山柰酚逆转MCF-7/阿霉素细胞耐药机制研究

Study on the mechanism of reversal effect of kaempferol on MCF-7/adriamycin cells

目的研究山柰酚逆转人乳腺癌耐阿霉素细胞MCF-7/阿霉素(ADR)耐药机制。方法(1)将MCF-7和MCF-7/ADR细胞分为空白组(培养基)、对照组(细胞、培养基)和实验组。实验组分别用含不同浓度(20,50,100,150,200,300,400,500μmol·L^(-1))山柰酚的培养基培养,每组分别干预24,48和72 h。干预后MTT法测定细胞存活率。(2)将MCF-7和MCF-7/ADR细胞分为空白组(培养基)、对照组(细胞、培养基)和联合组,联合组分别用含有不同浓度(3,6,12,24,48μmol·L^(-1))阿霉素、不同浓度(3,6,12,24,48μmol·L^(-1))紫杉醇和不同浓度(10,30,60,90,120μmol·L^(-1))顺铂的培养基培养,干预48 h。MTT法测定细胞存活率,计算IC50和耐药倍数。(3)将MCF-7/ADR细胞分为ADR组和低、中2个浓度ADR联用组(ADR+山柰酚20,50μmol·L^(-1));紫杉醇组和低、中2个浓度紫杉醇联用组(紫杉醇+山柰酚20,50μmol·L^(-1));顺铂组和低、中2个浓度顺铂联用组(顺铂+山柰酚20,50μmol·L^(-1)),计算IC50和逆转耐药倍数。(4)细胞分为MCF-7组与MCF-7/ADR组;再将MCF-7/ADR细胞分为4组:空白组和低、中、高3个浓度山柰酚组(山柰酚:20,50,100μmol·L^(-1))。蛋白质印迹法测定核因子κB(NF-κB)、NF-κB抑制剂(I-κB)、多药耐药蛋白(ABCB1)和乳腺癌耐药蛋白(ABCG2)蛋白表达水平(灰度值比值)。结果(1)山柰酚显著抑制MCF-7和MCF-7/ADR细胞存活率,与空白组相比,差异有统计学意义(P<0.05)。(2)MCF-7细胞:ADR组、紫杉醇组和顺铂组的IC50分别为(1.84±0.22),(1.76±0.12)和(0.78±0.53)μmol·L^(-1)。MCF-7/ADR细胞:这3组的IC50分别为(43.68±0.76),(85.93±9.04)和(203.20±5.08)μmol·L^(-1)。2种细胞的IC50相比差异均有统计学意义(均P<0.01)。上述3种药物在MCF-7/ADR细胞的耐药倍数分别为23.66,9.50和257.86。(3)MCF-7/ADR细胞:ADR组和低、中2个浓度联合组的IC50分别为(43.68±0.76),(17.68±3.14)和(23.51±0.06)μmol·L^(-1),低、中2个浓度联合组与ADR组相比差异有统计学意义(P<0.01);这3组的耐药逆转倍数分别为1.00,2.47和1.86。紫杉醇组和低、中2个浓度联用组的IC50分别为(85.93±9.04),(43.50±1.11)和(37.80±1.06)μmol·L^(-1),与紫杉醇组相比差异有统计学意义(P<0.01),这3组耐药逆转倍数分别为1.00,1.98,2.27。顺铂组和低、中2个浓度联用组IC50分别为(203.20±5.08),(54.81±4.11)和(30.57±0.70)μmol·L^(-1),与顺铂组相比差异有统计学意义(P<0.01),这3组耐药逆转倍数分别为1.00,3.71和6.65。(4)NF-κB在MCF-7组与MCF-7/ADR组的表达分别为3.10±0.50,8.40±1.95;ABCB1在2组的表达分别为1.27±0.26,3.39±0.44;ABCG2在2组的表达分别为0.65±0.27,1.62±0.23,上述指标在2组比较差异均有统计学意义(P<0.05,P<0.01)。MCF-7/ADR细胞:空白组、低、中、高3个浓度组的NF-κB表达分别为1.33±0.29,0.98±0.14,0.81±0.11和0.64±0.05;ABCB1在上述4组中表达分别为0.74±0.02,0.69±0.06,0.58±0.02和0.52±0.05,与空白组相比,中、高2个浓度组差异均有统计学意义(P<0.05,P<0.01)。I-κB在上述4组中表达分别为0.99±0.06,1.16±0.09,1.23±0.03和0.96±0.11,与空白组相比,中浓度组差异有统计学意义(P<0.05);ABCG2在上述4组中表达分别为0.13±0.07,0.47±0.15,0.35±0.09和0.16±0.21,与空白组相比,高浓度组差异有统计学意义(P<0.05)。结论山柰酚能够逆转MCF-7/ADR细胞对不同药物的耐药性,其作用机制可能与山柰酚降低ABCB1蛋白的表达和抑制NF-κB表达有关。

