Knockdown Of Bcl-2 Research Articles

Abstract 477: Bcl-2 suppresses cell spreading, adhesion and migration in non-small cell lung cancer cells

Abstract Bcl-2 is mainly thought to regulate cell apoptosis. However, little is known about its regulation associated with cell metastasis. Here, we demonstrate that Bcl-2 plays an important role in the cell spreading, adhesion and migration of non-small cell lung cancer cells. To study the role of Bcl-2, mutant cell lines exhibiting stably up-regulated and down-regulated Bcl-2 were generated using Bcl-2 plasmid and RNA interference. As compared to control H460 cells, Bcl-2 overexpressed cells exhibited significantly decreased cell spreading, adhesion and migration. Consistent with these results, the loss of Bcl-2 in Bcl-2 knockdown cells increased cell spreading, adhesion and migration. The morphology characteristics study has shown that Bcl-2 increased mesenchymal-epithelial transition phenotype whereas the loss of Bcl-2 increased epithelial-mesenchymal transition phenotype. The increase of fluorescent intensity of phalloidin staining in Bcl-2 overexpressed cells suggesting actin polymerization might be responsible in the supression of cell spreading, adhesion and migration. This result may explain why Bcl-2 has been suggested as a prognostic factor for survival in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 477. doi:1538-7445.AM2012-477

Nrf2 Protein Up-regulates Antiapoptotic Protein Bcl-2 and Prevents Cellular Apoptosis

Nuclear transcription factor Nrf2 regulates the expression and coordinated induction of a battery of genes encoding cytoprotective and drug transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival, and increased drug resistance. In this study, we investigated the role of Nrf2 in up-regulation of antiapoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Exposure of mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells to antioxidant tert-butylhydroquinone led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element between nucleotides -3148 and -3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/supershift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 antioxidant response element. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2, resulting in an increased Bcl-2 level and decreased etoposide- and UV/γ radiation-mediated DNA fragmentation. In addition, siRNA-mediated down-regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knockdown of Bcl-2 in Nrf2-activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and growth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance.

MiR-181a sensitizes resistant leukaemia HL-60/Ara-C cells to Ara-C by inducing apoptosis

Ara-C is one of the most commonly used drugs in the treatment of AML. However, the development of drug resistance always prevented its further use. It has been shown that miR-181a is associated with the clinical outcome of AML patients. Here, we investigated the possible role of miR-181a in AML Ara-C resistance. miR-181a expression was measured by real-time PCR. Cell viability was detected by MTT assay. Protein expressions were measured by western blotting. Caspase activity was examined by fluorescence assay. We found that miR-181a expression was downregulated in the Ara-C-resistant cell line HL-60/Ara-C compared with its parental cell line HL-60. Overexpression of miR-181a in HL-60/Ara-C cells sensitized the cells to Ara-C treatment. Furthermore, Bcl-2 was confirmed as a direct miR-181a target by immunoblot analysis and reporter gene assays. Knockdown of Bcl-2 mimicked the effect of enforced miR-181a expression by reducing cell viability. In addition, the apoptosis pathway was activated by cytochrome C release and caspase 9/caspase 3 activation after miR-181a overexpression. This study for the first time demonstrated that downregulation of miR-181a and upregulation of Bcl-2 in leukaemia cells confer resistance to Ara-C-based therapy. These results suggest that restoration of miR-181a expression might provide a promising therapeutic in drug resistance of leukaemia.

Abstract C149: STAT3 is a critical mediator of the prosurvival effect of Bcl-2-Rac1 interaction in human cancer cells.

Abstract The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. Gene silencing and functional inhibition of Rac1 blocked Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. We provide evidence for a co-localization and physical interaction between the 2 proteins, which could be blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. In an attempt to study the functional implication of this interaction, we carried out a computer simulation driven virtual predictive experiments based on the protein pathway dynamic network created by Cellworks Group Inc. The base line used for the study was a KRAS over-activated, PI3K overexpressed, CDKN2A deleted, β-catenin overexpressed and Bcl-2 overexpressed system aligned to HCT116 human colorectal cancer cell line. When a variant of the above base line was created with Rac1 overexpression, phenotypic indices of angiogenesis, proliferation, viability, metastasis as well as tumor volume were all upregulated. Strikingly, the expression levels of STAT3 and β-catenin were significantly amplified in the computer modeled variant cell line; however upon Rac1 or Bcl-2 knockdown in both the base and the variant cell lines, apoptotic markers such as Bax, caspase 3 and cleaved PARP1 were increased while STAT3, β-catenin, NF-κB and AGER were significantly down-regulated. Intrigued by these predictions, we set out to verify the link between STAT3 signaling and Bcl-2-Rac1 interaction. Interestingly, the activation status of STAT3 (pY705) strongly correlated with Bcl-2 expression as well as Rac1 activation status. Furthermore, STAT3 was found localized to mitochondria and preliminary results suggest an interaction with Bcl-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C149.

