Up-regulation Of Pro-survival Proteins Research Articles

New synthetic phenylquinazoline derivatives induce apoptosis by targeting the pro-survival members of the BCL-2 family

Chemo-resistant cancer cells acquire robust growth potential through cell signaling mechanisms such as the down-regulation of tumor suppressors and the up-regulation of pro-survival proteins, respectively. To overcome chemo-resistance of cancer, small molecule drugs that interact with the cell signaling proteins to enhance sensitization of cancer cells toward cancer therapies are likely to be effective for the treatment of chemo-drug resistant cancer. To identify high potency small molecules, a series of ten novel phenylquinazoline derivatives were synthesized to determine their cellular effects in MCF-7 and MCF-7- cisplatin-resistant (CR) human breast cancer cells which led to the identification of two bioactive compounds, SMS-IV-20 and SMS-IV-40, that exhibited an elevated level of cytotoxicity against the human breast cancer cells and spheroid cells. In addition, both compounds enhanced chemo-sensitization of the human breast cancer cells that were genetically engineered to express the tumor suppressor and pro-apoptotic proteins, MOAP-1, Bax, and RASSF1a (MBR), suggesting that the compounds interact with the MBR signaling pathway. Furthermore, when MCF-7-CR cells were treated with SMS-IV-20 and SMS-IV-40 in the presence of ABT-737, a BCL-XL and BCL-2 inhibitor, enhanced chemo-sensitization was observed, suggesting SMS-IV-20 and SMS-IV-40 exert antagonistic activity to regulate the functional activity of BCL-2 and BCL-XL. Western blot analysis showed that both SMS-IV-20 and SMS-IV-40 induced down-regulation of BCL-2 or both BCl-2 and BCL-XL expression, respectively while promoting the release of mitochondrial Cytochrome C. Taken together, the data showed that SMS-IV-20 and SMS-IV-40 are potent activators of apoptosis that enhance chemo-sensitization through their antagonistic actions on the pro-survival activity of the BCl-2 family in human cancer cells.

Chronic IL-1 Exposed AR+ PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

Inflammation drives prostate cancer (PCa) progression. While inflammation is a cancer hallmark, the underlying mechanisms mediating inflammation-induced PCa are still under investigation. Interleukin-1 (IL-1) is an inflammatory cytokine that promotes cancer progression, including PCa metastasis and castration resistance. We previously found that acute IL-1 exposure represses PCa androgen receptor (AR) expression concomitant with the upregulation of pro-survival proteins, causing de novo accumulation of castration-resistant PCa cells. However, acute inflammation is primarily anti-tumorigenic, while chronic inflammation is pro-tumorigenic. Thus, using the LNCaP PCa cell line as model, we found that PCa cells can evolve insensitivity to chronic IL-1 exposure, restoring AR and AR activity and acquiring castration resistance. In this paper we expanded our chronic IL-1 model to include the MDA-PCa-2b PCa cell line to investigate the response to acute versus chronic IL-1 exposure and to compare the gene expression patterns that evolve in the LNCaP and MDA-PCa-2b cells chronically exposed to IL-1. We chronically exposed MDA-PCa-2b cells to IL-1α or IL-1β for several months to establish sublines. Once established, we determined subline sensitivity to exogenous IL-1 using cell viability assay, RT-qPCR and western blot. RNA sequencing was performed for parental and subline cells and over representation analysis (ORA) for geneset enrichment of biological process/pathway was performed. MDA-PCa-2b cells repress AR and AR activity in response to acute IL-1 exposure and evolve insensitivity to chronic IL-1 exposure. While cell biological and molecular response to acute IL-1 signaling is primarily conserved in LNCaP and MDA-PCa-2b cells, including upregulation of NF-κB signaling and downregulation of cell proliferation, the LNCaP and MDA-PCa-2b cells evolve conserved and unique molecular responses to chronic IL-1 signaling that may promote or support tumor progression. Our chronic IL-1 subline models can be used to identify underlying molecular mechanisms that mediate IL-1-induced PCa progression.

