3-甲基腺嘌呤对人耐药大肠癌SW480细胞增殖及自噬活性的影响

Effect of 3-methyladenine on autophagy and cell proliferation in 5-Fu-resistant colon cancer cell line SW480

目的 :研究自噬抑制剂3-甲基腺嘌呤(3-MA)对耐5-氟尿嘧啶(5-Fu)结肠癌SW480细胞增殖及自噬活性的影响,探讨自噬抑制剂在逆转结直肠癌耐药中的作用。方法:浓度递增筛选法诱导耐5-Fu(100μg/L)的SW480细胞株,用10μmol/L浓度的3-MA处理后,再用5-Fu(浓度200μg/L)作用24 h。采用CCK-8法检测细胞增殖情况,流式细胞仪检测细胞凋亡率,丹酰戊二胺(MDC)染色和电镜检测细胞自噬活性变化。结果:3-MA对耐药细胞自噬活性的抑制率达89.7%,增殖抑制率为9.6%,凋亡率为5.7%,与对照组比较差异具有统计学意义(P<0.05)。经3-MA处理后,5-Fu对耐药SW480细胞增殖抑制率达71.6%±2.9%,凋亡率达36.6%±2.9%,而单用5-Fu细胞增殖抑制率仅为23.6%±2.9%,凋亡率仅为10.3%±2.5%,3-MA预处理后5-Fu对耐药细胞的增殖抑制率及凋亡率均明显增强(P<0.05),自噬泡数量显著减少,自噬活性明显降低(P<0.05)。结论:3-MA单用时仅有较弱的抗肿瘤作用,与5-FU联用时耐药细胞的增殖及自噬活性被显著抑制,增强了SW480耐药细胞对5-Fu的敏感性,提高了化疗效果。

Objective： To investigate the effects of 3-methyladenine on chemotherapy sensitivity of 5-Furesistant colon cancer cell line SW480 and reversing the 5-Furesistant in colon cancer. Methods： Stepwise 5-Fu selection was used to establish the 5-Fu resistant colon cancer cells （100 μg/L）. The cells were dealed with 3- methyladenine （10 μmol/L） fistly and 5-Fu （200 μg/L） for 24 hours. Cell proliferation inhibition rate was mea- sured with CCK-8. Apoptosis rate was detected with flow cytometer. Autophagic vacuoles in cells were observed by means of transmission electron microscopy and monodansylcadaverine （MDC）. Results： The inhibition rate of 3-methyladenine in the 5-Fu resistant cell autophagy activity was 89.7%, cell proliferation was 9.6%, but apoptosis rate was just 5.7%, were obviously higher than that in 5-Fu resistant cells （P〈0.05）. After dealing with 3-methyladenine（10 μmol/L） fistly and 5-Fu （200 μg/L） for 24 hours, the level of cell proliferation inhibition rate was 71.6% ± 2.9% and the apoptosis rate was 36.6% ± 2.9%, while dealing with 5-Fu （200 μg/L） for 24 hou rs, the level of cell proliferation inhibition rate was 23.6% ± 2.9% and the apoptosis rate was 10.3% ± 2.5%. After dealing with 3-methyladenine, the cell proliferation inhibition rate and apoptosis rate were markedly higher than that of 5-Fu only. Conclusion： The anticancer capacity of 3-methyladenine in colon cancer is weak, but it can reverse the 5-Fu-resistant in colon cancer partly, 5-Fu-induced apoptosis in 5-Fu resistant colon cancer cells may be enhanced by the inhibitor of autophagy.