山萘酚对MCF-7细胞增殖的影响

Effect of kaempferol on MCF-7 cell proliferation

目的研究山萘酚对乳腺癌细胞增殖与凋亡的影响及其作用机制。方法 (1)将细胞分为8组:空白组(培养基)、阿霉素组(阿霉素0.1μmol·L-1)和低、中、高3个浓度山奈酚组(山萘酚:20,50,100μmol·L-1,处理48 h)以及低、中、高3个联合组(阿霉素0.1μmol·L-1+分别加山萘酚20,50,100μmol·L-1)。干预后,MTT法测定细胞增值水平,流式细胞术检测细胞凋亡率。(2)将细胞分为4组:空白组和低、中、高3个浓度山萘酚组。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)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)、核因子κB(NF-κB)和NF-κB抑制剂(I-κB)蛋白表达水平(灰度值比值)。结果 (1)山萘酚均显著抑制MCF-7和Hepg-2细胞增殖(均P<0.01),且有时间和浓度依赖性。空白组、阿霉素组和低、中、高3个浓度山萘酚组以及低、中、高3个联合组的细胞凋亡率分别为(0.05±0.05)%,(4.30±0.30)%,(4.70±0.50)%,(14.55±1.15)%,(21.80±1.50)%,(5.90±2.20)%,(29.45±2.35)%和(17.95±2.25)%。阿霉素组或3个浓度实验组与空白组相比,差异均有统计学意义(均P<0.05);中、高2个浓度联合组与阿霉素组相比,差异均有统计学意义(均P<0.05)。(2)空白组和低、中、高3个浓度山萘酚组的Bcl-2蛋白表达分别为5.57±0.26,4.92±0.49,3.38±0.41和2.58±0.25;这4组的Caspase-9蛋白表达分别为2.89±0.38,1.87±0.10,1.73±0.06和1.23±0.23;这4组的Bax蛋白表达分别为1.72±0.11,1.93±0.07,2.30±0.14和2.75±0.25;这4组的NF-κB蛋白表达分别为4.42±0.92,3.38±0.55,2.93±0.47和2.54±0.16;这4组的I-κB蛋白表达分别为1.21±0.15,1.68±0.10,0.79±0.11和0.45±0.17。上述蛋白表达:中、高2个浓度组与空白组相比,差异均有统计学意义(均P<0.01)。结论山萘酚可能通过下调Bcl-2和Caspase-9的表达、升高Bax的表达,诱导细胞凋亡,从而抑制MCF-7的增殖,该过程还可能与NF-κB信号通路有关。

Objective To study the effect of kaempferol on proliferation and apoptosis of breast cancer cells MCF-7 and its mechanism. Methods(1)The cells was divided into 8 groups:blank group(without any treatment),adriamycin group(0.1 μmol·L-1 adriamycin for 48 h),kaempferol-L,-M,-H groups(50, 100, 200 μmol·L-1 kaempferol, respectively), and joint-L,-M,-H groups(0.1 μmol·L-1 adriamycin +50, 100, 200 μmol·L-1 kaempferol, respectively).After administration,cell proliferation was measured by MTT assay;apoptosis was detected by flow cytometry.(2)The cells was divided into 4 groups: blank group and kaempferol-L,-M,-H groups. After administration,the expression(ratio of gray value) of B-cell lymphoma-2(Bcl-2),Bcl-2 related protein X(Bax),Caspase-9,nuclear factor kappa-B(NF-κB) and inhibitor of NF-κB(I-κB) proteins were determined by Western blot. Results(1) Kaempferol significantly inhibited the proliferation of MCF-7 and Hepg-2 cells(P < 0. 01). It was time and concentration dependent. The apoptosis rate in blank group,model group,experimental-L,-M,-H groups and joint-L,-M,-H groups were(0. 05 ± 0. 05) %,(4. 30 ± 0. 30) %,(4. 70 ± 0. 50) %,(14. 55 ± 1. 15) %,(21. 80 ± 1. 50) %,(5. 90 ± 2. 20) %,(29. 45 ± 2. 35) % and(17. 95 ± 2. 25) %,respectively. There were significant difference between adriamycin group,kaempferol-L,-M,-H groups and black group(all P < 0. 05);there were significant difference between joint-M,-H groups group and adriamycin group(all P < 0. 05).(2) The expression of Bcl-2 in blank group,kaempferol-L,-M,-H groups were 5. 57 ± 0. 26,4. 92 ± 0. 49,3. 38 ± 0. 41,2. 58 ± 0. 25,respectively;the expression of Caspase-9 protein in the 4 groups were 2. 89 ± 0. 38,1. 87 ± 0. 10,1. 73 ± 0. 06,1. 23 ± 0. 23,respectively;The expression of Bax protein in the 4 groups were 1. 72 ± 0. 11,1. 93 ± 0. 07,2. 30 ± 0. 14,2. 75 ± 0. 25,respectively;the expression of NF-κB protein in the 4 groups were 4. 42 ± 0. 92,3. 38 ± 0. 55,2. 93 ± 0. 47,2. 54 ± 0. 16,respectively;The expression of I-κB protein in the 4 groups were 1. 21 ± 0. 15,1. 68 ± 0. 10,0. 79 ± 0. 11,0. 45 ± 0. 17,,respectively. There were significant difference of the factors between the experimental group-M,-H groups and black group(all P < 0. 01). Conclusion Kaempferol is capable of inhibiting breast cancer cells proliferation and promoted apoptosis in MCF-7 cells,the potential mechanism may be associated with down-regulating the expression of anti-apoptotic proteins Bcl-2 and Caspase-9,which may also be related to NF-κB signaling pathway.