Co-Operation of MCL-1 and BCL-X L Anti-Apoptotic Proteins in Stromal Protection of MM Cells from Carfilzomib Mediated Cytotoxicity

Introduction: Expression and function of BCL-2 family proteins in multiple myeloma (MM) is important for tumour cell survival and drug resistance, and may partly mediate the protective effect of bone marrow (BM) stroma. Proteasome inhibitors (PIs) are a major class of anti-myeloma drugs, however some patients are resistant and almost all eventually relapse. Carfilzomib is a second generation, irreversible proteasome inhibitor, which is effective in both newly diagnosed and relapsed/refractory disease, but drug resistance and relapse occur.Aim: We aimed to study the role of BCL-2 family proteins in Carfilzomib (CFZ) mediated cytotoxicity in the presence of stroma, focusing on priming of the pro-apoptotic protein, BIM.Methods: A panel of Human Myeloma Cell Lines (HMCL) and primary MM cells were exposed to Carfilzomib, and cell death assessed using Annexin V/PI staining and flow cytometry. Co-cultures employing the HS5 stromal cell line were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1hr pulse, to recapitulate the pharmacokinetics of drug exposure in patients. The expression of BCL-2 family proteins was assessed by flow cytometry and Western Blot (WB). The compounds S63845, ABT-199 (Venetoclax) and A-1331852 were used to inhibit MCL-1, BCL-2 and BCL-X L pro-survival proteins respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB.Results: CFZ induced dose and time-dependent cell death of HMCL and primary MM cells. This was primarily mediated by apoptosis as shown by AnV/PI staining (eg. MM1.s at 25nM CFZ: 34±6% apoptosis at 24h, 47±9% at 48h, p=0.016; 50nM CFZ: 60 ±3.8% at 24h vs 79±6% at 48h, p=0.004, mean ±SEM, n=3), and by reduced cytotoxicity in MM1.s BAX/BAK KO cells (100nM CFZ: 10±1% cell death vs 42±5% in WT at 6h, 33±14% vs 99±0.5% at 24h; p=0.0005, n=3). CFZ cytotoxicity was significantly reduced when MM1.s cells were co-cultured with HS-5 (50nM CFZ: 53±4% apoptosis vs 79±4% in suspension, at 48h, p=0.02, n=3) confirming the protective effect of stromal cells. Similar results were seen with H929, KMS27 and KMS12BM HMCL (Figure 1). Co-culture of MM1.s with HS-5 cells induced upregulation of MCL-1 and BCL-X L and downregulation of BCL-2. Combined inhibition of BCL-X L and MCL-1 at concentrations where single agents had minimal effect, resulted ina marked increase in apoptosis on stroma (Ctrl: 9±1%, S63845 100nM:12±1.4%, A-1331852 1nM: 17±3%, S63845+A-1331852: 60±9%; 48h, p=0.0001, n=3) (Figure 3). BIM immunoprecipitation showed that in the presence of the MCL-1 inhibitor, S63845, there was increased BCL-X L binding to BIM; conversely when BCL-X L was inhibited by A-1331852, an increase in MCL-1/BIM complexes was seen. Exposure of MM cells to CFZ in stromal co-cultures led to a further increase in the expression of BCL-X L and MCL-1. Because BCL-X L and MCL-1 were upregulated both by stromal interaction and by CFZ (Figure 2), we tested the cooperativity of these anti-apoptotic proteins on resistance to CFZ. CFZ-induced cytotoxicity was significantly enhanced by the addition of single BH3 mimetics in the presence of HS-5: cell death 48h, BCL-X L A-133182 5nM: 40±12%, CFZ 25nM: 57±10%, CFZ+A-1331852: 94±2%, p=0.0065, n=3. Cell death with MCL-1 inhibitor S63845 200nM: 12±3%, CFZ 25nM:40±13%, S63845+CFZ: 54±18%, p=0.02, n=3). However, combined BCL-X L and MCL-1 inhibition, using lower concentrations of BH3 mimetics, potently sensitized MM cells to CFZ-mediated cytotoxicity in the presence of stroma (% killing at CFZ 25nM + A-1331852 1nM: 48±8%; CFZ+S63845 100nM: 38±5%; CFZ+A-1331852+S63845: 92±2%, p<0.0001, n=3, Figure 3).Conclusion: BCL-X L and MCL-1 are upregulated in MM cells by stromal interaction, and also in response to CFZ. The upregulation of pro-survival proteins by stroma may mediate MM cell resistance to PIs, and blockade of both BCL-X L and MCL-1 is required in order to abrogate the protective effect of stroma and restore sensitivity to CFZ. Combination of BH-3 mimetic drugs at subtherapeutic doses with proteasome inhibitors may be a potential therapeutic strategy. [Display omitted] DisclosuresHuang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Khwaja: Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yong: Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding.