Upregulation of Cellular Bcl-2 by the KSHV Encoded RTA Promotes Virion Production

Apoptosis of virus infected cells can restrict or dampen full blown virus propagation and this can serve as a protective mechanism against virus infection. Consequently, viruses can also delay programmed cell death by enhancing the expression of anti-apoptotic proteins. Human Bcl-2 is expressed on the surface of the mitochondrial membrane and functions as the regulator of the delicate balance between cell survival and apoptosis. In this report, we showed that the replication and transcription activator (RTA) encoded by KSHV ORF 50, a key regulator for KSHV reactivation from latent to lytic infection, upregulates the mRNA and protein levels of Bcl-2 in 293 cells, and TPA-induced KSHV-infected cells. Further analysis revealed that upregulation of the cellular Bcl-2 promoter by RTA is dose-dependent and acts through targeting of the CCN9GG motifs within the Bcl-2 promoter. The Bcl-2 P2 but not the P1 promoter is primarily responsive to RTA. The results of ChIP confirmed the direct interaction of RTA protein with the CCN9GG motifs. Knockdown of cellular Bcl-2 by lentivirus-delivered small hairpin RNA (shRNA) resulted in increased cell apoptosis and decreased virion production in KSHV-infected cells. These findings provide an insight into another mechanism by which KSHV utilizes the intrinsic apoptosis signaling pathways for prolonging the survival of lytically infected host cells to allow for maximum production of virus progeny.

The Role of p27 Kip1 in Dasatinib-Enhanced Paclitaxel Cytotoxicity in Human Ovarian Cancer Cells

Less than 50% of ovarian cancers respond to paclitaxel. Effective strategies are needed to enhance paclitaxel sensitivity. A library of silencing RNAs (siRNAs) was used to identify kinases that regulate paclitaxel sensitivity in human ovarian cancer SKOv3 cells. The effect of dasatinib, an inhibitor of Src and Abl kinases, on paclitaxel sensitivity was measured in ovarian cancer cells and HEY xenografts. The roles of p27(Kip1), Bcl-2, and Cdk1 in apoptosis induced by dasatinib and paclitaxel were assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, siRNA knockdown of gene expression, transfection with Bcl-2 and Cdk1 expression vectors, and flow cytometry. All statistical tests were two-sided. Src family and Abl kinases were identified as modulators of paclitaxel sensitivity in SKOv3 cells. The siRNA knockdown of Src, Fyn, or Abl1 enhanced paclitaxel-mediated growth inhibition in ovarian cancer cells compared with a control siRNA. HEY cells treated with dasatinib plus paclitaxel formed fewer colonies than did cells treated with either agent alone. Treatment of HEY xenograft-bearing mice with dasatinib plus paclitaxel inhibited tumor growth more than treatment with either agent alone (average tumor volume per mouse, dasatinib + paclitaxel vs paclitaxel: 0.28 vs. 0.81 cm3, difference = 0.53 cm3, 95% confidence interval [CI] = 0.44 to 0.62 cm3, P = .014); dasatinib + paclitaxel vs. dasatinib: 0.28 vs. 0.55 cm3, difference = 0.27 cm3, 95% CI = 0.21 to 0.33 cm3, P = .035). Combined treatment induced more TUNEL-positive apoptotic cells than did either agent alone. The siRNA knockdown of p27(Kip1) decreased dasatinib- and paclitaxel-induced apoptosis compared with a negative control siRNA (sub-G1 fraction, control siRNA vs. p27(Kip1) siRNA: 42.5% vs. 20.1%, difference = 22.4%, 95% CI = 20.1% to 24.7%, P = .017). Studies with forced expression and siRNA knockdown of Bcl-2 and Cdk1 suggest that dasatinib-mediated induction of p27(Kip1) enhanced paclitaxel-induced apoptosis by negatively regulating Bcl-2 and Cdk1 expression. Inhibition of Src family and Abl kinases with either siRNAs or dasatinib enhances paclitaxel sensitivity of ovarian cancer cells through p27(Kip1)-mediated suppression of Bcl-2 and Cdk1 expression.

