Expression Of Bcl-2 Family Proteins Research Articles

Enhancement of Antiangiogenic Efficacy of Iron(II) Complex by Selenium Substitution

Antiangiogenesis therapy is a proven strategy for the treatment of cancers. Herein, we demonstrate that iron(II) complexes containing 1,10-phenanthroline(phen) derivatives were capable of suppressing angiogenesis in vitro in a dose-dependent manner. Interestingly, the introduction of selenium into an iron(II) complex ((Fe(phenSe)3 (ClO4 )2 (phenSe=2-selenoimidazole[4,5-f]1,10-phenanthroline)) could enhance its antiangiogenic efficacy. Mechanistic studies demonstrated that the complex potently induced endothelial cell apoptosis as evidenced by activation of caspases and PARP cleavage. The iron(II) complex activated p53-mediated mitochondrial dysfunction as can be seen by the upregulation in the expression of p53 and proapoptotic Bcl-2 family proteins, and downregulation in the expression of Bcl-2 family proteins. Additionally, the complex inhibited VEGF expression and gave rise to dephosphorylated AKT, which suppressed the transmission of the mitogenic signaling pathway. Taken together, this study could provide a strategy for the rational design of high-efficacy antivascular agents.

Centchroman induces redox-dependent apoptosis and cell-cycle arrest in human endometrial cancer cells.

Centchroman (CC) or Ormeloxifene has been shown to induce apoptosis and cell cycle arrest in various types of cancer cells. This has, however, not been addressed for endometrial cancer cells where its (CC) mechanism of action remains unclear. This study focuses on the basis of antineoplasticity of CC by blocking the targets involved in the cell cycle, survival and apoptosis in endometrial cancer cells. Ishikawa Human Endometrial Cancer Cells were cultured under estrogen deprived medium, exposed to CC and analyzed for proliferation and apoptosis. Additionally, we also analyzed oxidative stress induced by CC. Cell viability studies confirmed the IC50 of CC in Ishikawa cells to be 20µM after 48h treatment. CC arrests the cells in G0/G1 phase through cyclin D1 and cyclin E mediated pathways. Phosphatidylserine externalization, nuclear morphology changes, DNA fragmentation, PARP cleavage, and alteration of Bcl-2 family protein expression clearly suggest ongoing apoptosis in the CC treated cells. Activation of caspase 3 & 9, up-regulation of AIF and inhibition of apoptosis by z-VAD-fmk clearly explains the participation of the intrinsic pathway of programmed cell death. Further, the increase of ROS, loss of MMP, inhibition of antioxidant (MnSOD, Cu/Zn-SOD and GST) and inhibition of apoptosis with L-NAC suggests CC induced oxidative stress leading to apoptosis via mitochondria mediated pathway. Therefore, CC could be a potential therapeutic agent for the treatment of Endometrial Cancer adjunct to its utility as a contraceptive and an anti-breast cancer agent.

Novel derivative of aminobenzenesulfonamide (3c) induces apoptosis in colorectal cancer cells through ROS generation and inhibits cell migration

BackgroundColorectal cancer (CRC) is the 3rd most common type of cancer worldwide. New anti-cancer agents are needed for treating late stage colorectal cancer as most of the deaths occur due to cancer metastasis. A recently developed compound, 3c has shown to have potent antitumor effect; however the mechanism underlying the antitumor effect remains unknown.Methods3c-induced inhibition of proliferation was measured in the absence and presence NAC using MTT in HT-29 and SW620 cells and xCELLigence RTCA DP instrument. 3c-induced apoptotic studies were performed using flow cytometry. 3c-induced redox alterations were measured by ROS production using fluorescence plate reader and flow cytometry and mitochondrial membrane potential by flow cytometry; NADPH and GSH levels were determined by colorimetric assays. Bcl2 family protein expression and cytochrome c release and PARP activation was done by western blotting. Caspase activation was measured by ELISA. Cell migration assay was done using the real time xCELLigence RTCA DP system in SW620 cells and wound healing assay in HT-29.ResultsMany anticancer therapeutics exert their effects by inducing reactive oxygen species (ROS). In this study, we demonstrate that 3c-induced inhibition of cell proliferation is reversed by the antioxidant, N-acetylcysteine, suggesting that 3c acts via increased production of ROS in HT-29 cells. This was confirmed by the direct measurement of ROS in 3c-treated colorectal cancer cells. Additionally, treatment with 3c resulted in decreased NADPH and glutathione levels in HT-29 cells. Further, investigation of the apoptotic pathway showed increased release of cytochrome c resulting in the activation of caspase-9, which in turn activated caspase-3 and −6. 3c also (i) increased p53 and Bax expression, (ii) decreased Bcl2 and BclxL expression and (iii) induced PARP cleavage in human colorectal cancer cells. Confirming our observations, NAC significantly inhibited induction of apoptosis, ROS production, cytochrome c release and PARP cleavage. The results further demonstrate that 3c inhibits cell migration by modulating EMT markers and inhibiting TGFβ-induced phosphorylation of Smad2 and Samd3.ConclusionsOur findings thus demonstrate that 3c disrupts redox balance in colorectal cancer cells and support the notion that this agent may be effective for the treatment of colorectal cancer.