Effects of curcumin, D-pinitol alone or in combination in cytotoxicity induced by arsenic in PC12 cells

Arsenic is a well-known potent toxicant affecting people by causing various human diseases. Long-term exposure to arsenic has strong adverse health effects on liver and kidney disorders, and various forms of cancer. Contrarily, curcumin and D-pinitol are bioactive dietary compounds that have antioxidant properties. Both are used to treat a broad variety of human diseases. Thus, we hypothesized that both may have synergistic effects against arsenic-induced toxicity in PC12 cells. Cells were pretreated with curcumin (1, 2.5, 5 and 10 μM), D-pinitol (1, 2.5, 5 and 10 μM) alone or in combination, then exposed to sodium arsenite (10 μM). The final concentration of curcumin 2.5 μM and D-pinitol 5 μM was selected for combination treatment based on their highest protection at lowest concentration against arsenic toxicity. Results demonstrated that pretreatment of curcumin and D-pinitol and their combined treatment with arsenic rescued PC12 cells. Western blot analysis results showed that pretreatment of curcumin and D-pinitol and their combined treatment with arsenic significantly inhibited arsenic-induced cell death through up-regulation of pro-survival proteins and down-regulation of cell death-related proteins, although these protein expressions were negatively regulated by arsenic. Furthermore, the effect of combined treatment with curcumin and D-pinitol was stronger than individual treatment.

Doxorubicin resistance in breast cancer cells is mediated by extracellular matrix proteins

BackgroundCancer cell resistance to therapeutics can result from acquired or de novo-mediated factors. Here, we have utilised advanced breast cancer cell culture models to elucidate de novo doxorubicin resistance mechanisms.MethodsThe response of breast cancer cell lines (MCF-7 and MDA-MB-231) to doxorubicin was examined in an in vitro three-dimensional (3D) cell culture model. Cells were cultured with Matrigel™ enabling cellular arrangements into a 3D architecture in conjunction with cell-to-extracellular matrix (ECM) contact.ResultsBreast cancer cells cultured in a 3D ECM-based model demonstrated altered sensitivity to doxorubicin, when compared to those grown in corresponding two-dimensional (2D) monolayer culture conditions. Investigations into the factors triggering the observed doxorubicin resistance revealed that cell-to-ECM interactions played a pivotal role. This finding correlated with the up-regulation of pro-survival proteins in 3D ECM-containing cell culture conditions following exposure to doxorubicin. Inhibition of integrin signalling in combination with doxorubicin significantly reduced breast cancer cell viability. Furthermore, breast cancer cells grown in a 3D ECM-based model demonstrated a significantly reduced proliferation rate in comparison to cells cultured in 2D conditions.ConclusionCollectively, these novel findings reveal resistance mechanisms which may contribute to reduced doxorubicin sensitivity.