MET-Independent Lung Cancer Cells Evading EGFR Kinase Inhibitors Are Therapeutically Susceptible to BH3 Mimetic Agents

Targeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after initial responses. To define early molecular changes within residual tumor cells that persist after treatment, we analyzed drug-sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible epidermal growth factor receptor (EGFR) inhibitors, alone or in combination with MET-kinase inhibitors, to characterize the adaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependence on MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversed after withdrawal of targeted inhibitors. Findings were validated in a xenograft model, showing BCL-2 induction and p-STAT3[Y705] activation within the residual tumor cells surviving the initial antitumor response to targeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cells using BCL-2 Homology Domain 3 (BH3) mimetic agents such as ABT-737, or by dual RNAi-mediated knockdown of BCL-2/BCL-XL, was sufficient to eradicate the early-resistant lung-tumor-cells evading targeted inhibitors. Similarly, in a xenograft model the preemptive cotreatment of lung tumor cells with an EGFR inhibitor and a BH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response with prolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combined with targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung-cancer treatment.

Abstract 17: Bcl-2 is a downstream target of ATF5 that mediates ATF5's pro-survival function in a cell type-dependent manner

Abstract The activating transcription factor 5 (ATF5) is a member of the ATF/CREB family of basic zipper (bZIP) proteins. Recent reports showed that ATF5 loss-of-function causes apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for ATF5's cell type-dependent survival function is unknown. We report here that the anti-apoptotic factor Bcl-2 is a downstream target that mediates ATF5-promoted cancer-cell survival in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element (ARE) that is downstream of and adjacent to the negative regulatory element (NRE) in the Bcl-2 P2 promoter, stimulating Bcl-2 expression. Highlighting the importance of Bcl-2 in ATF5-dependent cancer-cell survival, expression of Bcl-2 blocks death of C6 and MCF-7 cells induced by d/nATF5 and depletion of Bcl-2 impairs ATF5-promoted cell survival. Moreover, we found that Bcl-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, MEFs, and human breast epithelial cells (HBEC), where knock-down of Bcl-2 but not that of ATF5 evokes cell death. These findings identify Bcl-2 as an essential mediator for ATF5's cancer-specific cell survival function in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from the differential regulation of downstream targets by ATF5 in different types of cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 17. doi:10.1158/1538-7445.AM2011-17

Radiosensitization of Head and Neck Squamous Cell Carcinoma by a SMAC-Mimetic Compound, SM-164, Requires Activation of Caspases

Chemoradiation is the treatment of choice for locally advanced head and neck squamous cell carcinoma (HNSCC). However, radioresistance, which contributes to local recurrence, remains a significant therapeutic problem. In this study, we characterized SM-164, a small second mitochondria-derived activator of caspase -mimetic compound that promotes degradation of cellular inhibitor of apoptosis-1(cIAP-1; also known as baculoviral IAP repeat-containing protein 2, BIRC2) and releases active caspases from the X-linked inhibitor of apoptosis inhibitory binding as a radiosensitizing agent in HNSCC cells. We found that SM-164 at nanomolar concentrations induced radiosensitization in some HNSCC cell lines in a manner dependent on intrinsic sensitivity to caspase activation and apoptosis induction. Blockage of caspase activation via short interfering RNA knockdown or a pan-caspase inhibitor, z-VAD-fmk, largely abrogated SM-164 radiosensitization. On the other hand, the resistant lines with a high level of Bcl-2 that blocks caspase activation and apoptosis induction became sensitive to radiation on Bcl-2 knockdown. Mechanistic studies revealed that SM-164 radiosensitization in sensitive cells was associated with NF-κB activation and TNFα secretion, followed by activation of caspase-8 and -9, leading to enhanced apoptosis. Finally, SM-164 also radiosensitized human tumor xenograft while causing minimal toxicity. Thus, SM-164 is a potent radiosensitizer via a mechanism involving caspase activation and holds promise for future clinical development as a novel class of radiosensitizer for the treatment of a subset of head and neck cancer patients.