Abstract B19: Targeting dependencies within apoptotic pathways through inhibition of BET bromodomains

Abstract Cancer is driven in large part by dysregulated transcriptional programs that allow for the acquisition of the many ‘Hallmarks of Cancer.' Multiple regulatory factors are essential for the establishment and maintenance of these cancer-specific transcriptional programs, and this dependence creates vulnerabilities that can be therapeutically targeted. Here we describe a selective dependence on the bromodomain and extraterminal (BET) family of proteins for the reprogramming of apoptotic signaling networks and demonstrate how this dependence can be predicted prior to therapeutic intervention. We demonstrate in phenotypically sensitive cell lines that BET inhibition results in a rapid and robust transcriptional response among regulators of apoptosis, and that this transcriptional response is correlated with changes in the apoptotic threshold of target cells and subsequent apoptosis. We show that the robustness of the apoptotic response, and not that of the cytostatic response, predicts phenotypic sensitivity to BETi. Consistent with this, we observed that acquired BETi tolerance in two disparate cellular models is driven by dysregulated expression of anti-apoptotic BCL2 family proteins, and that genetic or pharmacological manipulation of apoptotic signaling can modify the phenotypic response to BETi. We further demonstrate that the basal expression levels of a set of apoptotic factors significantly predict preclinical response to BETi, and in particular note that BETi preferentially targets those cells that are dependent on BCL2 for survival. Our findings suggest that tumor cells have acquired a dependence on BET bromodomain function to evade apoptosis, and highlight opportunities to exploit this dependence in the clinic through rational patient selection and drug combination strategies. Citation Format: Andrew R. Conery, Richard C. Centore, Kerry L. Spillane, Nicole E. Follmer, Archana Bommi-Reddy, Charlie Hatton, Barbara M. Bryant, Patricia Greninger, Arnaud Amzallag, Cyril H. Benes, Jennifer A. Mertz, Robert J. Sims, III. Targeting dependencies within apoptotic pathways through inhibition of BET bromodomains. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B19.

Selenadiazole derivatives antagonize hyperglycemia-induced drug resistance in breast cancer cells by activation of AMPK pathways.

Hyperglycemia is an important factor for chemoresistance of breast cancer patients with diabetes. In the present study, a novel selenadiazole derivative has been evaluated and found to be able to antagonize the doxorubicin (DOX) resistance of MCF-7 cells under simulated diabetes conditions. Hyperglycemia promotes the proliferation, invasion and migration of MCF-7 cells through activation of ERK and AKT pathways, which could be inhibited by the synthetic selenadiazole derivative. The antitumor effects of the selenadiazole derivative were attributed to its ability to activate AMPK pathways. Furthermore, the high lipophilicity (log P = 1.9) of the synthetic selenadiazole derivative facilitated its uptake by cancer cells and subsequently potentiated the cellular uptake of DOX, leading to a strong enhancment of the antiproliferative activity of DOX on MCF-7 cells by induction of apoptosis. The apoptosis was initiated by the ROS overproduction induced by the cooperation of the selenadiazole derivative and DOX. The excessive ROS then caused damage to DNA, which upregulated the expression of proapoptosis Bcl-2 family proteins and led to fragmentation of mitochondria, which finally caused apoptosis of the cancer cells. Taken together, this study provides a rational strategy for using selenadiazole derivatives to overcome hyperglycemia-induced drug resistance in breast cancer by activation of AMPK-mediated pathways.

Targeting MAPK Signaling Pathway with Cobimetinib (GDC-0973) Enhances Anti-Leukemia Efficacy of Venetoclax (ABT-199/GDC-0199) in Acute Myeloid Leukemia Models