Objective To investigate the reversal activity and mechanism of kaempferol against MCF-7/adriamycin(ADR)cells.Methods(1)MCF-7 and MCF-7/ADR cells were divided into blank group(containing medium only),control group(not containing drugs)and experimental group.The experimental group was treated with different concentrations of kaempferol(0,20,50,100,150,200,300,400,500μmol·L^(-1))in 24,48,72 h,respectively.After administration,cell viability was measured by MTT assay.(2)Again,the experimental group of MCF-7 and MCF-7/ADR cells were treated with different concentrations of ADR(3,6,12,24,48μmol·L^(-1)),paclitaxel(3,6,12,24,48μmol·L^(-1))and cisplatin(10,30,60,90,120μmol·L^(-1)),after treatment of 48 h,cell viability was measured by MTT assay,and the IC50 and fold of resistance was calculated.(3)MCF-7 cells were divided into 3 groups:adriamycin group,and joint-L,-M groups(adriamycin+20,50μmol·L^(-1)kaempferol);paclitaxel,joint-L,-M groups(paclitaxel+20,50μmol·L^(-1)kaempferol);cisplatin,joint-L,-M groups(cisplatin+20,50μmol·L^(-1)kaempferol).The IC50 and the reverse fold was calculated.(4)The cells were divided into two groups:MCF-7 group and MCF-7/ADR group;the MCF-7/ADR cells were divided into 4 groups:blank group,kaempferol-L,-M,-H groups(kaempferol 20,50,100μmol·L^(-1)),respectively.The expression(ratio of gray value)of nuclear factor-κB(NF-κB),inhibitor of NF-κB(I-κB),multidrug resistance protein(ABCB1),and breast cancer resistance protein(ABCG2)was determined by Western blot.Results(1)Kaempferol significantly inhibited the proliferation of MCF-7 in different time(P<0.01).(2)The IC50 of adriamycin group,paclitaxel group,cisplatin group in MCF-7cells were(1.84±0.22),(1.76±0.12)and(0.78±0.53)μmol·L^(-1),respectively.The IC50 of the 3 groups in MCF-7/ADR cells were(43.68±0.76),(85.93±9.04)and(203.20±5.08)μmol·L^(-1),respectively.There are significant difference between these two groups(P<0.01).The fold of resistance of adriamycin,paclitaxel,cisplatin were 23.66,9.50 and 257.86,respectively.(3)The IC50 of adriamycin group,joint-L,-M groups were(43.68±0.76),(17.68±3.14)and(23.51±0.06)μmol·L^(-1).There are significant difference between these groups and adriamycin(P<0.01).The reverse fold of these groups were 1.00,2.47 and 1.86 respectively.The IC50 of paclitaxel,joint-L,-M groups were(85.93±9.04),(43.50±1.11)and(37.80±1.06)μmol·L^(-1);the reverse fold of these groups are 1.00,1.98 and 2.27 respectively.The IC50 of cisplatin,joint-L,-M groups were(203.20±5.08),(54.81±4.11)and(30.57±0.70)μmol·L^(-1).There was significant difference between these groups and paclitaxel group(P<0.01).The reverse fold of these groups were 1.00,3.71,6.65 respectively.(4)NF-κB expression in MCF-7 group and MCF-7/ADR group were 3.10±0.50,8.40±1.95;NF-κB expression in these two groups were 1.27±0.26,3.39±0.44;ABCG2 expression in these two groups were 0.65±0.27,1.62±0.23.There was significant difference between these two groups(P<0.05,P<0.01).MCF-7/ADR cells:NF-κB expression in blank,kaempferol-L,-M,-H groups were 1.33±0.29,0.98±0.14,0.81±0.11,0.64±0.05;ABCB1 expression in the 4 groups were 0.74±0.02,0.69±0.06,0.58±0.02,0.52±0.05.There are significant difference between-M,-H group and blank group(P<0.05,P<0.01).I-κB expression in the 4 groups were 0.99±0.06,1.16±0.09,1.23±0.03,0.96±0.11.There was significant difference between-M group and blank group(P<0.05).ABCG2 expression in the 4 groups were 0.13±0.07,0.47±0.15,0.35±0.09,0.16±0.21.There was significant difference between-H group and blank group(P<0.05).Conclusion Kaempferol could reverse the drug resistance of MCF-7/ADR cells in different drugs.The mechanism may be related to inhibiting the activation of NF-κB signaling and reducing the expression of ABCB1 in MCF-7/ADR cells.