MiR-34a in Age and Tissue Related Radio-Sensitivity and Serum miR-34a as a Novel Indicator of Radiation Injury

MiR-34a, a direct target of p53, has shown to exert potent anti-proliferative effects. It has also been found that miR-34a can be induced by irradiation in vitro and in vivo. However, the relationship between miR-34a and radio-sensitivity, and its potential diagnostic significance in radiation biology, remain unclear. This study found that differing responses to ionizing radiation (IR) of young and adult mice were related to miR-34a. First, we found that miR-34a could be induced in many organs by radiation of both young and adult mice. However, the level of miR-34a induced by young mice was much higher when compared to adult mice. Next, we found that miR-34a played a critical role in radio-sensitivity variations of different tissues by enhancing cell apoptosis and decreasing cell viability. We also found that the induction of miR-34a by radiation was in a p53 dependent manner and that one possible downstream target of miR-34a that lead to different radio-sensitivity was the anti-apoptosis molecular Bcl-2. However, over-expression of miR-34a and knockdown of Bcl-2 could significantly enhance the radio-sensitivity of different cells while inhibition of miR-34a could protect cells from radiation injury. Finally, we concluded that miR-34a could be stable in serum after IR and serve as a novel indicator of radiation injury. Taken together, this data strongly suggests that miR-34a may be a novel indicator, mediator and target of radiation injury, radio-sensitivity and radioprotection.

Helicobacter pylori VacA Reduces the Cellular Expression of STAT3 and Pro-survival Bcl-2 Family Proteins, Bcl-2 and Bcl-XL, Leading to Apoptosis in Gastric Epithelial Cells

Helicobacter pylori vacuolating cytotoxin, VacA, stimulates apoptosis via a mitochondria-dependent pathway. VacA induces apoptosis via activation of the pro-apoptotic B-cell lymphoma (Bcl)-2 family proteins, Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), while the implication of such pro-survival Bcl-2 family members as Bcl-2 and Bcl-XL in the VacA-induced apoptosis remains unknown. Signal transduction and activator of transcription 3 (STAT3) is a pivotal transcription factor that upregulates Bcl-2 and Bcl-XL. This study was conducted to elicit the implication of STAT3 and pro-survival Bcl-2 and Bcl-XL in the intrinsic apoptosis. Immunoblot and reverse transcriptase real-time polymerase chain reaction (RT-PCR) were employed to assess the cellular expression of STAT3, Bcl-2, and Bcl-XL in response to purified VacA in gastric adenocarcinoma cell lines. VacA-induced apoptosis was quantitated morphologically following knockdown by each specific small interfering RNA (siRNA) or in the presence of pharmacological inhibitors. VacA reduced STAT3, Bcl-2, and Bcl-XL expression in a dose-dependent manner. Knockdown of STAT3, Bcl-2, and Bcl-XL by siRNA induced apoptosis to a similar extent in the case of sufficient VacA inoculation. The VacA-mediated reduction of STAT3 expression was independent of cellular vacuolization, since a vacuolar-type ATPase inhibitor, bafilomycin A1, did not inhibit VacA-induced reduction of STAT3, Bcl-2, and Bcl-XL expression. Instead, a c-JUN NH2-terminal kinase (JNK) inhibitor, SP600125, restored the VacA-induced reduction of STAT3 expression to the basal level. VacA-induced apoptosis may be, in part, implicated in the reduction of STAT3 linking to the downregulation of Bcl-2 and Bcl-XL, in association with JNK activity.

E-cadherin contributes to the bystander effect of TK/GCV suicide therapy and enhances its antitumoral activity in pancreatic cancer models.

The thymidine kinase/ganciclovir (TK/GCV) cancer gene therapy approach is based on inducing GCV metabolite cytotoxicity in tumor cells expressing the herpes simplex virus TK gene and exposed to GCV. A bystander effect, mediated by gap junctions, accounts for the transfer of toxic metabolites from TK-expressing cells to neighboring cells. It has been proposed that E-cadherin participates in the formation and function of such gap junctions. In this study we investigate the influence of E-cadherin on TK/GCV suicide therapy with a panel of cellular and in vivo models of pancreatic ductal adenocarcinoma. We observed a strong correlation of E-cadherin expression and the TK/GCV bystander effect, associated with the modulation of gap junction communication and connexin expression or localization. Importantly, the co-expression of TK and E-cadherin genes in the adenoviral vector AdTat8TKIE improved TK/GCV cytotoxicity and triggered a potent antitumoral effect, superior to standard AdTat8TK/GCV in MIAPaCa-2 xenografts. The increased expression of E-cadherin resulted in the reduction of the bcl-2 content. Interestingly, the knockdown of bcl-2 sensitized cells to TK/GCV. Thus, we propose that by restoring E-cadherin in pancreatic tumor cells we will improve TK/GCV therapy, both by enhancing the bystander effect and by facilitating the induction of apoptosis.