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. Pro-survival molecules of BCL-2 family play critical roles in leukemia transformation and chemoresistance. The anti-leukemia potency of selective BCL-2 inhibitor venetoclax (ABT-199/GDC-0199) has been demonstrated in AML models (Pan et al. Cancer Discovery 2014). However, venetoclax is often associated with resistance due to its poor inhibition of MCL-1. RAF/MEK/ERK (MAPK) pathway is commonly activated in AML, and can stabilize anti-apoptotic MCL-1 and inactivate the pro-apoptotic BIM. In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib).First, anti-leukemia activity of cobimetinib and venetoclax was examined in 18 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death, as compared to the single agent treatment (Fig 1A). Cobimetinib inhibited cell proliferation in the majority of AML cases (34.2 ± 23.7%) and the cell growth suppression was more profound in the combination group (60.2 ± 28.8%, p<0.001) (Fig 1A). The clonogenic potential of myeloid progenitors was significantly suppressed by the combination (82.5 ± 20.0%), as compared to cobimetinib (38.3 ± 14.6%, p=0.01) or venetoclax (41.9 ± 18.6%, p<0.05). The normal progenitor function was minimally affected.We next investigated signaling patterns and BCL-2 family protein expression in AML stem/progenitor cells using a 34-antibody panel for CyTOF. In AML 4400240, we identified 10 distinct subpopulations based on cell surface marker expression that were used to build the SPADE tree. BCL-2 and MCL-1 were both enriched in progenitor populations phenotypically positive for CD34, CD38, CD123 and HLA-DR (A2-5 and A9-10, Fig 1B and 1C). G-CSF-induced p-ERK and SCF-induced p-S6 signaling pathways were efficiently suppressed by cobimetinib (Fig 1D). The inhibition of p-S6 may reflect the sensitivity to cobimetinib of this sample (IC50=1.68 nM); consistent with previous study that suppression of TORC1 predicted response to MEK inhibitor (Corcoran et al. Sci Transl Med 2013).We have previously reported activity of venetoclax/cobimetinib combination in a panel of myeloid leukemia cell lines and revealed distinct response patterns to single agents and combination (Han et al. ASH 2015). Functional proteomics RPPA data indicated that p-ERK, p-S6 and p-RSK pathways were significantly down-regulated in response to the combination in cells showing synergy. Western blotting was performed to validate the RPPA data in 4 cell lines representing different response patterns. Cobimetinib inhibited p-ERK when used at high dose (1 _M) in all cell lines. In turn, inhibition of p-S6 at both ser235/236 and ser240/244 sites occurred at low dose of cobimetinib in sensitive cell lines (OCI-AML3 and MV4-11) and required high dose in resistant lines. In OCI-AML3 cells that showed synergy to combination, elevated levels of cleaved PARP was observed in the combination group, which was likely due to suppression of both BCL2:BIM and MCL-1:BIM complex, followed by release of free BIM to induce cell death.To test the efficacy in vivo, we injected NSGS mice with genetically engineered MOLM3/Luc/GFP cells. Bioluminescent imaging demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the venetoclax (100 mg/kg/d) group and in venetoclax plus cobimetinib (10 mg/kg/d) co-treated mice (Fig 1E and 1F). Human CD45 engraftment and cell counts in both bone marrow and spleen demonstrated a trend towards decreased tumor burden when venetoclax was combined with cobimetinib in vivo (Fig 1G and 1H).In summary, combinatorial blockade of MAPK and BCL-2 pathways promotes cell death and suppresses proliferation in the majority of primary AML cells. The anti-leukemia efficacy of combined blockade of signaling and pro-survival pathways is associated with downregulation of MCL-1, release of pro-death BIM and suppression of p-S6. Additional novel transcriptional biomarkers of response are now being analyzed and will be presented. Combined efficacy of these agents is under clinical investigation in a Phase I trial in elderly relapsed/refractory AML (NCT02670044). [Display omitted] DisclosuresLeverson:AbbVie: Employment, Other: Shareholder in AbbVie. Monique:Genentech: Employment. Phillips:AbbVie Inc.: Employment. Chen:AbbVie: Employment. Jin:AbbVie Inc.: Employment. Daver:Ariad: Research Funding; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Kiromic: Research Funding; BMS: Research Funding; Otsuka: Consultancy, Honoraria; Karyopharm: Honoraria, Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Sampath:Genentech: Employment. Konopleva:AbbVie: Research Funding; Genentech: Research Funding.

Alvocidib Potentiates the Activity of Venetoclax in Preclinical Models of Acute Myeloid Leukemia

