Abstract
Doxorubicin is one of chemotherapeutic drug in treatment with a board range of cancers (such as breast, bladder, ovaries and uterine cancer) for decades. However, the side effect of doxorubicin in producing ROS to cause cell dysfunction leading to cell apoptosis and myocyte damage to limit its usage in clinics. In current study, in order to monitor the doxorubicin-mediated cardiopathy-induced by ROS, a cysteine-labeling version of 2D-DIGE was employed to monitor the thiol modifications of redox-modulated proteins associated with doxorubicin treatment. In addition, recent studies indicated that reduction of intracellular anti-oxidants could result in drug resistance. However, the redox-modifications of resistance-associated cellular targets have not been reported. For this, we conduct ICy dyes-based labeling and MALDI-TOF MS to investigate how resistant cell protect itself from doxorubicin induced ROS in comparison with sensitive cell according to their differences in redox regulation. The other limited application of doxorubicin is drug resistance. For this, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin resistant MES-SA/Dx5 as a model system to examine resistance-induced cellular responses. Furthermore, in order to explore more drug resistance-associated markers in higher resistance cell model with resistance-dependent manner, we developed a panel of differential doxorubicin resistant levels of uterine sarcoma cell lines: MES-SA, MES-SA/DxR-2μM and MES-SA/DxR-8μM. First, we applied 2D-DIGE combined with MALDI-TOF/TOF MS to examine the global protein expression changes in doxorubicin resistant cells. Second, subcellular quantitative proteomic analysis has been performed to enrich low-abundant resistance-associated proteins from subcellular organelles, such as mitochondrial, nuclear and secreted fractions. From these identified proteins, we investigated the possible role of Asparagine synthetase (ASNS), progesterone receptor membrane component 1 (PGRMC1), Reticulocalbin-1 (RCN1), mitochondrial acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) and nuclear XRCC3 in drug resistance by RNA interference technique. Importantly, we further focus on PGRMC1 by overexpression and knockdown methods to double confirm its important role in uterine cancer proliferation, anti-apoptosis and invasion ability. To sum up, we utilized ICy dye labeling strategy to elucidate the cell responses to doxorubicin-induced cardiotoxicity; additionally, we compared the differentially redox-modulated proteins between sensitive and resistant cell lines to explore potential cellular proteins in the formation of doxorubicin resistance. Moreover, our proteomic approaches allowed us to identify numerous low abundant proteins involved in drug-resistance-forming mechanism. This study have been provided numerous potential resistance-associated proteins as therapeutic targets to overcome chemotherapy-induced drug resistance.