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A Smac mimetic augments the response of urothelial cancer cells to gemcitabine and cisplatin.

ABSTRACT: Cisplatin-based chemotherapy is considered the gold standard for patients with advanced bladder cancer. However, despite initial response, many patients will relapse; therefore, novel salvage treatment strategies are desperately needed. Herein, we studied a mechanism based treatment combination using a Smac mimetic with standard chemotherapy. Using a panel of 10 urothelial cancer cell lines, we exposed them to a combination of gemcitabine, cisplatin, and a Smac mimetic. Sensitivity was determined using a DNA fragmentation assay. We determined that three cell lines (UMUC-3, UMUC-13, and RT4v6) were considered sensitive to the combination of gemcitabine and cisplatin and an additional three cell lines were sensitized to gemcitabine and cisplatin with the addition of the Smac mimetic (UMUC-6, UMUC-12, and UMUC-18). We next explored the constitutive expression of selected members of the IAP family (XIAP, cIAP-1, cIAP-2, and Survivin), the BCL family (BCL-2, BCLXL, and BAX) and Smac using gene expression profiling and western blotting. We determined that RNA and protein expression of SMAC, selected members of the IAP family and members of the BCL family did not correlate to drug sensitivity. Lastly, using an in vivo mouse model, we determined that treatment with the Smac mimetic in combination with gemcitabine and cisplatin resulted in increased apoptosis, decreased microvessel density and decreased cellular proliferation. This novel treatment strategy may be effective in patients with advanced urothelial carcinoma and warrants further investigation.

SUBMITTER: Lee EK

PROVIDER: S-EPMC3909550 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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A Smac mimetic augments the response of urothelial cancer cells to gemcitabine and cisplatin.

Lee Eugene K EK Jinesh G Goodwin G Laing Naomi M NM Choi Woonyoung W McConkey David J DJ Kamat Ashish M AM

Cancer biology & therapy 20130619 9

Cisplatin-based chemotherapy is considered the gold standard for patients with advanced bladder cancer. However, despite initial response, many patients will relapse; therefore, novel salvage treatment strategies are desperately needed. Herein, we studied a mechanism based treatment combination using a Smac mimetic with standard chemotherapy. Using a panel of 10 urothelial cancer cell lines, we exposed them to a combination of gemcitabine, cisplatin, and a Smac mimetic. Sensitivity was determine ...[more]

PMID: 23792592

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Project description:Purposeinhibitors of apoptosis proteins (IAPs) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy. Experimental design: A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL, and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting.Resultssingle-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis.Experimental designa panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-Alone, TRAIL alone, Chemotherapy alone, compound-A plus TRAIL and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting.Conclusionour results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.

Project description:BackgroundOvarian cancer is initially responsive to frontline chemotherapy. Unfortunately, it often recurs and becomes resistant to available therapies and the survival rate for advanced and recurrent ovarian cancer is unacceptably low. We thus hypothesized that it would be possible to achieve more durable treatment responses by combining cisplatin chemotherapy with SW IV-134, a cancer-targeted peptide mimetic and inducer of cell death. SW IV-134 is a recently developed small molecule conjugate linking a sigma-2 ligand with a peptide analog (mimetic) of the intrinsic death pathway activator SMAC (second-mitochondria activator of caspases). The sigma-2 receptor is overexpressed in ovarian cancer and the sigma-2 ligand portion of the conjugate facilitates cancer selectivity. The effector portion of the conjugate is expected to synergize with cisplatin chemotherapy and the cancer selectivity is expected to reduce putative off-target toxicities.MethodsOvarian cancer cell lines were treated with cisplatin alone, SW IV-134 alone and a combination of the two drugs. Treatment efficacy was determined using luminescent cell viability assays. Caspase-3/7, - 8 and - 9 activities were measured as complementary indicators of death pathway activation. Syngeneic mouse models and patient-derived xenograft (PDX) models of human ovarian cancer were studied for response to SW IV-134 and cisplatin monotherapy as well as combination therapy. Efficacy of the therapy was measured by tumor growth rate and survival as the primary readouts. Potential drug related toxicities were assessed at necropsy.ResultsThe combination treatment was consistently superior in multiple cell lines when compared to the single agents in vitro. The expected mechanism of tumor cell death, such as caspase activation, was confirmed using luminescent and flow cytometry-based assay systems. Combination therapy proved to be superior in both syngeneic and PDX-based murine models of ovarian cancer. Most notably, combination therapy resulted in a complete resolution of established tumors in all study animals in a patient-derived xenograft model of ovarian cancer.ConclusionsThe addition of SW IV-134 in combination with cisplatin chemotherapy represents a promising treatment option that warrants further pre-clinical development and evaluation as a therapy for women with advanced ovarian cancer.