IntroductionVenetoclax (ABT-199) is an approved BCL-2 inhibitor for the treatment of patients with chronic lymphocytic leukemia (CLL). Multiple clinical trials are underway to explore its efficacy in additional indications. While venetoclax demonstrated high remission rates in combination with azacitidine in early stage clinical trials, the question of durability of responses and primary and acquired resistance remain, especially given the modest activity and rapid development of resistance as a single agent. One reported mechanism of intrinsic resistance is high expression of other BCL-2 family proteins, including MCL-1. We and others have demonstrated that the CDK9 inhibitor, alvocidib, can mediate transcriptional repression of anti-apoptotic MCL-1. It has also been shown that alvocidib can increase pro-apoptotic BIM, a dual activator and sensitizer BH3-only protein that can directly induce apoptosis and simultaneously inactivate anti-apoptotic BCL-2 family proteins such as MCL-1 and BCL-2, thus having the same effect on mitochondria-associated apoptosis as MCL-1 down-regulation, with the potential to directly induce apoptosis. An alvocidib-containing cytotoxic chemotherapy regimen demonstrated favorable remission rates in high-risk AML patients over standard therapy in a randomized Phase 2 trial indicating its potential role and safety in AML. We hypothesized that alvocidib and venetoclax would synergize against AML cells by shifting the overall balance of pro- and anti-apoptotic BCL-2 proteins in favor of apoptosis and thus represent a novel active treatment regimen in AML.AimsThis study seeks to examine the efficacy of a treatment regimen containing alvocidib and venetoclax in multiple preclinical studies, including in vivo models of AML.MethodsCell viability assays interrogating alvocidib and venetoclax activity in cell lines were performed using CellTiter-Glo according to manufacturer's protocol. mRNA/miRNA expression changes were assessed using standard RT-PCR technique. Protein expression changes were assessed using standard western immunoblotting technique. To assess the efficacy of an alvocidib and venetoclax combination on tumor growth in an in vivo model, the OCI-AML3 xenograft mouse model and ex vivo studies with AML patient samples were performed.ResultsHerein we demonstrate that alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion in 3 out of 4 AML cell lines analyzed, while in cells where alvocidib did not reduce MCL-1 protein levels (i.e. MOLM-13) a dose-dependent decrease in miR17-92, and concomitant increase in BIM protein was observed after 24 hours of alvocidib treatment. The alvocidib-venetoclax combination resulted in very strong synergistic reductions of cell viability (with combination indices [CI] of 0.4 to 0.7), both in venetoclax-sensitive and resistant cells. The venetoclax-sensitive lines, MV4-11 and MOLM-13, exhibited 5- to 10-fold reduction of venetoclax EC50 values in the low nM range when combined with only 80 nM alvocidib. Importantly, venetoclax-resistant lines, OCI-AML3 and THP-1, exhibited at least 20-fold reduction of venetoclax EC50 values from near 1 µM to 10-50 nM, when combined with 80 nM alvocidib.In the venetoclax-resistant OCI-AML3 xenograft model, single agent alvocidib and venetoclax achieved tumor growth inhibition (TGI) of 9.7 and 31.5%, respectively, while the combination achieved 87.9% TGI at the same dose levels of individual drugs.ConclusionsTaken together, our data suggest that the combination of alvocidib with venetoclax is highly synergistic in vitro and in vivo, in both venetoclax-sensitive and -resistant AML across a heterogeneous genomic background. The particularly high level of synergy achieved in venetoclax-resistant cell lines highlights the central importance of both BCL-2 and MCL-1-mediated cell survival in AML. Importantly, the addition of alvocidib to venetoclax treatment reduced IC50s to clinically achievable concentrations. Therefore, we conclude that an alvocidib/venetoclax combination may be a novel approach for the treatment of AML and warrants further pre-clinical and clinical validation. DisclosuresWhatcott:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Mesa:Celgene: Research Funding; Galena: Consultancy; Promedior: Research Funding; Ariad: Consultancy; Novartis: Consultancy; CTI: Research Funding; Incyte: Research Funding; Gilead: Research Funding. Peterson:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Inhibition of Cyclin Dependent Kinase 9 Synergistically Enhanced Venetoclax Activity in Mantle Cell Lymphoma Cells

Better therapeutic strategies are needed for patients with mantle cell lymphoma (MCL), an aggressive and largely incurable subtype of Non-Hodgkin Lymphoma. Concurrent expression of anti-apoptotic BCL2 family proteins in lymphoma cells contribute to their evasion of apoptosis. Therefore, targeting only one anti-apoptotic protein may lead to or uncover resistance associated with activity of other anti-apoptotic BCL2 family members. A variety of cyclin-dependent kinase (CDK) inhibitors are undergoing clinical trials either as a single agent or in combination with other approved drugs. CDK9, a portion of the elongation factor P-TEFb, phosphorylates Ser-2 in the C-terminal domain of RNA Polymerase II, which is required for transcript elongation. The effect of CDK9 inhibition is observed most immediately on those proteins with rapid turnover rates such as the BCL2 family protein MCL1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. Here we report the responses of 4 MCL cell lines (Mino, Jeko-1, CCMCL1 and JVM2) and 5 primary MCL samples (representing de novo and relapsed cases, including two relapsed cases after ibrutinib failure and a relapsed case harbor Myc rearrangement) to venetoclax and a novelCDK9 inhibitor A-1467729.Exposure of Mino and Jeko-1 cells to venetoclax rapidly induced apoptosis (IC50 at 5 hours were 235 and 955 nM, respectively). In contrast, CCMCL1 and JVM2 cells were not sensitive to venetoclax with IC50s > 3000 nM. However, CCMCL1 cells were more responsive to A-1467729 alone than the other 3 lines, while JVM2 cells were much less sensitive to A-1467729. All primary samples were sensitive to venetoclax, ex vivo, at the doses between 1 - 100 nM, although their IC50s were variable (range: 2-90 nM). A-1467729 at doses of 1-20 nM had modest single agent effects on the primary samples; however, its combination with venetoclax synergistically induced apoptosis and decreased the IC50 of venetoclax by 2-10 times in all cell lines and primary samples. The strongest synergy was observed in Jeko-1 cells with all combined indexes < 0.1. Studies on mechanisms through immunoblotting and immunohistochemical staining demonstrated that A-1467729 quickly down-regulated phospho-RNA Polymerase II (Ser2) and MCL1 protein levels. CCMCL1 cells lack BCL2 expression, while JVM2 displayed higher expression of MCL1 than other cells. The expression levels of BCL2 and MCL1 in primary samples were case-dependent as well. The expression pattern and level of anti-apoptotic BCL2 family proteins in cell lines and primary cases may be responsible for their variable reactions to these two agents. To further confirm that CDK9 inhibition was affecting cell viability at least partially through its function on MCL1, A-1210477, a MCL1 inhibitor, was applied to the same study. Strong synergistic apoptotic induction was also observed when A-1210477 was combined with venetoclax, especially in MCL1-"dependent" CCMCL1 cells as evidenced by flow cytometry based apoptotic assay and PARP cleavage. Further mechanism studies aiming the effects of CDK9 inhibitor/venetoclax on MCL1/BIM association is being under investigation. MCL mouse xenograft study for such a combined effect has been planned. In summary, the combination of a CDK9 inhibitor and venetoclax showed synergistic induction of apoptosis in both MCL cell lines and primary patient samples. These findings support further evaluation of the efficacy of such a combination in MCL, including ibrutinib-resistant MCL. DisclosuresSouers:AbbVie: Employment. Phillips:AbbVie Inc.: Employment. Hsi:HTG Molecular Diagnostics: Consultancy; Abbvie: Honoraria, Research Funding; Seattle Genetics: Honoraria; Cellerant: Honoraria, Research Funding; Eli Lilly: Research Funding; Onyx Pharmaceuticals: Honoraria.

