微波热疗协同吉西他滨抑制人肺鳞癌细胞增殖及诱导凋亡机制研究

Microwave Hyperthermia Combined with Gemcitabine Inhibits Proliferation and Induces Apoptosis of Human Lung Squamous Carcinoma Cells

背景与目的肺癌是全球发病率及死亡率最高的恶性肿瘤之一,寻找有效的抗肿瘤方法显得很重要,微波热疗作为一种新的治疗技术越来越受到重视。本研究旨在探讨微波热疗联合吉西他滨对人肺鳞癌细胞NCI-H1703和NCI-H2170体外增殖的影响及诱导凋亡的机制。方法 CCK-8法分别检测微波热疗、吉西他滨对细胞增殖影响以及微波热疗与吉西他滨不同序贯方式对细胞增殖的影响;克隆形成实验观察阴性对照组、微波热疗组、吉西他滨组及热化联合组对细胞克隆形成的影响;流式细胞术检测不同处理组细胞的总凋亡率;Caspase-3、Caspase-8活性检测试验检测各组细胞Caspase-3、Caspase-8酶活性;CCK-8法检测AC-DEVD(Caspase-3抑制剂)对细胞增殖的影响;Western blot检测微波热疗和吉西他滨对肺鳞癌细胞凋亡相关蛋白的表达。结果微波热疗对肺鳞癌有增殖抑制作用;吉西他滨对两株细胞的IC_(50)值分别为8.89μmol/L和44.18μmol/L;先化疗后微波热疗对两株肺鳞癌细胞有协同作用且可明显抑制细胞的克隆形成(P<0.001)、促进细胞凋亡(P<0.001)以及显著提高Caspase-3酶活性(P<0.001),但对Caspase-8酶活性影响不大(P>0.05);加入AC-DEVD(Caspase-3抑制剂)后,热化联合组细胞增殖率提高(P<0.001);Western blot检测显示微波热疗联合吉西他滨可上调p53、Caspase-3、Cleaved-Caspase-3、Cleaved-PARP以及Bax蛋白的表达。结论先吉西他滨后微波热疗可协同抑制人肺鳞癌细胞的体外增殖以及促进凋亡,其机制可能是通过活化p53、切割PARP蛋白诱导癌细胞发生Caspase-3依赖性凋亡,从而发挥其协同抑制癌细胞增殖的作用。

Background and objective Lung cancer is one of the highest morbidity and mortality in the world and it is very important to find an effective anti-tumor method.Microwave hyperthermia,a new treatment technology,has been getting more and more attention.This studywas designed to investigate the effects of microwave hyperthermia combined with gemcitabine on the proliferation and apoptosis of human lung squamous cell carcinoma (NCI-H1703and NCI-H2170)in vi- tro.Methods The proliferation of ceUs treated with microwave hyperthermia,the effect of gemcitabine on cellproliferation and the proliferation of cells treated with different methods of microwave hyperthermia and gemcitabine were detected by CCK-8 assay.Colony formation assay was used to measure the colony formation of human lung squamous cell carcinoma calls.Flow cytometry assay was used to detect the total apoptosis rates of the treated calls.Caspase-3,Caspase-8activity assay was used to detect the activity of Caspase-3,Caspase-8enzyme in each group of cells.CCK-8assay was used to detect the effect of control group,AC-DEVD (Caspase-3inhibitor)group,thermalization combined group,and thermal AC-DEVD combined group on cell proliferation.The levels of p53,Caspase-3,C1eaved-Caspase-3,PARP,Bax and BCL-2protein expression were detected using Western blot assay.Results Our results demonstrated that microwave hyperthermia inhibited the proliferation of lung squamous cell carcinoma.The IC50values of gemcitabine for the two ceils were 8.89 μtmol/L and 44.18μmol/L,respectively.The first chemotherapy after microwave hyperthermia has synergistic effect on the two lung sqnamous cell carcinoma cells and can significantly inhibit the cell clone formation (P<0.001),promote ceil apoptosis (P<0.001)and increase Caspase-3enzyme activity (P<0.001).However,it has no effect on Caspase-8enzyme activity (P>0.05).Furthermore,Western blot analysis showed that microwave hyperthermia combined with gemcitabine could up-regulate the p53,Caspase-3,Cleaved-Caspase-3, Cleaved-PARP and Bax protein expression.Conclusion Microwave hyperthermia combined with gemcitabine remarkably in- hibit the proliferation and induce apoptosis of human lung squamous cell carcinoma in vitro.This effect may be associated with the activation of p53,cleavage of PARP protein,and induced the Caspase-3dependent apoptosis.