二氟甲基鸟氨酸对不同鸟氨酸脱羧酶基因G316A遗传学类型的乳腺癌细胞系增殖、凋亡和细胞周期的影响

Effect of difluoromethylorithine proliferation, apoptosis and cell cycle in different breast cell lines with different ODC G316A

目的观察二氟甲基鸟氨酸（DFMO）对不同鸟氨酸脱羧酶（ODC） 基因G316A遗传学类型的乳腺癌细胞系增殖、凋亡和细胞周期的影响及可能的机制。方法利用四甲基偶氮唑蓝（MTT）法、流式细胞术检测DFMO处理的乳腺癌细胞系MDA-435（ODC GG）和SK-br3（ODC AA）的增殖抑制、凋亡以及细胞周期变化,采用Taqman探针法检测DFMO处理的MCF-7细胞系ODC G316A等位基因A和G表达量的变化。结果10 mmol／L DFMO对MDA-435和SK-br3作用48 h的生长抑制率分别为24.1 %、33.6 %,20 mmol／L DFMO作用48 h分别为46.3 %、53.5 %,差异均具有统计学意义（t=2.134,P=0.021;t=2.213,P=0.019）;10 mmol／L DFMO对MDA-435和SK-br3作用72 h的生长抑制率分别为28.9 %、35.7 %,20 mmol／L DFMO作用72 h分别为54.3 %、65.4 %,差异均具有统计学意义（t=2.434,P=0.015;t=2.489,P=0.013）;20 mmol／L DFMO作用24、48、72 h后,MDA-435（ODC GG）和SK-br3（ODC AA）细胞凋亡率分别为（7.58±2.06）% 和（13.88±3.45）%（t=2.047,P=0.041）、（43.28±14.28）%和（59.96±16.42）%（t=3.680,P=0.000）、（77.87±30.25）%和（93.08±32.15）%（t=3.293,P=0.000 1）,差异具有统计学意义（P〈0.05）。MDA-435（ODC GG）细胞和SK-br3（ODC AA）细胞培养24、48、72 h后S期细胞所占比例分别为（13.25±2.38）%和（12.89±2.21）%（P〉0.05）,（21.43±3.12）%和（12.24±3.55）%（t=2.638,P=0.012）,（16.32±3.23）%和（15.24±3.01）%（P〉0.05）。经DFMO处理的乳腺癌MCF-7（ODC AG）细胞系ODC G316A等位基因A的表达量降低（t=3.708,P=0.000）,而G的表达量无明显变化。结论DFMO对不同ODC G316A遗传学类型的乳腺癌细胞系增殖、凋亡和细胞周期的影响不同;DFMO抑制ODC的活性可能是通过与ODC G316A的等位基因A相结合而实现的。

ObjectiveTo investigate the proliferation, apoptosis and cell cycle and possible mechanisms of different breast cell lines by difluoromethylorithine （DMFO）. MethodsThe growth of breast cancer MDA-435 （ODC GG） cell lines and SK-br3 （ODC AA） cell lines treated with DFMO were observed. The apoptosis and cell cycle were detected by flow cytometry. PCR was applied to detect the changes of A and G alleles of ODC G316A in MCF-7 cells treated with DFMO. ResultsThe growth inhibition rates of MDA-435 and SK-br3 cells treated with 10 mmol/L and 20 mmol/L DFMO after 48 h were 24.1 % and 33.6 %, 46.3 % and 53.5 %, respectively, and there was statistical significance （t = 2.134, P = 0.021, t = 2.213, P = 0.019）. The growth inhibition rates of MDA-435 and SK-br3 treated with 10 mmol/L and 20 mmol/L DFMO after 72 h were 28.9 % and 35.7 %, 54.3 % and 65.4 %, respectively, and there was statistical significance （t = 2.434, P = 0.015, t = 2.489, P = 0.013）. The apoptosis rates of MDA-435 （ODC GG） and SK-br3 （ODC AA） cells both dealt with 20 mmol/L of DFMO after 24 h, 48 h and 72 h were （7.58±2.06） % and （13.88±3.45） % （t = 2.047, P = 0.041）, （43.28±14.28） % and （59.96±16.42） % （t = 3.680, P = 0.000）, （77.87±30.25） % and （93.08±32.15） % （t = 3.293, P = 0.000 1）, respectively. The proportions of S stage cells MDA-435 （ODC GG） and SK-br3 （ODC AA） cells under the same condition after 24 h, 48 h and 72 h were （13.25±2.38） % and （12.89±2.21） % （P 〉 0.05）, （21.43±3.12） % and （12.24±3.55） % （t = 2.638, P = 0.012）, （16.32±3.23） % and （15.24±3.01） % （P 〉 0.05）, respectively. After the treatment by DFMO, the expression of ODC G316A allele A in breast cancer cell line MCF-7 （ODC AG） was reduced （t = 3.708, P = 0.000）, and the expression of G had no significant changes. ConclusionThe proliferation inhibition and apoptosis in breast cancer cells treated by DFMO is different in breast cancer cells with different genetic type of ODC G316A. DFMO can inhibit the activity of ODC, and the mechanism may be that DFMO could selectively bind to ODC G316A allele A.