Targeting the Apoptotic Pathway in Chondrosarcoma Using Recombinant Human Apo2L/TRAIL (Dulanermin), a Dual Proapoptotic Receptor (DR4/DR5) Agonist

Recombinant human Apo2L/TRAIL (dulanermin) is based on the ligand for death receptors (DR4 and DR5), which promotes apoptosis. We report a patient with refractory chondrosarcoma who showed a prolonged response to dulanermin and explore mechanisms of response and resistance. This heavily pretreated patient had progressive metastatic chondrosarcoma to the lung. On dulanermin (8 mg/kg i.v. on days 1-5 in a 21-day cycle), the patient achieved a sustained partial response with only subcentimeter nodules remaining. After 62 months of dulanermin treatment, progressive disease in the lungs was noted, and the patient underwent a resection that confirmed chondrosarcoma. DR4 was detected (immunohistochemistry) in the patient's tumor, which may have enabled the response. However, upregulation of prosurvival proteins, namely, phosphorylated (p)-NF-κBp65 (Ser 536), p-STAT3 (Tyr 705), p-ERK 1/2 (Thr 202/Tyr 204), p-mTOR (Ser 2448), FASN, and Bcl-2, were also detected, which may have provided the underlying mechanisms for acquired dulanermin resistance. The patient was restarted on dulanermin and has continued on this treatment for an additional 16 months since surgery (78 months since initiation of treatment), with his most recent computed tomography (CT) scans showing no evidence of disease.

Abstract A60: Late stage intestinal polyps in ApcMin/+ mice are resistant to treatment with retinoids and TRAIL due to up-regulation of pro-survival proteins.

Abstract The purpose of this study is to determine the mechanism of apoptotic resistance observed in advanced polyps in a mouse model of Familial Adenomatous Polyposis (FAP). FAP is an autosomal dominantly inherited disorder caused by mutations in the tumor suppressor Apc (adenomatous polyposis coli) gene, which results in the formation of hundreds to thousands of polyps, predominantly in the colon and rectum. Left untreated, these polyps will form colorectal adenocarcinomas. Although surgical removal of the colon eliminates the colon cancer risk, these individuals are also at increased risk for other primary cancers, including tumors of the small intestine and stomach. Systemic chemopreventive interventions to specifically remove the pre-cancerous cells in these individuals could greatly improve their outcomes and quality of life. Recently, we have developed a new method based on the ability of combined retinoid and TRAIL (tumor necrosis factor related apoptosis inducing ligand) treatment to induce apoptosis and regression of intestinal polyps in ApcMin/+ mice, a mouse model of FAP. Treatment of these mice with retinoid causes an increase in TRAIL death receptors DR4 and DR5 as well as a concomitant decrease in the decoy receptors. APC deficiency leads to a decreased level of the anti-apoptotic protein FLIP (flice-like inhibitory protein). Thus, the combination of TRAIL and retinoid specifically targets APC deficient cells for apoptosis. We have further determined that early stage intestinal polyps in the ApcMin/+ mouse respond readily to this treatment combination, while late stage polyps are resistant to this treatment. Following up on these findings, we have tested the hypothesis that late stage adenomas of the intestine are resistant to TRAIL and retinoid treatment due to increased inflammatory signaling, resulting in increased anti-apoptotic signaling. We used ApcMin/+ mice at either two months of age or five-six months of age. Adenomas from young mice and older mice were subjected to immunoblot and immunohistochemistry comparing the levels of anti-apoptotic proteins as well as markers of inflammation. Results: Polyps from older mice expressed markedly increased levels of the anti-apoptotic proteins FLIP (flice-like inhibitory protein), XIAP (x-linked inhibitor of apoptosis) and Bcl-2. These polyps also exhibited increased staining for the pro-inflammatory protein cox-2 (cyclooxygenase-2) and the transcription factor NFκB (nuclear factor kappa B). Conclusion: The increased inflammation known to occur in older ApcMin/+ mice leads to an increase in pro-survival proteins that inhibits induction of apoptosis. This suggests that pre-treatment with an anti-inflammatory agent such as sulindac might sensitize adenomas in older mice to retinoid and TRAIL treatment for the induction of apoptosis. Citation Format: Jennifer S. Davis, Shaoyi Huang, Xiaoyang Ren, Zhengming Xu, Ernest Hawk, Xiangwei Wu. Late-stage intestinal polyps in ApcMin/+ mice are resistant to treatment with retinoids and TRAIL due to upregulation of prosurvival proteins. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A60.