17-(dimethylaminoethylamino)-17-demethoxygeldanamycin and Ovarian-Neoplasms

Ovarian carcinoma is initially sensitive to platinum-based therapy, but become resistant over time. The study of cancer sensitizing substance is therefore the major challenge for a number of scientific groups. Our experiments were carried out on human ovarian adenocarcinoma A2780cis cells resistant to cisplatin and their response to 2-(4'fluoro-phenylamino)-4H-1,3-thiazine[6,5-b]indole (thiazine[6,5-b]indole) and/or heat shock protein (Hsp) 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) using proliferation assay, cell cycle analysis and monitoring of apoptosis were examined. A2780cis cells revealed the same fold of resistance to Hsp90 inhibitor 17-DMAG as it is declared for cisplatin (18 times), but only 3.2 times for thiazine[6,5-b]indole. Our results showed that the combination of thiazine[6,5-b]indole and 17-DMAG significantly reduced proliferation of A2780cis cells and led to their accumulation in G2/M phase of the cell cycle. Moreover, both thiazine[6,5-b]indole as well as 17-DMAG increased the number of annexin V positive A2780cis cells in time dependent manner. Interestingly, thiazine[6,5-b]indole treatment significantly activated also caspase-3 compared to untreated or 17-DMAG-treated cells and reduced mitochondrial membrane potential (MMP) of A2780cis cells with more significant decline after combined treatment. In this regard, the incubation of A2780cis cells with thiazine[6,5-b]indole induced PARP protein cleavage as well as an increased level of Bad protein with more pronounced changes after combined treatment. Importantly, Hsp70 protein was not upregulated in A2780cis cells neither by individual treatment nor by mutual combination. Our results signify antiproliferative and pro-apoptotic effects of novel thiazine[6,5-b]indole potentiated by Hsp90 inhibitor 17-DMAG in ovarian adenocarcinoma cells resistant to cisplatin and therefore represents new strategy in cancer treatment.

Topics: Adenocarcinoma; Antineoplastic Agents; Benzoquinones; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Dose-Response Relationship, Drug; Female; HSP90 Heat-Shock Proteins; Humans; Indoles; Lactams, Macrocyclic; Ovarian Neoplasms; Thiazines

Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance.

Topics: Antineoplastic Agents; Benzoquinones; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Fanconi Anemia Complementation Group N Protein; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mutation; Nuclear Proteins; Ovarian Neoplasms; Platinum; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Structure, Tertiary; Rad51 Recombinase; Tumor Suppressor Proteins

17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat-shock protein 90 (Hsp90) inhibitor, has been intensively investigated for cancer therapy and is undergoing clinical trials. Human epidermal growth factor receptor 2 (HER-2) is one of the client proteins of Hsp90 and its expression is decreased upon 17-DMAG treatment. In this study, we aimed to noninvasively monitor the HER-2 response to 17-DMAG treatment in xenografted mice.. The sensitivity of human ovarian cancer SKOV-3 cells to 17-DMAG in vitro was measured by MTT assay. HER-2 expression in SKOV-3 cells was determined by flow cytometry. Nude mice bearing SKOV-3 tumors were treated with 17-DMAG and the therapeutic efficacy was evaluated by tumor size measurement. Both treated and control mice were imaged with microPET using (64)Cu-DOTA-trastuzumab and (18)F-FDG. Biodistribution studies and immunofluorescence staining were performed to validate the microPET results.. SKOV-3 cells are sensitive to 17-DMAG treatment, in a dose-dependent manner, with an IC(50) value of 24.72 nM after 72 h incubation. The tumor growth curve supported the inhibition effect of 17-DMAG on SKOV-3 tumors. Quantitative microPET imaging showed that (64)Cu-DOTA-trastuzumab had prominent tumor accumulation in untreated SKOV-3 tumors, which was significantly reduced in 17-DMAG-treated tumors. There was no uptake difference detected by FDG PET. Immunofluorescence staining confirmed the significant reduction in tumor HER-2 level upon 17-DMAG treatment.. The early response to anti-Hsp90 therapy was successfully monitored by quantitative PET using (64)Cu-DOTA-trastuzumab. This approach may be valuable in monitoring the therapeutic response in HER-2-positive cancer patients under 17-DMAG treatment.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Benzoquinones; Cell Line, Tumor; Copper Radioisotopes; Female; Fluorodeoxyglucose F18; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mice; Mice, Nude; Neoplasm Transplantation; Organometallic Compounds; Ovarian Neoplasms; Positron-Emission Tomography; Receptor, ErbB-2; Tissue Distribution; Trastuzumab