间隙连接蛋白43的磷酸化调节在卵巢上皮性癌化疗耐药中的作用

目的 通过检测卵巢上皮性癌(卵巢癌)间隙连接蛋白43(Cx43)、非磷酸化Cx43及蛋白激酶C(PKC)的表达,探讨Cx43的磷酸化调节在卵巢癌化疗耐药中的作用.方法 采用免疫组化法检测29例化疗敏感(化疗敏感组)和28例化疗耐药(化疗耐药组)卵巢癌患者癌组织中以及PKC抑制剂--星孢菌素处理后的卵巢癌顺铂耐药细胞株SKOV3/DDP细胞中Cx43、非磷酸化Cx43及PKC蛋白的表达,并采用三磷酸腺苷-生物荧光肿瘤药敏实验(ATP-TCA)检测SKOV3/DDP细胞对化疗药物的敏感性.结果 (1)免疫组化法检测显示,化疗耐药组Cx43和非磷酸化Cx43蛋白的阳性表达率(分别为54%和14%)明显低于化疗敏感组(分别为83%和59%,P<0.05);化疗耐药组PKC蛋白的阳性表达率明显高于化疗敏感组(分别为64%和31%,P<0.05).卵巢癌组织中,PKC蛋白的阳性表达率与Cx43、非磷酸化Cx43蛋白的阳性表达率呈明显负相关关系(r=-0.626和-0.714,P<0.05).(2)免疫组化法检测显示,星孢菌素处理后SKOV3/DDP细胞中PKC蛋白的表达强度减弱,Cx43及非磷酸化Cx43蛋白的表达强度增强,随着星孢菌素处理时间的延长,Cx43蛋白的表达强度进一步增强.(3)ATP-TCA检测显示,体外培养的SKOV3/DDP细胞对紫杉醇和顺铂均耐药;紫杉醇和顺铂分别联合星孢菌素处理后细胞敏感度增加,其中联合低浓度(1×10-8 mol/L)星孢菌素者为中度敏感.联合高浓度(1×10-7 moL/L)星孢菌素者为高度敏感.结论 PKC通过对Cx43的磷酸化作用导致Cx43蛋白的表达下降,降低卵巢癌对化疗药物的敏感性,这种效应可以被星孢菌素逆转.

Expression of connexin 43 in ovarian cancer and its relationship with chemoresistance

Objective To examine the expression of protein kinase C (PKC), connexin43(Cx43) and non-phosphorylated Cx43 in ovarian cancer, and discuss the role of phosphorylated of Cx43 in chemoresistance in ovarian cancer. Methods We examined the expression of Cx43, non-phosphorylated Cx43 and PKC in ovarian cancer tissue by immunohistochemistry, and compared their expression in chemosensitivity group and ehemoresistance group. Cisplatin resistant ovarian cancer cell line SKOV3/DDP cells were treated by staurosporine (a kind of PKC inhibitors). Then expression of Cx43, non-phosphorylated Cx43 and PKC were tested. Meanwhile, we tested chemosensitivity of SKOV3/DDP cells by ATP bioluminescence tumor chemosensifivity assay (ATP-TCA). Results (1) Immunohistochemically,the rates of positive expression of Cx43 and non-phospharylated Cx43 were 54%, 14% respectively in the chemoresistance group, which were 83%, 59% in the chemosensitivity group respectively (P<0.05). The rate of positive expression of PKC in 28 chemoresistance ovarian cancer cases (64%) was higher than that in 29 chemosensitivity cases (31% ,P<0.05). Both of them were significantly lower in ehemoresistanee group than in chemosensitivity group (P<0.05). In addition, the expression of PKC was negatively correlated with the expression of Cx43 and non-phosphorylated Cx43. The correlation coefficients were -0. 626 and -0. 714, respectively (P<0.05). (2) Immunohistochemically, PKC was down regulated, and Cx43 and non-phosphorylated Cx43 were up regulated in SKOV3/DDP cells after staurosporine treatment. The longer the staurosporine worked, the more expression of Cx43 was. (3) By ATP-TCA, SKOV3/DDP cells were resistant to paclitaxel and cisplatin. The tumor growth inhibition was higher in the group of paclitaxel or cisplatin combined staurosporine than in the group of paclitaxel or cisplatin alone. The sensitivity was intermediate in the group combined with low concentration staurosporine (1×10-8 moL/L), and the sensitivity was high in the group combined with high concentration staurosporine (1×10-7 mol/L). Conclusions Phosphorylation of Cx43 caused by PKC leads to decrease in the expression of Cx43. This effect makes ovarian cancer cells less chemosensitive. Phosphorylation of Cx43 caused by PKC can be inhibited by staurosporine.