Angiogenin prevents serum withdrawal‐induced apoptosis of P19 embryonal carcinoma cells

Angiogenin is a 14 kDa protein originally identified as an angiogenic protein. Recent development has shown that angiogenin acts on both endothelial cells and neuronal cells. Loss-of-function mutations in the coding region of the ANG gene have recently been identified in patients with amyotrophic lateral sclerosis. Angiogenin has been shown to control motor neuron survival and protect neurons from apoptosis under various stress conditions. In this article, we characterize the anti-apoptotic activity of angiogenin in pluripotent P19 mouse embryonal carcinoma cells. Angiogenin prevents serum withdrawal-induced apoptosis. Angiogenin upregulates anti-apoptotic genes, including Bag1, Bcl-2, Hells, Nf-kappab and Ripk1, and downregulates pro-apoptotic genes, such as Bak1, Tnf, Tnfr, Traf1 and Trp63. Knockdown of Bcl-2 largely abolishes the anti-apoptotic activity of angiogenin, whereas the inhibition of Nf-kappab activity results in a partial, but significant, inhibition of the protective activity of angiogenin. Thus, angiogenin prevents stress-induced cell death through both the Bcl-2 and Nf-kappab pathways.

Glutaredoxin Regulates Apoptosis in CardiomyocytesviaNFκB Targets Bcl-2 and Bcl-xL: Implications for Cardiac Aging

Cardiomyocyte apoptosis is a well-established contributor to irreversible injury following myocardial infarction (MI). Increased cardiomyocyte apoptosis is associated also with aging in animal models, exacerbated by MI; however, mechanisms for this increased sensitivity to oxidative stress are unknown. Protein mixed-disulfide formation with glutathione (protein glutathionylation) is known to change the function of intermediates that regulate apoptosis. Since glutaredoxin (Grx) specifically catalyzes protein deglutathionylation, we examined its status with aging and its influence on regulation of apoptosis. Grx1 content and activity are decreased by approximately 40% in elderly (24-mo) Fischer 344 rat hearts compared to adult (6-mo) controls. A similar extent of Grx1 knockdown in H9c2 cardiomyocytes led to increased apoptosis, decreased NFkappaB-dependent transcriptional activity, and decreased production (mRNA and protein) of anti-apoptotic NFkappaB target genes, Bcl-2 and Bcl-xL. Knockdown of Bcl-2 and/or Bcl-xL in wild-type H9c2 cells to the same extent ( approximately 50%) as observed in Grx1-knockdown cells increased baseline apoptosis; and knockdown of Bcl-xL, but not Bcl-2, also increased oxidant-induced apoptosis analogous to Grx1-knockdown cells. Natural Grx1-deficient cardiomyocytes isolated from elderly rats also displayed diminished NFkappaB activity and Bcl-xL content. Taken together, these data indicate diminution of Grx1 in elderly animals contributes to increased apoptotic susceptibility via regulation of NFkappaB function.

Elevated XIAP expression alone does not confer chemoresistance

Background:In various tumour types, elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been observed and XIAP targeting in diverse tumour entities enhanced the susceptibility to chemotherapeutic agents. Therefore, XIAP has been described and reviewed repeatedly as a chemoresistance factor in different tumour entities. However, rather than being an adverse prognostic marker, recent data suggest that elevated XIAP expression may be associated with a favourable clinical outcome. These somewhat conflicting findings, and the fact that in early studies XIAP suppressed apoptosis only when expressed transiently at levels far in excess of its physiological concentration, argue that the function of XIAP as an anti-apoptotic factor in tumour cells is both more complex and diverse than previously appreciated.Methods:To better understand the impact of long-term elevated XIAP expression on resistance to chemotherapy, we generated cell lines stably overexpressing XIAP. The role of mitochondria was examined by stable expression of Bcl2 or stable knockdown of second mitochondria-derived activator of caspase (SMAC) in combination with up- or downregulation of XIAP expression.Results:Our data show that long-term expression of XIAP at concentrations comparable to that in tumour cells (two- to five-fold increase) resulted in little or no resistance towards chemotherapeutic drugs. The XIAP overexpression only in conjunction with stable knockdown of a single XIAP-antagonising factor such as SMAC resulted in severe resistance to cytostatic agents demonstrating XIAP as a potent chemoresistance factor only in cells lacking functional XIAP regulatory circuits.Conclusion:Our results demonstrated that elevated XIAP expression alone cannot serve as a predictive marker of chemoresistance. Our data suggest that in order to predict the impact of XIAP on chemosusceptibility for a given tumour entity, the expression levels and functional states of all XIAP modulators need to be taken into account.

