关键词: 卵巢癌；α2?巨球蛋白；顺铂；耐药；TGF?β/SMADs信号通路')">">卵巢癌；α2?巨球蛋白；顺铂；耐药；TGF?β/SMADs信号通路

Abstract: Objective To investigate the relationship between the expression levels of alpha⁃2⁃macroglobulin (A2M) in ovarian cancer patients and their prognosis and platinum resistance, and to analyze the regulation of A2M on the TGF⁃β/SMADs signaling pathway and its impact on the apoptosis of tumor cells induced by Cisplatin (DDP). Methods The Western blot was used to detect A2M expression in xenogeneic subcutaneous tumor models of ovarian cancer treated by DDP in nude mice, and the relationship between A2M expression and DDP resistance was analyzed. Immunohistochemistry was used to detect the expression of A2M in 129 ovarian cancer tissues and analyze its relationship with prognosis. ELISA was used to detect A2M content and analyze its relationship with  DDP resistance in serum samples of 103 cases of ovarian cancer. The 0, 0.5, 1.0, 2.0, 4.0 μmol/L A2M protein was added to drug⁃resistant ovarian cancer cells SKOV3/DDPⅡ, A2M content in co⁃culture system was detected by ELASA and analyzed its relationship with cytokines, and the apoptosis of DDP drug⁃resistant ovarian cancer cells was detected by flow cytometry.  The expression of A2M gene in sensitive cells SKOV3 was knocked down by the RNA interference technology, CCK⁃8 assay was used to detect the cell activity of each group after treated exogenous A2M and knocking down A2M, and the half maximal inhibitory concentration (IC₅₀) of DDP was calculated; The Western blot was used to detect the key nodes of TGF⁃β/SMADs signal transduction, TGF⁃β1, TGFβR2, SMAD2/3, and their phosphorylated expression, and the expression of cell apoptosis⁃related proteins BCL⁃2 and PARP1. A xenograft model of ovarian cancer in nude mice was used to compare the effect of DDP treatment on the tumorigenesis volume of SKOV3 cells with A2M gene knockdown and control cells. Results The expression of A2M protein in xenografts of drug⁃resistant mice decreased with the increase of DDP treatment, whereas the expression of PARP1 protein was up⁃regulated. A2M protein expression was positively correlated with overall survival and progression⁃free survival in ovarian cancer patients (all P<0.05), and the serum A2M content in platinum⁃resistant ovarian cancer patients decreased (P=0.031). Exogenous addition of A2M protein could significantly decrease the cytokine contents of TGF⁃β1, MMP1, MMP2, MMP7 and MMP9 compared with those without A2M protein (all P<0.05), and the total apoptosis rates of SKVO3/DDP Ⅱ cells and A2780/DDP Ⅱ cells increased and the IC₅₀ of DDP decreased (P<0.0001). At the same time, the expression of TGF⁃β1 and PKCa were decreased, the phosphorylation levels of TGFβR2 and SAMD2/3 in SKVO3/DDP Ⅱ cells were down⁃regulated, and TGF⁃β1⁃SMAD2/3 complex was inhibited to play the role of transcription factor, thereby inhibited the phosphorylation of apoptosis⁃related protein BCL⁃2 and induced the inactivation of PARP1 protein, and promoted the apoptosis of ovarian cancer cells. However, the results of DDP IC₅₀ and TGF⁃β1, SMAD2/3, PKCa, BCL⁃2, PARP1 protein expression in SKVO3 cells with A2M gene knockdown were opposite. The tumor volume of the established mouse xenograft model was significantly higher than the control group after 8 DDP treatments. Conclusions A2M is involved in the sensitivity of ovarian cancer cells to DDP treatment by negatively regulating the TGF⁃β/SMADs signaling pathway, and its knockdown can activate the BCL⁃2 dependent cell anti⁃apoptotic pathway, which leads to platinum resistance and poor prognosis of ovarian cancer.

Key words: Ovarian cancer')">Ovarian cancer, α2?macroglobulin, Cisplatin, Resistance； TGF?β/SMADs signaling pathway