3-aminopyridine-2-carboxaldehyde-thiosemicarbazone and Ovarian-Neoplasms

The potent ribonucleotide reductase (RNR) inhibitor 3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) was tested as a chemosensitizer for restored cisplatin-mediated cytotoxicity in platinum-resistant ovarian cancer.. Preclinical in vitro platinum-resistant ovarian cancer cell survival, RNR activity, and DNA damage assays were done after cisplatin or cisplatin plus 3-AP treatments. Six women with platinum-resistant ovarian cancer underwent four-day 3-AP (96 mg/m(2), day one to four) and cisplatin (25 mg/m(2), day two and three) infusions every 21 days until disease progression or adverse effects prohibited further therapy. Pre-therapy ovarian cancer tissues were analyzed by immunohistochemistry for RNR subunit expression as an indicator of cisplatin plus 3-AP treatment response.. 3-AP preceding cisplatin exposure in platinum-resistant ovarian cancer cells was not as effective as sequencing cisplatin plus 3-AP together in cell survival assays. Platinum-mediated DNA damage (i.e., γH2AX foci) resolved quickly after cisplatin-alone or 3-AP preceding cisplatin exposure, but persisted after a cisplatin plus 3-AP sequence. On trial, 25 four-day overlapping 3-AP and cisplatin cycles were administered to six women (median 4.2 cycles per patient). 3-AP-related methemoglobinemia (range seven to 10%) occurred in two (33%) of six women, halting trial accrual.. When sequenced cisplatin plus 3-AP, RNR inhibition restored platinum-sensitivity in platinum-resistant ovarian cancers. 3-AP (96 mg/m(2)) infusions produced modest methemoglobinemia, the expected consequence of ribonucleotide reductase inhibitors disrupting collateral proteins containing iron.. ClinicalTrials.gov NCT00081276.

Topics: Adult; Aged; Cell Line, Tumor; Cell Survival; Deoxycytosine Nucleotides; DNA Damage; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Gynecology; Histones; Humans; Immunoblotting; Medical Oncology; Middle Aged; Ovarian Neoplasms; Platinum; Pyridines; Ribonucleotide Reductases; Thiosemicarbazones; Treatment Outcome

PARP inhibitors exploit synthetic lethality to target epithelial ovarian cancer (EOC) with hereditary BRCA mutations and defects in homologous recombination repair (HRR). However, such an approach is limited to a small subset of EOC patients and compromised by restored HRR due to secondary mutations in BRCA genes. Here, it was demonstrated that triapine, a small-molecule inhibitor of ribonucleotide reductase, enhances the sensitivity of BRCA wild-type EOC cells to the PARP inhibitor olaparib and the topoisomerase II inhibitor etoposide. Triapine abolishes olaparib-induced BRCA1 and Rad51 foci, and disrupts the BRCA1 interaction with the Mre11-Rad50-Nbs1 (MRN) complex in BRCA1 wild-type EOC cells. It has been shown that phosphorylation of CtIP (RBBP8) is required for the interaction with BRCA1 and with MRN to promote DNA double-strand break (DSB) resection during S and G(2) phases of the cell cycle. Mechanistic studies within reveal that triapine inhibits cyclin-dependent kinase (CDK) activity and blocks olaparib-induced CtIP phosphorylation through Chk1 activation. Furthermore, triapine abrogates etoposide-induced CtIP phosphorylation and DSB resection as evidenced by marked attenuation of RPA32 phosphorylation. Concurrently, triapine obliterates etoposide-induced BRCA1 foci and sensitizes BRCA1 wild-type EOC cells to etoposide. Using a GFP-based HRR assay, it was determined that triapine suppresses HRR activity induced by an I-SceI-generated DSB. These results suggest that triapine augments the sensitivity of BRCA wild-type EOC cells to drug-induced DSBs by disrupting CtIP-mediated HRR.. These findings provide a strong rationale for combining triapine with PARP or topoisomerase inhibitors to target HRR-proficient EOC cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Ovarian Epithelial; Carrier Proteins; Cell Line, Tumor; Drug Synergism; Female; Humans; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Pyridines; Recombination, Genetic; Recombinational DNA Repair; Thiosemicarbazones; Topoisomerase Inhibitors; Transfection

Triapine (Vion Pharmaceuticals, New Haven, CT) is a potent ribonucleotide reductase inhibitor which exerts its antineoplastic activity by inhibiting DNA synthesis and repair. The objectives of this study were: (1) to determine whether Triapine has cytotoxic effects on epithelial ovarian cancer (EOC) cells; (2) to characterize the apoptotic cascade induced in response to this agent; and (3) to determine its utility in combination treatment with carboplatin and paclitaxel.. Five EOC cell lines were treated with tenfold dilutions of Triapine (0.1 to 100 microM) for 24 and 48 hours. Cell viability was determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corp, Madison, WI) and the morphologic features of apoptosis were observed using Hoechst staining. The apoptotic cascade was characterized by Western blot analyses.. All EOC cell lines treated with Triapine showed decreased cell viability in a time- and dose-dependent manner. Hoechst staining revealed nuclear shrinkage and chromatin condensation and fragmentation, which correlated with the occurrence of apoptosis. Western blots demonstrated that Bid activation was one of the initiating signals involved in the cascade. In addition, cleavage of XIAP and down-regulation of Akt were observed. We also demonstrated that Triapine enhances the cytotoxic effects of carboplatin and paclitaxel.. The present findings demonstrate that Triapine induces cell death through the induction of apoptosis. The initial activation of Bid indicates the involvement of the mitochondrial pathway. The demonstration that Triapine is an effective addition to a carboplatin regimen suggests the possibility of a new combination therapy for ovarian cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carboplatin; Caspase 2; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Mitochondria; Ovarian Neoplasms; Paclitaxel; Pyridines; Thiosemicarbazones