尼古丁抑制穿孔素/颗粒酶B诱导的A549肺癌细胞凋亡

Nicotine inhibits perforin/granzyme B-induced apoptosis in A549 lung cancer cells

目的:首次探讨尼古丁对穿孔素/颗粒酶B(perforin/granzyme B)诱导的A549肺癌细胞凋亡的抑制作用及其机制。方法:以人肺癌细胞株A549为研究对象,采用四甲基偶氮噻唑蓝(MTT)比色法检测细胞活性,吖啶橙/溴化乙啶(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)染色后流式细胞术定量检测细胞凋亡率,RT-PCR法检测X连锁凋亡抑制蛋白(XIAP)及存活素(survivin)mRNA的表达量。结果:(1)不同浓度尼古丁单独作用A549细胞时未见明显诱导凋亡现象,可显著抑制细胞生长(P<0.01),其抑制率与作用时间和剂量呈正相关。(2)0.125 mg/L perforin作用于A549细胞(1×106个)时,PI染核率可达到90%以上。而当加入1μL granzyme B(1 mg/L)孵育6 h时,perforin/granzyme B组细胞可呈现90%以上的凋亡现象。(3)6μmol/L尼古丁作用于perforin/granzyme B预处理的细胞6 h时可见明显凋亡抑制现象,当检测其凋亡抑制蛋白XIAP和survivin mRNA的含量时,发现尼古丁组表达量较高,perforin/granzyme B组表达量明显降低,而当尼古丁联合perforin/granzyme B时表达量最高。结论:尼古丁可明显抑制perforin/granzyme B诱导的A549肺癌细胞凋亡现象,这种作用可能与凋亡抑制蛋白XIAP和survivin的表达上调有关。

AIM： To investigate the mechanism that nicotine inhibits perforin/granzyme B -induced apeptosis in A549 lung cancer cells. METHODS ： MTY assay, acridine orange （AO）/ethidium bromide （EB） staining, Annexin V - FITC/PI staining and RT - PCR were applied to identify the viability, the morphological characteristics of apoptosis, the apeptotic rate and the mRNA expression of X - linked inhibitor of apeptosis protein （XIAP） and survivin, respectively. RESULTS ： No significant apoptosis was observed when nicotine was used alone, and the proliferation of A.549 lung cancer cells was inhibited by nicotine in a concentration - and time - dependent manner （P 〈 0. 01 ） , The positive rate of PI staining in A.549 cells treated with 0. 125 mg/L perforin was up to 90%. When 1μL of granzyme B （ 1 mg/L） was added to the ceils for 6 h, more than 90% apeptotic cells were found. Combination of nicotine （6 μmol/L） with perforin/granzyme B for 6 h significantly inhibited apeptosis. Furthermore, higher XIAP and survivin mRNA was detected when used nicotine alone, and the lowest XIAP and survivin mRNA was measured when used perforin/granzyme B alone. However, the highest XIAP and survivin mRNA was observed when used perforin/granzyme B combined with nicotine. CONCLUSION： Nicotine inhibits perforin/granzyme B - induced apoptosis in A.549 lung cancer cells. This effect may be related to up - regula, tion of the inhibitor of apeptosis proteins, XIAP and survivin.