卡非佐米联合米托坦对人肾上腺皮质癌细胞H295R、SW13增殖及细胞周期的影响

Effects of carfilzomib combined with mitotane on proliferation and cell cycle of human adrenocortical carcinoma H295R and SW13 cells

目的探讨卡非佐米联合米托坦对人肾上腺皮质癌(ACC)细胞H295R、SW13增殖及细胞周期的影响。方法体外培养人ACC细胞H295R、SW13,采用MTT法确定米托坦作用H295R、SW13细胞48 h时的半数有效抑制浓度(IC_(50))值分别为18.42、62.37μmol/L,卡非佐米分别为3.86、11.62μmol/L。将两种对数生长期细胞随机分为对照组、卡非佐米组、米托坦组及序贯给药的卡非佐米→米托坦组、卡非佐米+米托坦组、米托坦→卡非佐米组,给药浓度分别为两种药物IC_(50)值的1/8、1/4、1/2、1、2倍,检测各浓度干预48 h时各组细胞增殖抑制率。根据Chou-Talaly公式计算IC_(50)及2倍IC_(50)作用下两种药物序贯给药时的联合指数(CI)。将两种对数生长期细胞随机分为对照组、米托坦单药组(25、50μmol/L)、卡非佐米单药组(1μmol/L)、卡非佐米(1μmol/L)→米托坦(25、50μmol/L)组,药物干预48 h时检测细胞增殖抑制率。将两种对数生长期细胞随机分为空白组、米托坦组、卡非佐米组、卡非佐米→米托坦组,后三组以IC_(50)药物浓度干预96 h,采用流式细胞仪检测细胞周期。结果 H295R及SW13细胞在IC_(50)及2倍IC_(50)浓度下,卡非佐米→米托坦组细胞增殖抑制率均高于米托坦→卡非佐米组及卡非佐米+米托坦组,米托坦→卡非佐米组均高于卡非佐米+米托坦组,组间比较P均<0.05。H295R和SW13细胞在米托坦、卡非佐米IC_(50)及2倍IC_(50)浓度条件下,卡非佐米→米托坦组和米托坦+卡非佐米组中两药的CI值均<1,且卡非佐米→米托坦组两药的CI值均小于米托坦+卡非佐米组(P均<0.05);米托坦→卡非佐米组中两药的CI值均<1,且均高于卡非佐米→米托坦组和米托坦+卡非佐米组(P均<0.05)。1μmol/L卡非佐米与25、50μmol/L米托坦联合的卡非佐米→米托坦组H295R及SW13增殖抑制率分别高于25、50μmol/L米托坦组(P均<0.05)。四组G1、G2/M、S期细胞百分比比较差异均无统计学意义(P均>0.05)。结论卡非佐米联合米托坦对ACC细胞存在序贯依赖性的协同抑制细胞增殖作用,给予卡非佐米后给予米托坦的抗增殖作用最佳,但两药联合对细胞周期无明显影响。

Objective To investigate the effects of carfilzomib combined with mitotane on the proliferation and cell cycle of human adrenocortical carcinoma（ ACC） H295 R and SW13 cells. Methods Human ACC H295 R and SW13 cells were cultured in vitro. Using MTT to determine the IC50 values of mitotane on H295 R and SW13 cells at 48 h,which were 18.42 and 62.37 μmol/L,respectively,and those of carfilzomib were 3. 86 and 11. 62 μmol/L. These two cell lines in the logarithmic growth phase were randomly divided into the control group,carfilzomib group,mitotane group,and the carfilzomib → mitotane group,carfilzomib ＋ mitotane group,and the mitotane →carfilzomib group（ namely according to the sequential administration）. The concentrations of the two drugs were 1/8,1/4,1/2,1,2 times of IC50 values,respectively. The inhibitory rate of cell proliferation was measured at 48 h. The combine index（ CI） of the two drugs was calculated according to the Chou-Talalay formula. These two cells in the logarithmic growth phase were randomly divided into the control group,mitotane monotherapy group（ 25 and 50 μmol/L）,carfilzomib monotherapy group（ 1 μmol/L）,carfilzomib（ 1μmol/L） → mitotane（ 25 and 50 μmol/L） group. The inhibitory rate of cell proliferation at 48 h after drug intervention was detected. These two cells in the logarithmic growth phase were randomly divided into the blank group,mitotane group,carfilzomib group,carfilzomib → mitotae group,and the latter three groups were intervened at the drug concentration of IC50 for 96 h,and flow cytometry was used to detect the cell cycle. Results The proliferation inhibitory rates of H295 R and SW13 cells were significantly higher in the carfilzomib→mitotane group than in the mitotane→carfilzomib group and the carfilzomib ＋ mitotane group at the concentrations of IC50 and 2-fold IC50; the mitotane→carfilzomib group was higher than the carfilzomib ＋ mitotane group; there were significant differences between every two groups（ all P〈0. 05）. CI value 〉1 was found in both the carfilzomib→mitotane group and the mitotane ＋ carfilzomib group at the concentrations of IC50 and 2-fold IC50 in H295 R and SW13 cell lines,and the CI value in the carfilzomib→mitotane group was lower than that in the mitotane ＋ carfilzomib group（ all P〈0. 05）; CI value〈 1 was found in the mitotane→carfilzomib group,which was greater than that of the carfilzomib→mitotane group and the mitotane ＋ carfilzomib group（ all P〈0. 05）. The proliferation inhibitory rates of H295 R and SW13 cells were significantly higher in the carfilzomib（ 1 μmol/L） →mitotane（ 25 and 50 μmol/L） group than in the mitotane group（ 25 and 50 μmol/L）,respectively. There was no significant difference in the percentage of cells in the G1,G2/M,and S phases between these four groups（ P〉0. 05）. Conclusion There are sequence-dependent antiproliferative effects of mitotane and carfilzomib on H295 R and SW13 cell lines,and the sequential administration of carfilzomib followed by mitotane exhibits the strongest anticancer activity,but the combination of these two drugs has no significant effect on the cell cycle.