山柰酚诱导人乳腺癌耐阿霉素细胞MCF-7/阿霉素凋亡的研究

Mechanism of kaempferol-induced apoptosis of adriamycin MCF-7/adriamycin resistant cells

目的研究山柰酚对人乳腺癌耐阿霉素细胞MCF-7/阿霉素(ADR)的增殖与凋亡的作用机制。方法(1)将人乳腺细胞MCF-7分为2组:空白组(培养基)和实验组。实验组分别用含不同浓度的山柰酚(20,50,100,150,200,300,400,500μmol·L^(-1))的培养基干预。干预48 h后,噻唑蓝(MTT)法测定细胞存活率。(2)将MCF-7/ADR细胞分为3组:ADR组(ADR 0.1μmol·L^(-1))和低、中2个浓度山柰酚联合组(ADR 0.1μmol·L^(-1)+分别加山柰酚20,50μmol·L^(-1)),MTT法测定细胞存活率,计算半抑制浓度(IC_(50))。(3)将MCF-7/ADR细胞分为8组:空白组(培养基)、ADR组(ADR 0.1μmol·L^(-1))、低、中、高3个浓度的山柰酚组(山柰酚:20,50,100μmol·L^(-1))和低、中、高3个联合组(ADR 0.1μmol·L^(-1)+分别加山柰酚20,50,100μmol·L^(-1))。干预后,流式细胞术检测细胞凋亡率。(4)将细胞分为2组:MCF-7组与MCF-7/ADR组;再将MCF-7/ADR细胞分为4组:空白组合低、中、高3个浓度山柰酚组(山柰酚:20,50,100μmol·L^(-1))。干预后,蛋白质印迹法测定B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)蛋白表达水平(灰度值比值)。结果(1)根据细胞存活率选择低、中、高3个浓度(20,50,100μmol·L^(-1))的山柰酚进行以下实验。(2)ADR组和低、中2个浓度山柰酚联合组的IC_(50)分别为(43.68±0.71),(17.68±3.14)和(23.51±0.07)μmol·L^(-1)。低、中2个浓度山柰酚联合组与ADR组相比,差异均有统计学意义(均P<0.01)。(3)空白组、ADR组、3个浓度(由低到高)的山柰酚组和低、中、高3个联合组的细胞凋亡率分别为(0.17±0.24)%,(23.00±1.14)%,(9.53±1.24)%,(17.07±1.04)%,(22.20±1.15)%,(16.03±1.74)%,(21.43±2.21)%和(21.93±3.57)%。ADR组、山柰酚组和联合组与空白组相比,差异均有统计学意义(均P<0.01),低剂量联合组与ADR相比,差异有统计学意义(P<0.05)。(4)MCF-7组与MCF-7/ADR组的Bcl-2蛋白表达分别为1.47±0.33和0.56±0.20,这2组的Caspase-9蛋白表达分别为0.89±0.67和4.12±1.25。2组相比,上述蛋白表达的差异均有统计学意义(P<0.05)。空白组和低、中、高3个浓度山柰酚组的Bcl-2蛋白表达分别为1.30±0.19,1.20±0.05,1.45±0.15和0.81±0.16;这4组的Caspase-9蛋白表达分别为1.02±0.09,0.96±0.07,0.79±0.07和0.40±0.18。高剂量组与空白组相比,上述2种蛋白表达的差异有统计学意义(P<0.05和P<0.01)。结论山柰酚对MCF-7细胞有毒性作用,与ADR联合用药后降低ADR在MCF-7/ADR中的IC_(50),山奈酚提高MCF-7/ADR细胞的凋亡,可能与降低Bcl-2蛋白表达与升高Caspase-9蛋白表达有关。

Objective To study the effect of kaempferol on the proliferation and apoptosis of adriamycin resistant MCF-7/adriamycin(ADR)breast cancer cells and its mechanism.Methods(1)MCF-7 cells were divided into two groups:blank group(containing medium only)and experimental group.The experimental group was treated with different concentrations of kaempferol(20,50,100,150,200,300,400,500μmol·L^(-1)).After administration for 48 h,cell viability was measured by MTT assay.(2)MCF-7/ADR cells were divided into 3 groups:ADR group(0.1μmol·L^(-1)ADR)and joint-L,-M groups(0.1μmol·L^(-1)ADR+20,50μmol·L^(-1)kaempferol).Cell viability was measured by MTT assay,and half inhibiting concentration(IC_(50))was calculated.(3)MCF-7/ADR cells were divided into 8 groups:black group(without any treatment),ADR group(0.1μmol·L^(-1)adriamycin),kaempferol-L,-M,-H groups(20,50,100μmol·L^(-1)kaempferol),and joint-L,-M,-H groups(0.1μmol·L^(-1)ADR+20,50,100μmol·L^(-1)kaempferol).After administration,apoptosis was detected by flow cytometry.(4)Cells were divided into 2 groups:MCF-7 groups,MCF-7/ADR groups;and the MCF-7/ADR were divided into 4 groups:blank group and kaempferol-L,-M,-H(20,50,100μmol·L^(-1))groups.After administration,the expression(ratio of gray value)of B-cell lymphoma-2(Bcl-1),Bcl-2 related protein X(Bax),Caspase-9 was determined by Western blot.Results(1)The low,middle and high concentration(20,50,100μmol·L^(-1))kaempferol was chosen for further investigation according to the cell survival rate.(2)The IC_(50) of ADR group and joint-L,-M groups were(43.68±0.71)μmol·L^(-1),(17.68±3.14)μmol·L^(-1)and(23.51±0.07)μmol·L^(-1),respectively.Compared between joint-L,-M groups and ADR group,the difference were significant(all P<0.01).(3)Apoptosis rate in the black group,ADR group,kaempferol-L,-M,-H groups,and joint-L,-M,-H groups were(0.17±0.24)%,(23.00±1.14)%,(9.53±1.24)%,(17.07±1.04)%,(22.20±1.15)%,(16.03±1.74)%,(21.43±2.21)%and(21.93±3.57)%.There are significant difference between ADR group,kaempferol-L,-M,-H groups,joint-L,-M,-H groups and black groups(all P<0.01),the difference is also significant between joint-L group and ADR group(P<0.05).(4)The expression of Bcl-2 in MCF-7 and MCF-7/ADR groups were 1.47±0.33 and 0.56±0.20,respectively;the expression of Caspase-9 in this two groups were 0.89±0.67 and 4.12±1.25,respectively.There are significant difference of the factors in MCF-7 and MCF-7/ADR groups(all P<0.05).The expression of Bcl-2 in black group,kaempferol-L,-M,-H groups were 1.30±0.19,1.20±0.05,1.45±0.15,0.81±0.16,respectively;the expression of Caspase-9 in this 4 groups were 1.02±0.09,0.96±0.07,0.79±0.07,0.40±0.18,respectively.The expressions of Bcl-2 and Caspase-9 are significant between kaempferol-H group and blank group(P<0.05 and P<0.01).Conclusion Kaempferol has a toxic effect on MCF-7 cells.When combined with ADR kaempferol can reduce the IC_(50) of ADR in MCF-7/ADR and increase the apoptosis of MCF-7/ADR cells,which may be related to the decrease of Bcl-2 expression and the increase of Caspase-9 expression.