NRBP2 Overexpression Increases the Chemosensitivity of Hepatocellular Carcinoma Cells via Akt Signaling.

Hepatocellular carcinoma is highly resistant to chemotherapy. Research data supported that cancer stem cells (CSC) may be responsible for the chemoresistance and strategies that suppress CSCs stemness could also inhibit the drug resistance. In this study, we found that nuclear receptor binding protein 2 (NRBP2) expression was downregulated in the CD133+ hepatocellular carcinoma CSCs. Most adjacent noncancerous liver tissue analyzed expressed higher level of NRBP2 compared with cancerous tissue in hepatocellular carcinoma patients, and high NRBP2 expression indicated a better prognosis. Real-time PCR results showed that NRBP2 negatively correlated with stemness-related genes, including Oct3/4, Nanog, Notch1, Ep300, and CD133 mRNA expression. High NRBP2 expression in hepatocellular carcinoma cells downregulated CK19 protein expression, inhibited tumorsphere formation, and tumorigenesis ability, indicating that high NRBP2 expression restrains the hepatocellular carcinoma cell stemness. Overexpression of NRBP2 reduced the IC50 of sorafenib in hepatocellular carcinoma cells, and NRBP2 expression was negatively correlated with hepatocellular carcinoma cell resistance to the chemotherapy agents, including cisplatin and the Akt signaling inhibitor perifosine. Coimmunoprecipitation results showed that NRBP2 could bind with Annexin A2 (ANXA2) and inhibit ANXA2 expression. Coexpression of ANXA2 restored the chemoresistant ability in NRBP2-overexpressing hepatocellular carcinoma cells. Further analysis showed that NRBP2 downregulated Akt and its downstream signaling target Bad phosphorylation level. ANXA2 coexpression partially restored the Akt phosphorylation. Analysis of the expression of Bcl2 family proteins showed that NRBP2 may increase hepatocellular carcinoma cell chemosensitivity by regulating expression of survival proteins involved in the Akt and Bcl2 pathway. These results suggest that NRBP2 plays an important role in the tumor progression and chemotherapeutic resistance of hepatocellular carcinoma. Cancer Res; 76(23); 7059-71. ©2016 AACR.

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins.

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.

The induction of apoptosis in SGC-7901 cells through the ROS-mediated mitochondrial dysfunction pathway by a Ir(III) complex.

A new ligand BTCP and its iridium(III) complex [Ir(ppy)2(BTCP)]PF6 (Ir-1) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1H NMR and 13C NMR. The cytotoxic activity in vitro of the ligand and its complex against SGC-7901, HeLa, HOS, PC-12, BEL-7402, MG-63, SiHa, A549, HepG2 and normal cell LO2 were evaluated by MTT method [MTT=(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]. The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The reactive oxygen species (ROS), mitochondrial membrane potential, autophagy and cell invasion were studied under fluorescent microscope. The expression of caspases and Bcl-2 family proteins were investigated by western blot. The IC50 values of complex toward SGC-7901, BEL-7402 and MG-63 cells are 3.9±0.5, 5.4±1.2 and 4.2±0.6µM. The complex can increase the levels of ROS, and induce a decrease in the mitochondrial membrane potential. Ir-1 inhibits the cell growth at G0/G1 phase in SGC-7901 cells, and the complex can induce both autophagy and apoptosis and inhibit the cell invasion. And the complex induces apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Amsacrine-induced apoptosis of human leukemia U937 cells is mediated by the inhibition of AKT- and ERK-induced stabilization of MCL1.