Diazoxide Suppresses Hepatic Ischemia/Reperfusion Injury After Mouse Liver Transplantation by a BCL-2-Dependent Mechanism

Ischemia-reperfusion injury (IRI) is responsible for primary liver dysfunction and failure after transplantation. The mitochondrial pathway appears to be involved in liver ischemia-reperfusion injury. Mitochondrial ATP-sensitive K (mitoK(ATP)) channels play a central role in protecting the heart from injury in ischemic preconditioning. The selective mitoK(ATP) channel agonist diazoxide potently reduced mitochondrial injury by preventing cytochrome c loss from the intermembrane space. Therefore, this study sought to determine whether diazoxide can attenuate ischemia-reperfusion injury induced by orthotopic liver transplantation (OLT) in mice. Furthermore, it was found that up-regulation of the Bcl-2 gene is a mechanism of diazoxide cytoprotection. Donors were treated with diazoxide, Bcl-2 siRNA, or diazoxide + Bcl-2 siRNA and vehicle 10 min or 24 h before liver harvesting. Liver grafts were then orthotopically transplanted into their corresponding recipients. Liver injury, as judged by transaminase level and histologic examination, was significantly lower in the diazoxide group compared with vehicle controls. The percentage of apoptotic cells and the amount of cytochrome c in the cytosol 6 h after transplant were also markedly reduced in diazoxide-treated grafts compared with vehicle-treated controls. Diazoxide notably up-regulated expression of Bcl-2, while siRNA knockdown of Bcl-2 abolished the cytoprotective effects of diazoxide. Diazoxide attenuated graft injury after mouse liver transplantation. One mechanism of diazoxide protection involves the induction of Bcl-2, an anti-apoptotic protein. Diazoxide might be useful clinically in hepatic surgery and transplantation.

Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs’ proliferation and migration. Over-expression of Bcl-2 increased HAECs’ tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation. Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.

Targeted knockdown of Bcl2 in tumor cells using a synthetic TRAIL 3′‐UTR microRNA

Targeting tumor-related overexpression of anti-apoptotic Bcl2 protein by RNAi has been suggested as a potential treatment for cancer. However, the stability of RNAi and its delivery are still major obstacles to the clinical testing of Bcl2 RNAi. Here, we explore a novel strategy of expressing a synthetic Bcl2 microRNA (smRNA) in the 3' untranslated region (UTR) of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis-inducing protein without apparent toxic effects in normal cells. TRAIL was specifically expressed from the human telomerase reverse transcriptase promoter (pTRT) that is active in many human tumors. Using this approach, we demonstrated that pTRT drove the tumor-specific expression of Bcl2 smRNA, which was processed by the host RNAi machinery and silenced endogenous Bcl2 expression in tumor cells. Bcl2 smRNA induced tumor cell apoptosis by activating caspase-3 and led to significant sensitization of tumor cells to TRAIL-induced apoptosis, while normal cells were spared. We also showed that the combined therapy of TRAIL-induced apoptosis and Bcl2 downregulation was superior to the mono-therapy of TRAIL or Bcl2 smRNA alone. This study proves a general paradigm for cancer therapy by using 3' UTR microRNA technology.

