SERPINB3 inibisce il poro di transizione della permeabilità mitocondriale attraverso la regolazione della produzione di ROS mitocondriali e incrementa la resistenza ai trattamenti chemioterapici

Abstract

Resistance to chemotherapeutic agents is well known in patients with hepatocarcinoma. Inhibition of the mitochondrial permeability transition pore (PTP) is a crucial step in tumor cell resistance to apoptosis induced by anticancer drugs. Since SERPINB3 is overexpressed in hepatocellular carcinoma and has an anti-apoptotic activity, aim of the study was to assess the role of this serpin on PTP modulation during treatment with chemotherapeutic agents. HepG2 cells stably transfected with SERPINB3 where assayed for cell death induced by Cisplatin, Doxorubicin, Etoposide and 5-Fluorine Uracil. Reactive Oxygen Species (ROS) were detected with dichlorofluorescein. Threshold of PTP opening was evaluated by CRC assay and Complex I activity was determined by spectrophotometric assay. After cell death induction by Cisplatin and Doxorubicin, HepG2 cells expressing SERPINB3 showed a significant increase in viability compared to controls, while there was no difference in cell death after Etoposide and 5-FU treatments. The addition of the antioxidant N-acetyl cysteine abrogated cell death induction by Cisplatin and Doxorubicin, suggesting that SERPINB3 protects from death through an antioxidant activity. Since Cisplatin and Doxorubicin induce mitochondrial oxidative stress and favor PTP opening, cells were treated with the PTP inducer EM20-25 that acts at mitochondrial level. In presence of SERPINB3 the effect of EM20-25 resulted in PTP opening inhibition and decreased ROS formation. Subcellular localization analysis revealed that a fraction of SERPINB3 was located in mitochondria and that it increased after death-promoting treatments. Mitochondrial SERPINB3 was found associated to respiratory chain Complex I by immunoprecipitation experiments. In vitro analysis confirmed the inhibition by SERPINB3 of Complex I activity, known as one of the main sources of mitochondrial ROS. In conclusion, SERPINB3 acts at mitochondrial level protecting cells from chemotherapeutic-induced oxidative stress and consequent cell death. This antioxidant function could represent a relevant advantage for a transformed cell to balance its ROS equilibrium and for SB3-expressing cells exposed to anticancer treatments