红花多糖联合环杷明对乳腺癌细胞增殖和凋亡的影响

Effect of safflower polysaccharide combine cyclopamine on the proliferation and apoptosis of breast cancer cells

目的观察红花多糖联合环杷明对乳腺癌细胞增殖、凋亡的影响。方法培养人乳腺癌细胞株MDA-MB-231，细胞计数试剂盒（CCK-8）检测分别加入0.05、0.10、0.20、0.40、0.80 mg/ml红花多糖及2.5、5.0、10.0、20.0、40.0 μmol/L的环杷明培养24、48、72 h的细胞增殖情况，计算半数抑制浓度（IC50）；根据IC50选择最佳的红花多糖及环杷明作用浓度；将后续实验分为对照组、红花多糖组、环杷明组、红花多糖＋环杷明组，CCK-8实验检测4组细胞的增殖水平，流式细胞仪检测4组细胞凋亡水平，Western blot检测4组细胞B细胞淋巴瘤/白血病-2（bcl-2）、bcl-2相关X蛋白（bax）、Fas、Gli蛋白表达。结果按照上述浓度处理后，红花多糖作用于细胞24、48、72 h后的IC50分别为（0.89±0.04）、（0.35±0.03）、（0.14±0.02） mg/ml，环靶明作用于细胞24、48、72 h后的IC50分别为（56.30±1.23）、（28.85±1.02）、（15.26±0.87） μmol/L，不同浓度及不同作用时间的红花多糖组和环靶明组的细胞存活率均低于对照组。选择0.8 mg/ml红花多糖和20 μmol/L的环杷明进行后续研究；红花多糖组、环杷明组、红花多糖＋环杷明组细胞存活率分别为58.76%、56.67%、24.29%，对照组细胞存活率显著高于红花多糖组（P＝0.003）、环杷明组（P＝0.003）及红花多糖＋环靶明组（P＝0.000）；而红花多糖＋环杷明组细胞存活率低于红花多糖组（P＝0.005）和环杷明组（P＝0.005）。红花多糖组、环杷明组、红花多糖＋环杷明组细胞凋亡率分别为19.26%、16.24%、26.76%，对照组细胞凋亡率显著低于红花多糖组（P＝0.001）、环杷明组（P＝0.001）及红花多糖＋环靶明组（P＝0.000）；而红花多糖＋环杷明组细胞凋亡率低于红花多糖组（P＝0.005）和环杷明组（P＝0.004）。红花多糖组、环杷明组、红花多糖＋环杷明组bcl-2、Gli1蛋白表达均显著低于对照组，bax、Fas蛋白表达均显著高于对照组；红花多糖＋环杷明组bcl-2、Gli1蛋白表达显著低于红花多糖组和环杷明组，而bax、Fas蛋白表达显著高于红花多糖组和环杷明组。结论红花多糖及Hedgehog信号通路抑制剂环杷明均能抑制人乳腺癌细胞株MDA-MB-231增殖，红花多糖及环杷明联用对细胞增殖、凋亡的作用强于单独用红花多糖和环杷明。

Objective To investigate the effect of safflower polysaccharide combine cyclopamine on the proliferation and apoptosis of breast cancer cells. Methods Human breast cancer cell line MDA- MB -231 were cultured, cell proliferation and half maximal inhibitory concentration （IC50） calcu- lation were detected after 0. 05, 0. 10, 0. 20, 0. 40, 0. 80 mg/ml safflower polysaecharide and 2. 5, 5.0, 10. 0, 20.0, 40. 0 μmol/L cyclopamine were added separately to cells for 24, 48, 72 h by cell counting kit - 8 （ CCK - 8 ） test; the best concentration of safflower polysaecharide and cyclopamine were choiced according to the IC50 ; and the next test will be divided into control group, safflower polysaccharide group, cyclopamine group, safflower polysaccharide ＋ cyclopamine group, ceils proliferation in four groups were detected by CCK - 8 test, apoptosis were detected by flow cytometry, and the expression of B cell lymphoma/leukemia - 2 （ bcl - 2 ）, bcl - 2 associated X protein （bax） , Fas, Gli protein was detected by Western blotting. Results IC50 calculation after safflower polysaccharide treated cells for 24, 48, 72 h cell were （0. 89 ±0. 04）, （0. 35 ±0. 03）, （0. 14 ±0. 02） mg/ml, IC50 calculation after safflower cyclopamine trea-ted cells for 24, 48, 72 h cell were （56. 30 ± 1.23 ） , （28.85 ± 1.02） , （ 15.26 ± 0. 87） μmol/L, survival rate after safflower polysaccharide and cyclopamine treated cells for 24, 48, 72 h were significantly lower than 0 hour According to the above concentration treatment, 0.8 mg/mL safflower polysaccharide and 20 μmol/L eyclopamine were used for a follow - up study ; the cell survival rate in safflower polysaccharide group, cyclopamine group and safflower polysaccharide ＋ cyclopamine group was 58.76%, 56. 67%, 24. 29%, the control group significantly higher than polysaccharide group （P = 0.003 ）, cyclopamine group （P =0. 003） and safflower polysaccharide ＋ cyclopamine group （P =0. 000）, the apoptosis rate in safflower polysaccharide group, cyclopamine group and safflower polysaccharide ＋ cyclopamine group was 19. 26%, 16. 24%, 26. 76%, the control group significantlyhigher than polysaccharide group （P= 0. 001 ）, cyclopamine group （ P = 0. 001 ） and safflower polysaccharide ＋ cyclopamine group （ P = 0. 000 ） o cell survival rate in safflower polysaecharide ＋ cyclopamine group was significantly lower than saf- flower polysaceharide group （P = 0. 005 ） and cyelopamine group （ P = 0.005 ） , the apoptosis rate in saf- flower polysaccharide ＋ eyclopamine group was significantly higher than safflower polysaccharide group （ P = 0.005 ） and cyclopamine group （ P = 0.004 ） ; the expression bcl - 2 and Glil protein in safflower polysaccharide group, eyclopamine group, safflower polysaccharide ＋ cyclopamine group was significantly lower than the control group, the expression of bax and Fas protein was significantly higher than the control group; the expression of bcl- 2, Glil protein in safflower polysaccharide ＋ cyclopamine group was signifi- cantly lower than the safflower polysaccharide group and the cyclopamine group, and bax and Fas protein expression was significantly higher than the safflower polysaccharide group and the cyclopamine group. Conclusion Safflower polysaccharide and Hedgehog pathway inhibitors were able to inhibit the proliferation of human breast cancer cell line MDA- MB -231, safflower polysaecharide and cyclopamine combination on cell oroliferation and anootosis were stronzer than separate safflower oolvsaccharide and cvclooamine.