Previous studies have attributed the anticancer activity of amsacrine to its inhibitory effect on topoisomerase II. However, 9-aminoacridine derivatives, which have the same structural scaffold as amsacrine, induce cancer cell apoptosis by altering the expression of BCL2 family proteins. Therefore, in the present study, we assessed whether BCL2 family proteins mediated the cytotoxic effects of amsacrine on human leukemia U937 cells. Amsacrine-induced apoptosis of U937 cells was characterized by caspase-9 and caspase-3 activation, increased intracellular Ca2+ concentration, mitochondrial depolarization, and MCL1 down-regulation. Amsacrine induced MCL1 down-regulation by decreasing its stability. Further, amsacrine-treated U937 cells showed AKT degradation and Ca2+-mediated ERK inactivation. Blockade of ERK-mediated phosphorylation of MCL1 inhibited the effect of Pin1 on the stabilization of MCL1, and AKT degradation promoted GSK3β-mediated degradation of MCL1. Restoration of ERK phosphorylation and AKT expression abrogated amsacrine-induced MCL1 down-regulation. Moreover, MCL1 over-expression inhibited amsacrine-induced depolarization of mitochondria membrane and increased the viability of amsacrine-treated cells. Taken together, our data indicate that amsacrine abolishes ERK- and Pin1-mediated stabilization of MCL1 and promotes GSK3β-mediated degradation of MCL1, leading to activate mitochondria-mediated apoptosis pathway in U937 cells.

Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP.

Synthesis, characterization and in vitro biological activities of ruthenium(II) polypyridyl complexes

The ruthenium(II) polypyridyl complexes [Ru(dmb)2(TCPI)](PF6)2 (1) and [Ru(ttbpy)2(TCPI)](PF6)2 (2) (dmb = 4,4′-dimethyl-2,2′-bipyridine, TCPI = 2-(3-(1H-1,3,7,8-tetraazacyclopenta[l]phenanthren-2-yl)phenyl)benzo[de]isoquinoline-1,3-dione, ttbpy = 4,4′-ditertiary butyl-2,2′-bipyridine) were synthesized and characterized. The in vitro cytotoxicities of the complexes were examined against a panel of cancer cell lines including SGC-7901, PC-12, HepG-2, SiHa, Eca-109, HeLa, Eca-9706, HOS and LO2 by 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide (MTT) method. Both complexes show higher activities against PC-12 cells, with IC50 values of 34.4 ± 1.3 and 26.8 ± 2.4 μM for 1 and 2, respectively. Cell apoptosis was assayed with acridine orange (AO) and ethidium bromide (EB) and annexin V/PI staining methods using fluorescence microscopy and flow cytometry. The reactive oxygen species, mitochondrial membrane potential and cell cycle distribution were assessed. Cell invasion was determined by Matrigel invasion assay, and the proteins associated with cell apoptosis were analyzed by western blot. The results suggest that the complexes induce the apoptosis of PC-12 cells through a ROS-mediated mitochondrial dysfunction pathway, accompanied by regulation of the expression of caspases and Bcl-2 family proteins.

Houttuynia cordataThunb Promotes Activation of HIF-1A–FOXO3 and MEF2A Pathways to Induce Apoptosis in Human HepG2 Hepatocellular Carcinoma Cells

Houttuynia cordata Thunb (H cordata), a medicinal plant, has anticancer activity, as it inhibits cell growth and induces cell apoptosis in cancer. However, the potential anti-cancer activity and mechanism of H cordata for human liver cancer cells is not well understood. Recently, we identified hypoxia-inducible factor (HIF)-1A, Forkhead box (FOX)O3, and MEF2A as proapoptotic factors induced by H cordata, suggesting that HIF-1A, FOXO3, and MEF2A contribute to the apoptosis of HepG2 hepatocellular carcinoma cells. FOXO3 transcription factors regulate target genes involved in apoptosis. H cordata significantly increased the mRNA and protein expression of HIF-1A and FOXO3 and stimulated MEF2A expression in addition to increased apoptosis in HepG2 cells within 24 hours. Therefore, we determined the potential role of FOXO3 on apoptosis and on H cordata–induced MEF2A in HepG2 cells. HIF-1A silencing by siRNA attenuated MEF2A and H cordata–mediated FOXO3 upregulation in HepG2 cells. Furthermore, H cordata–mediated MEF2A expression enhanced caspase-3 and caspase-7, which were abolished on silencing FOXO3 with siRNA. In addition, H cordata inhibited growth of human hepatocellular carcinoma xenografts in nude mice. Taken together, our results demonstrate that H cordata enhances HIF-1A/FOXO3 signaling, leading to MEF2A upregulation in HepG2 cells, and in parallel, it disturbs the expression of Bcl-2 family proteins (Bax, Bcl-2, and Bcl-xL), which results in apoptosis. Taken together, these findings demonstrate that H cordata promotes the activation of HIF-1A–FOXO3 and MEF2A pathways to induce apoptosis in human HepG2 hepatocellular carcinoma cells and is, therefore, a promising candidate for antitumor drug development.

