可替宁对穿孔素/粒酶B诱导的NCI-H460肺癌细胞凋亡的抑制作用

Cotinine Inhibits Perforin/Granzyme B-Induced Apoptosis in NCI-H460 Lung Cancer Cells

目的探讨可替宁对穿孔素/粒酶B(perforin/granzyme B)诱导的NCI-H460非小细胞肺癌细胞凋亡的抑制作用及其机制。方法以人肺癌细胞株NCI-H460为研究对象,采用四甲基偶氮噻唑蓝(MTT)比色检测细胞生长增殖活性,吖啶橙/溴化乙锭(AO/EB)双染荧光显微镜下观察细胞凋亡形态学变化,异硫氰酸荧光素标记的膜联蛋白V/碘化丙啶(Annexin V-FITC/PI)染色后流式细胞仪定量检测细胞凋亡率,RT-PCR及Western blot法检测凋亡抑制蛋白XIAP及Survivin的表达量。结果 (1)不同浓度可替宁单独作用NCI-H460细胞时未见明显诱导凋亡现象,可显著抑制细胞生长,其抑制率与作用时间和剂量呈正相关。(2)0.25μg/ml浓度穿孔素作用NCI-H460细胞(1×106个)时,PI染核率可达到90%以上。而当加入1.5μl粒酶B(1μg/ml)孵育6h时,穿孔素/粒酶B组细胞可呈现90%以上的凋亡现象。(3)166ng/ml浓度可替宁作用穿孔素/粒酶B预处理的细胞6h时可见明显凋亡抑制现象,当检测其凋亡抑制蛋白XIAP、survivin表达量时,发现穿孔素/粒酶B组表达量明显降低,可替宁组表达量较高,而当可替宁联合穿孔素/粒酶B时表达量最高。结论可替宁可明显抑制穿孔素/粒酶B诱导的NCI-H460肺癌细胞凋亡现象,这种作用可能与凋亡抑制蛋白XIAP、survivin的表达上调有关。

Objective To study the mechanism that cotinine inhibits perforin/granzyme B - induced apoptosis in NCI - H460 lung cancer cells. Methods An MTT assay, AO/EB and Annexin V -FITC/PI staining, RT- PCR and Western blotting were applied to identify the viability of ceils, characteristic morphology and stages of apoptosis, expression of XIAP and Survivin respectively. Results First, we observed that cotinine induced no significant apoptosis when used alone, and inhibited cell proliferation in a concentration - and time - dependent manner （P 〈 0.01 ）. Second, when perforin （0.25 ~g/ml） worked on NCI - H460 cells （ 1 x 106 ） , PI staining rate was up to 90%. When adding 1.51xl of granzyme B （ 1 ixg/ml） of 6h incubation, the ceils could present more than 90% apoptosis. Third, we observed that when cotinine （166ng/ml） combined with perforin/granzyme B for 6h,it could inhibit significant apoptosis. Futhermore, the higher of XIAP and survivin was observed when cotinine was used alone, the lowest of XIAP and Survivin was observed when perforin/ granzyme B was used alone, but the highest of XIAP and Survivin was also observed when perforin/granzyme B was used combined with cotinine. Conclusion Cotinine could inhibit perforin/granzyme B - induced apoptosis in NCI - H460 lung cancer ceils. This effect may be related to the up - regulation of inhibitor of apoptosis protein XIAP and Survivin.