RESUMO
PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Neoplasias Ovarianas/terapia , Interferência de RNA , Ensaios Antitumorais Modelo de Xenoenxerto , Adenosina Trifosfatases/genética , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Sítios de Ligação , Western Blotting , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/metabolismo , Cisplatino/farmacologia , ATPases Transportadoras de Cobre , Adutos de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga TumoralRESUMO
beta-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved beta-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance.