Abstract 2444: TSLP regulates expression of Bcl2 family proteins in Ph-like ALL with CRLF2 alterations

Abstract B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood malignancy. A subset of children with B-ALL are at high risk for relapse and death. Gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome positive ALL. Approximately half of these Ph-like B-ALL are characterized by genetic alterations that result in overexpression of CRLF2. CRLF2, together with the IL-7 receptor α chain, forms a receptor complex for the cytokine, TSLP. When TSLP binds, the receptor initiates downstream JAK2/STAT5 and PI3/AKT/mTOR pathway activation. The activating JAK mutations found in some CRLF2 B-ALL led to speculation that TSLP stimulation is not a factor in this disease. However, we find that TSLP increases phosphorylation of STAT5, AKT and S6 (downstream of mTOR) in CRLF2 B-ALL cells, including those with JAK defects. Activation of these pathways has been associated with oncogenesis and chemoresistance and their downstream targets include members of the Bcl2 family. The Bcl2 family pro-survival molecule Bcl-XL is a down stream target of STAT5 in Ph+ B-ALL. Mcl-1, another BCL2 family pro-survival molecule is known to be upregulated by mTOR activation via post-translational mechanisms in B cell lymphoma. We hypothesized that TSLP-induced JAK2/STAT5 and PI3/AKT/mTOR pathway activation contribute to chemoresistance in high risk CRLF2 B-ALL by upregulating the expression of Bcl-XL and Mcl-1. To test this hypothesis we cultured human CRLF2 B-ALL cell lines (MUTZ5 and CALL4) with and without TSLP and evaluated expression of the Bcl2 family pro-survival proteins, Bcl-XL, Mcl-1, and Bcl2. We found that TSLP induced significant increases in Bcl-XL and Mcl-1 proteins, but not Bcl2 in CRLF2 B-ALL cells. These cell lines have activating Jak mutations and thus reflect the ability of TSLP to increase expression of the Bcl2 family proteins in cases where activating JAK mutations are present. Next we evaluated the effect of Mcl-1 inhibitor on MUTZ5 and CALL4 cells. Preliminary data from these experiments show that cell counts in cultures treated with Mcl-1 inhibitor are reduce by &amp;gt;90% and this reduction is maintained in the presence of TSLP. These data provide evidence that TSLP-induced CRLF2 signals increase expression of Bcl2 pro-survival proteins, even in CRLF2 B-ALL cells with activating JAK mutations. These data also suggest that Mcl-1 inhibitors could be an effective treatment for this disease. Ongoing studies will evaluate the effect of TSLP and Mcl-1 inhibitors in primary CRLF2 B-ALL samples. Citation Format: Cornelia Stoian, Muhammad Omair Kamal, Olivia Francis, Rhaya Johnson, Simone Montgomery, Jacqueline Coats, Hannah Choi, Shania Aponte-Paris, Micheal Reed, Shanalee Martinez, Karina Mayagoitia, Evgeny Chirshev, Chunhua Song, Sinisa Dovat, Kimberly J. Payne. TSLP regulates expression of Bcl2 family proteins in Ph-like ALL with CRLF2 alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2444.

Synthesis, characterization and anticancer activity studies of ruthenium(II) polypyridyl complexes on A549 cells

Four new ruthenium(II) polypyridyl complexes [Ru(N-N)2(bddp)](ClO4)21–4 (N-N=dmb: 4,4′-dimethyl-2,2′-bipyridine 1, bpy: 2,2′-bipyridine 2, phen: 1,10-phenanthroline 3 and dmp: 2,9-dimethyl-1,10-phenanthroline 4, bddp=benzilo[2,3-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine) were synthesized and characterized by elemental analysis, ESI-MS and 1H NMR. The cytotoxicity in vitro of the complexes against BEL-7402, HeLa, MG-63 and A549 cell lines was investigated by MTT method. The complexes show high cytotoxic activity toward the selected cell lines with an IC50 value ranging from 5.3±0.6 to 15.7±3.6μM. The apoptosis was studied with acridine orange (AO)/ethdium bromide (EB) and Hoechst 33258 staining methods. The cellular uptake was investigated with DAPI staining method. The reactive oxygen species (ROS) and mitochondrial membrane potential were performed under fluorescent microscope and flow cytometry. The complexes can induce an increase in the ROS levels and a decrease in the mitochondrial membrane potential. The comet assay was studied with fluorescent microscope. The percentage in apoptotic and necrotic cells and cell cycle arrest were assayed by flow cytometry. The effects of the complexes on the expression of caspases and Bcl-2 family proteins were studied by western blot analysis. The results show that the complexes induce apoptosis in A549 cells through an ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of Bcl-2 family proteins.

Imiquimod Induces Apoptosis in Human Endometrial Cancer CellsIn vitro and Prevents Tumor Progression In vivo.

