Herceptin与卡铂联合应用对宫颈癌细胞的抑制及其机制

Herceptin combined with carboplatin inhibiting cervical cancer cells and the related mechanism

目的:探讨Herceptin(Her)对宫颈腺癌HeLa、鳞癌SiHa细胞的抑制差异,以及与卡铂(carboplatin,CBP)的协同作用及其机制。方法:以Her5、10、20、40、80μg/ml和CBP0.5、1、2、4、8μg/ml分别组成单药组和Her+CBP联合用药组[(10+1)、(20+2)、(40+4)μg/ml],另设不加药对照组,分别作用于HeLa和SiHa细胞。MTT法检测Her对HeLa、SiHa细胞增殖的抑制作用及与CBP的协同作用,透射电镜观察肿瘤细胞的超微结构改变,流式细胞术检测用药后的细胞凋亡率及细胞周期,RT-PCR法检测细胞用药后her-2/neu和rasmRNA的表达,Westernblotting和免疫组化方法检测HER-2/neu和Ras蛋白的表达。结果:Her能明显抑制宫颈癌细胞生长,与CBP联合具有协同作用或相加作用(P<0.05,P<0.01)。Her能诱导细胞凋亡,使细胞周期阻滞于G1期;与CBP联合作用后凋亡率更高,使细胞周期进一步阻滞于G2期,同时降低S期比例。用药后her-2/neu和rasmRNA及其蛋白的表达也显著降低(P<0.05)。药物的抑制作用在宫颈腺癌Hela细胞中更明显些。结论:Her单药或与CBP联合应用能明显抑制宫颈癌HeLa、SiHa细胞生长,Her与CBP的协同作用与细胞周期的双重阻滞有关,且在腺癌HeLa细胞中更明显;Her通过抑制Ras/MAPK通路抑制宫颈癌细胞的增殖。

Objective： To evaluate the inhibitory effect of Herceptin on HeLa, SiHa cells and to investigate the synergistic mechanism of Herceptin and Carboplatin. Methods： HeLa and SiHa cells were treated with Herceptin （at 5, 10, 20, 40, and 80 μg/ml）, Carboplatin （at 0.5, 1, 2, 4, and 8 μg/ml）, and Her＋CBP[（10±1）, （20±2）, and （40±4） μg/ml] separately. The untreated cells were taken as control. SP immunohistochemical method was used to detect the protein expression of HER-2/neu and downstream Ras oncogene. MTT method was used to study the inhibition effect of Herceptin on HeLa, SiHa cells and its synergetic effect with carboplatin. FCM was used to detect the cell apoptosis and cell cycle. The mRNA expression of her-2/neu and ras were assessed by RT-PCR; the protein expression of HER-2/neuand Ras were studied by Western blotting. Results： Herceptin significantly inhibited cervical cancer cells proliferation, and there was a synergistic effect when combined with carboplatin （ P 〈 0.05, P 〈 0.01 ）. Herceptin induced cell apoptosis and arrested cell at G1 phase. When combined with Carboplatin, Herceptin induced more severe apoptosis and arrested cell at G2 phase; meanwhile, the cells of S and also decreased. The mRNA and protein expressions of HER-2/neu and Ras were decreased after treated with Herceptin alone and in combination with Carboplatin （ P 〈 0.05 ）. The inhibitory effect of Herceptin and Carboplatin was more obvious on HeLa cells. Conclusion： Herceptin alone or in combination with Carboplatin can greatly inhibit the growth to HeLa and SiHa cells. The synergistic mechanism of Herceptin with Carboplatin is related to the double blockage of cell cycle. The inhibitory effect is more obvious on HeLa cells. Herceptin inhibits proliferation of cervical cancer cells by restraining Ras/MAPK pathway.