteins that mediate and regulate the integration of mitochondrial functions with other cellular activities. As a metabolite transporter, VDAC1 contributes to the metabolic phenotype of cancer cells. This is reflected by VDAC1 over-expression in many cancer types, and by inhibition of tumor development upon silencing VDAC1 expression. Along with regulating cellular energy production and metabolism, VDAC1 is also a key protein in mitochondria-mediated apoptosis, participating in the release of apoptotic proteins and interacting with anti-apoptotic proteins. The involvement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space is discussed, as is VDAC1 oligomerization as an important step in apoptosis induction. VDAC also serves as an anchor point for mitochondria-interacting proteins, some of which are also highly expressed in many cancers, such as hexoki-nase (HK), Bcl2, and Bcl-xL. By binding to VDAC, HK provides both metabolic benefit and apoptosis-suppressive capacity that offers the cell a proliferative advantage and increases its resistance to chemotherapy. VDAC1-based peptides that bind specifically to HK, Bcl2, or Bcl-xL abolished the cell's abilities to bypass the apoptotic pathway. Moreover, these peptides promote cell death in a panel of genetically characterized cell lines derived from different human cancers. These and other functions point to VDAC1 as a rational target for the development of a new generation of therapeutics. [ABSTRACT FROM AUTHOR]

The Bcl-2-family proteins have long been known for their role as key regulators of apoptosis. Overexpression of various members of the family is associated with oncogenesis. Its founding member, anti-apoptotic Bcl-2 regulates cell death at different levels, whereby Bcl-2 emerged as a major drug target to eradicate cancers through cell death. This resulted in the development of venetoclax, a Bcl-2 antagonist that acts as a BH3 mimetic. Venetoclax already entered the clinic to treat relapse chronic lymphocytic leukemia patients. Here, we discuss the role of Bcl-2 as a decision-maker in cell death with focus on the recent advances in anti-cancer therapeutics that target the BH4 domain of Bcl-2, thereby interfering with non-canonical functions of Bcl-2 in Ca2+-signaling modulation. In particular, we critically discuss previously developed tools, including the peptide BIRD-2 (Bcl-2/IP 3 R-disrupter-2) and the small molecule BDA-366. In addition, we present a preliminary analysis of two recently identified molecules that emerged from a molecular modeling approach to target Bcl-2's BH4 domain, which however failed to induce cell death in two Bcl-2-dependent diffuse large B-cell lymphoma cell models. Overall, antagonizing the non-canonical functions of Bcl-2 by interfering with its BH4-domain biology holds promise to elicit cell death in cancer, though improved tools and on-target antagonizing small molecules remain necessary and ought to be designed. • Anti-apoptotic Bcl-2 prevents cell death through its mitochondrial and endoplasmic reticulum functions. • Anti-apoptotic Bcl-2 via its BH4 domain prevents pro-apoptotic, IP 3 receptor-driven Ca2+ signaling. • Tools that target Bcl-2's BH4 domain such as BIRD-2 elicit cell death in several cancer cell types and are complementary with BH3 mimetics. • BDA-366 was proposed as the first small molecule BH4-domain antagonist, yet triggers cell death independently of Bcl-2. • The first bona fide BH4-domain antagonist small molecule still needs to emerge. [ABSTRACT FROM AUTHOR]

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells' viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells. [ABSTRACT FROM AUTHOR]

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2's hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca 2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP 3 R). The cell-permeable Bcl-2/IP 3 R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2's BH4 domain, unleashing pro-apoptotic Ca 2+ -release events. We compared eight "primed to death" diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC 50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP 3 R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP 3 R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca 2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca 2+ -dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca 2+ -signaling events via its BH4 domain., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2β, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.

Our focus in the past several years has been on the identification of novel and effective pan-Bcl-2 antagonists. We have recently reported a series of Apogossypolone (ApoG2) derivatives, resulting in the chiral compound (±) BI97D6. We report here the synthesis and evaluation on its optically pure (-) and (+) atropisomers. Compound (-) BI97D6 potently inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 76 ± 5, 31 ± 2, 25 ± 8, and 122 ± 28 nM, respectively. In a cellular assay, compound (-) BI97D6 effectively inhibits cell growth in the PC-3 human prostate cancer and H23 human lung cancer cell lines with EC(50) values of 0.22 ± 0.08 and 0.14 ± 0.02 μM, respectively. Similarly, compound (-) BI97D6 effectively induces apoptosis in the BP3 human lymphoma cell line in a dose-dependent manner. The compound also shows little cytotoxicity against bax(-/-)/bak(-/-) cells, suggesting that it kills cancers cells predominantly via a Bcl-2 pathway. Moreover, compound (-) BI97D6 displays in vivo efficacy in both a Bcl-2-transgenic mouse model and in a prostate cancer xenograft model in mice. Therefore, compound (-) BI97D6 represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.