The increasing incidence of endometrial cancer (EC), in younger age at diagnosis, calls for new tissue-sparing treatment options. This work aims to evaluate the potential of imiquimod (IQ) in the treatment of low-grade EC. Effects of IQ on the viabilities of Ishikawa and HEC-1A cells were evaluated using MTT assay. The ability of IQ to induce apoptosis was evaluated by testing changes in caspase 3/7 levels and expression of cleaved caspase-3, using luminescence assay and western blot. Apoptosis was confirmed by flow cytometry and the expression of cleaved PARP. Western blot was used to evaluate the effect of IQ on expression levels of Bcl-2, Bcl-xL, and BAX. Finally, the in vivo efficacy of IQ was tested in an EC mouse model. There was a decrease in EC cell viability following IQ treatment as well as increased caspase 3/7 activities, cleaved caspase-3 expression, and Annexin-V/ 7AAD positive cell population. Western blot results showed the ability of IQ in cleaving PARP, decreasing Bcl-2 and Bcl-xL expressions, but not affecting BAX expression. In vivo study demonstrated IQ's ability to inhibit EC tumor growth and progression without significant toxicity. IQ induces apoptosis in low-grade EC cells in vitro, probably through its direct effect on Bcl-2 family protein expression. In, vivo, IQ attenuates EC tumor growth and progression, without an obvious toxicity. Our study provides the first building block for the potential role of IQ in the non-surgical management of low-grades EC and encouraging further investigations.

Phytoestrogen Bakuchiol Exhibits In Vitro and In Vivo Anti-breast Cancer Effects by Inducing S Phase Arrest and Apoptosis.

Phytoestrogen has been proposed as an alternative to hormone replacement therapy, which has been demonstrated to promote a high risk of breast cancer. However, the effect of phytoestrogen on breast cancer development has not been fully understood. Bakuchiol is an active ingredient of a traditional Chinese herbal medicine Fructus Psoraleae, the dried ripe fruit of Psoralea corylifolia L. (Fabaceae). The in vitro and in vivo estrogenic activities and anti-breast cancer effects of bakuchiol have not been well-studied. We found that bakuchiol induced the GFP expression in transgenic medaka (Oryzias melastigma, Tg, Chg:GFP) dose-dependently (0–1 μg/ml), demonstrating its in vivo estrogenic activity. Low dose of bakuchiol (1 μg/ml) induced the cell proliferation and ERα expression in MCF-7 cells, which could be blocked by the anti-estrogen ICI 182780, suggesting the in vitro estrogenic activity of bakuchiol. Our data indicated that high doses of bakuchiol (>2 μg/ml) inhibited breast cancer cell growth, with a stronger anti-proliferative effect than resveratrol, a widely studied analog of bakuchiol. High doses of bakuchiol (4, 7, and 10 μg/ml) were used for the further in vitro anti-breast cancer studies. Bakuchiol induced ERβ expression and suppressed ERα expression in MCF-7 cells. It also induced S phase arrest in both MCF-7 and MDA-MB-231 cells, which could be rescued by caffeine. Knock-down of p21 also marginally rescued S phase arrest in MCF-7 cells. The S phase arrest was accompanied by the upregulation of ATM, P-Cdc2 (Tyr15), Myt1, P-Wee1 (Ser642), p21 and Cyclin B1, suggesting that blocking of Cdc2 activation may play an important role in bakuchiol-induced S phase arrest. Furthermore, bakuchiol induced cell apoptosis and disturbed mitochondrial membrane potential in MCF-7 cells. The bakuchiol-induced apoptosis was associated with increased expression of Caspase family and Bcl-2 family proteins, suggesting that bakuchiol may induce apoptosis via intrinsic apoptotic pathway. The in vivo anti-breast cancer effect of bakuchiol was further proved in zebrafish (Danio rerio, wild-type AB) xenografts. 0.5 μg/ml of bakuchiol significantly reduced the MCF-7 cell mass in zebrafish xenografts. Overall, these results suggested the potential of using bakuchiol in HRT and breast cancer treatment.

Neonatal anoxia leads to time dependent progression of mitochondrial linked apoptosis in rat cortex and associated long term sensorimotor deficits

Neonatal anoxia arises due to oxygen deprivation at the time of birth and results in life long neurodevelopmental deficits and sometimes may lead to death. The present study investigated the time dependent cortical mitochondrial dysfunction linked apoptosis and related sensorimotor deficits in neonates. Neonates after 30h to birth (P2) were subjected to anoxia of two episodes (10min in each) at a time interval of 24h by passing 100% N2 into an enclosed chamber as confirmed by pulse oximetry. Sensorimotor activities like reflex latency and hanging latency were carried out 24h after last anoxic episode i.e. from P4 (day-1) and continued up to P10 (day-7). Mitochondrial dysfunction after anoxia was evident by the decrease in respiration states, respiratory control ratio (RCR), antioxidant enzyme activity but an increase in oxidative stress and lipid peroxidation and alteration in mitochondrial membrane potential (MMP) at different time points (30min, 24h and day-7). A change in expression of Bcl-2 family proteins and opening of mitochondrial transition pore (mPTP) in terms of mitochondrial swelling was observed resulting in release of cytochrome-C which further activated intrinsic (mitochondrial) pathway of apoptosis through increased expression of caspase-9/3 as confirmed by flow cytometry. In conclusion, anoxia injury leads to progressive activation of mitochondrial events leading to increase in apoptotic cell death following secondary pathological insult. Therefore, strategies in limiting mitochondrial-linked apoptosis during the secondary insult input process may be useful in treatment of long term sensorimotor deficits following anoxia.