Project description:Cholangiocarcinoma (CCA), a malignant tumor originating in the biliary tract, is well known to be associated with adverse clinical outcomes and high mortality rates due to the lack of effective therapy. Evasion of apoptosis is considered a key contributor to therapeutic success and chemotherapy resistance in CCA, highlighting the need for novel therapeutic strategies. In this study, we demonstrated that the induction of necroptosis, a novel regulated form of necrosis, could potentially serve as a novel therapeutic approach for CCA patients. The RNA sequencing data in The Cancer Genome Atlas (TCGA) database were analyzed and revealed that both receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), two essential mediators of necroptosis, were upregulated in CCA tissues when compared with the levels in normal bile ducts. We demonstrated in a panel of CCA cell lines that RIPK3 was differentially expressed in CCA cell lines, while MLKL was more highly expressed in CCA cell lines than in nontumor cholangiocytes. We therefore showed that treatment with both tumor necrosis factor-? (TNF-?) and Smac mimetic, an inhibitor of apoptosis protein (IAP) antagonist, induced RIPK1/RIPK3/MLKL-dependent necroptosis in CCA cells when caspases were blocked. The necroptotic induction in a panel of CCA cells was correlated with RIPK3 expression. Intriguingly, we demonstrated that Smac mimetic sensitized CCA cells to a low dose of standard chemotherapy, gemcitabine, and induced necroptosis in an RIPK1/RIPK3/MLKL-dependent manner upon caspase inhibition but not in nontumor cholangiocytes. We further demonstrated that Smac mimetic and gemcitabine synergistically induced an increase in TNF-? mRNA levels and that Smac mimetic reversed gemcitabine-induced cell cycle arrest, leading to cell killing. Collectively, our present study demonstrated that TNF-? and gemcitabine induced RIPK1/RIPK3/MLKL-dependent necroptosis upon IAP depletion and caspase inhibition; therefore, our findings have pivotal implications for designing a novel necroptosis-based therapeutic strategy for CCA patients.

Project description:Colorectal cancer is a molecularly heterogeneous disease. Responses to genotoxic chemotherapy in the adjuvant or palliative setting vary greatly between patients, and colorectal cancer cells often resist chemotherapy by evading apoptosis. Antagonists of an inhibitor of apoptosis proteins (IAPs) can restore defective apoptosis signaling by degrading cIAP1 and cIAP2 proteins and by inhibition of XIAP. Due to the multiple molecular mechanisms-of-action of these targets, responses to IAP antagonist may differ between molecularly distinct colon cancer cells. In this study, responses to the IAP antagonist Birinapant and oxaliplatin/5-fluorouracil (5-FU) were investigated in 14 colon cancer cell lines, representing the consensus molecular subtypes (CMS). Treatment with Birinapant alone did not result in a substantial increase in apoptotic cells in this cell line panel. Annexin-V/PI assays quantified by flow cytometry and high-content screening showed that Birinapant increased responses of CMS1 and partially CMS3 cell lines to oxaliplatin/5-FU, whereas CMS2 cells were not effectively sensitized. FRET-based imaging of caspase-8 and -3 activation validated these differences at the single-cell level, with CMS1 cells displaying sustained activation of caspase-8-like activity during Birinapant and oxaliplatin/5-FU co-treatment, ultimately activating the intrinsic mitochondrial apoptosis pathway. In CMS2 cell lines, Birinapant exhibited synergistic effects in combination with TNFα, suggesting that Birinapant can restore extrinsic apoptosis signaling in the context of inflammatory signals in this subtype. To explore this further, we co-cultured CMS2 and CMS1 colon cancer cells with peripheral blood mononuclear cells. We observed increased cell death during Birinapant single treatment in these co-cultures, which was abrogated by anti-TNFα-neutralizing antibodies. Collectively, our study demonstrates that IAP inhibition is a promising modulator of response to oxaliplatin/5-FU in colorectal cancers of the CMS1 subtype, and may show promise as in the CMS2 subtype, suggesting that molecular subtyping may aid as a patient stratification tool for IAP antagonists in this disease.

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A Smac mimetic augments the response of urothelial cancer cells to gemcitabine and cisplatin.

Publications

A Smac mimetic augments the response of urothelial cancer cells to gemcitabine and cisplatin.

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