Abstract B22: Dynamics of cancer cell survival dependency on Bcl-2 family members

Abstract Apoptosis is regulated through the interactions of the Bcl-2 family of proteins. Overexpression of anti-apoptotic Bcl-2 family members plays a key role in promoting survival for cells undergoing various insults that would normally trigger cell death. Consequently, these proteins are believed to serve as contributing factors to the processes of tumor initiation, tumor progression and the development of resistance to therapeutics. ABT-263, an antagonist of Bcl-2, Bcl-xL, and Bcl-w, is currently being tested in clinical trials. Some cancer cells express high amounts of Mcl-1, another Bcl-2 family anti-apoptotic protein that shares pro-survival function with Bcl-2 but is not targeted by ABT-263. Preclinical data indicates Mcl-1 overexpressing cells are not sensitive to ABT-263. To understand this mechanism of Mcl-1 dependence, A2058 melanoma cells were infected with a lentivirus encoding inducible shRNA against Mcl-1. Upon induction of MCL1 knockdown, massive cell death occurred. Interestingly, a small subset of the cell population managed to survive. Subsequent western blot analysis confirmed a sustained depletion of Mcl-1. Further study indicated A2058 cells deficient in Mcl-1 survived by upregulating expression of Bcl-2, a process we term “switching dependency”. In addition, these cells became sensitized to ABT-263 treatment. An A2058 xenograft model indicates that combined knockdown of both Mcl-1 and Bcl-2 significantly inhibited tumor growth when compared to tumors treated with ABT-263 lacking either protein individually. Real-time kinetic studies reveal a rapid sensitization of A2058 cells to ABT-263 upon sh-RNA induction. To understand whether de novo protein synthesis of Bcl-2 plays a mechanistic role in dependency switching, A2058 sh-Mcl-1 cells were treated with the protein synthesis inhibitor cycloheximide. Kinetic analysis indicates that de novo protein synthesis is not required for dependency switching. Real-time quantitative PCR was then employed to determine if a relationship exists between the decrease in Mcl-1 mRNA, and subsequent sensitivity to ABT-263, is a consequence of a change in Bcl-2 mRNA levels. Surprisingly, the results indicate as the loss of Mcl-1 message occurs, there is no concurrent change in Bcl-2 mRNA. Taken together these data indicate that dependency switching, and therefore survival, occurs without de novo Bcl-2 protein synthesis or alterations in protein turnover. Furthermore, dependency switching is a possible mechanism cancer cells employ to develop resistance toward various anti-cancer therapies with novel implications for both the concept of oncogene addiction and development of targeted cancer therapeutics. Therefore there is an immediate need for elucidation of this novel cancer resistance mechanism. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B22.

Downregulation of MiR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells through Modulation of NOTCH1 and BCL2.

Abstract 361▪▪This icon denotes an abstract that is clinically relevant.Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p<0.05) were shown to be related to the EVI1 expression status. Amongst these, miR-449a was selected for further study based on its homology to the known cancer associated miRNA miR-34a. Downregulation of miR-449a by EVI1 was further confirmed in the leukemic cell line U937 with tetracycline controllabel (tet-off) EVI1 overexpression. Next, direct transcriptional regulation of miR-449a expression by EVI1 was demonstrated by chromatin immunoprecipitation (ChIP). To test the functional consequences of downregulation of miR-449a in AML cells, reconstitution of the expression of miR-449a in the Kasumi-3 and UCSD-AML1 cell lines was performed, which resulted in significantly decreased cell viability, increased apoptosis and differentiation towards the megakaryocytic and monocytic lineages. Interestingly, siRNA mediated knockdown of EVI1 expression in Kasumi-3 or UCSD-AML1 almost completely abrogated the miR-449a induced reduction in cell viability, while electroporation of both cell lines with EVI1 siRNAs alone had essentially no effect on cell viability. These data strongly suggest that repression of miR-449a expression is essential for the survival and growth of EVI1 overexpressing cells and that this requirement is specifically imposed by EVI1 itself. We next demonstrated that the predicted miR-449a targets NOTCH1 and BCL2 were bona fide miR-449a targets using promoter reporter assays. To asses the contribution of these target genes to the observed phenotype upon miR-449a upregulation, knockdown of NOTCH1 and BCL2 was performed, revealing similar effects on cell viability and apoptosis. These results indicated that the effects seen upon treatment of cells with a precursor miR-449a are at least partly mediated through NOTCH1 and BCL2. In conclusion, we provided for the first time evidence that EVI1 mediated downregulation of miR-449a leads to NOTCH1 and BCL2 upregulation and is required for sustained proliferation and survival of EVI1 overexpressing cells. These data also open new perspectives for therapeutic intervention through modulation of miR-449a and/or its target genes. Disclosures:No relevant conflicts of interest to declare.