艾洛替尼耐药肝癌HepG2细胞系的建立及其耐药机制

目的体外建立艾洛替尼(erlotinib)耐药的人肝癌细胞系HepG2/erlotinib,鉴定其生物学特性,并对其耐药机制进行初步探讨。方法逐步递增艾洛替尼并最终给予艾洛替尼50μmol·L-1连续冲击HepG2细胞12个月,建立HepG2/erlotinib。磺酰罗丹明B法检测HepG2/erlotinib耐药倍数和耐药谱;测定细胞生长曲线并计算细胞增殖时间;流式细胞术检测细胞周期;RT-PCR和Western印迹法检测HepG2/erlotinib细胞乳腺癌耐药蛋白(BCRP)基因和蛋白的表达;流式细胞术检测细胞表面BCRP蛋白表达。结果经过12个月艾洛替尼诱导的HepG2耐药细胞,艾洛替尼的IC50值为(72.3±6.1)μmol·L-1,艾洛替尼对正常HepG2细胞的IC50值为(10.6±0.3)μmol·L-1,耐药倍数为6.8±0.7,表明HepG2/erlotinib为艾洛替尼耐药细胞系。HepG2/erlotinib对顺铂、多柔比星、米托蒽醌和拓扑替康的IC50均大于正常HepG2细胞的IC50,表明产生交叉耐药性。HepG2细胞和HepG2/erlotinib细胞的群体倍增时间分别为(20.7±3.3)h和(26.7±1.2)h,耐药细胞倍增时间延长。与HepG2细胞相比,HepG2/erlotinib的S期细胞比例明显增高(P<0.05),G0/G1期细胞比例明显降低(P<0.01);HepG2/erlotinib细胞中BCRP基因和BCRP蛋白表达显著升高(P<0.05)。结论建立的HepG2/erlotinib具有耐药细胞的特征和生物学特性,其耐药机制与BCRP蛋白表达增加有关。

Establishment of erlotinib-resistant HepG2 cell line and its resistant mechanisms

OBJECTIVE To establish a human hepatocellular carcinoma cell line with resistance to erlotinib, and to investigate its possible resistance mechanism. METHODS The cell line HepG2 was cultured by gradually increasing dose of erlotinib and final dose 50 μmol·L^-1 in vitro for 12 months to generate its resistance cell line HepG2/erlotinib. The resistant index of ertotinib was determined by sulforhodamine B (SRB); cell growth curve, doubling time and cell cycle phase distribution were measured; breast cancer resistance protein (BCRP) mRNA and its protein level were examined by RT-PCR, Western blotting and flow cytometry . RESULTS After induced for 12 months, a resistant cell line HepG2/erlotinib was established. The IC₅₀ value of HepG2/erlotinib to erlotinib was (72.3±6.1)μmol·L^-1 while that of HepG2 to erlotinib was (10.6±0.3)μmol·L^-1, with the resistant index of 6.84±0.7. The IC₅₀ values of HepG2/erlotinib to cisplatin, doxorubicin, mitoxantrone and topotecan were greater than that of HepG2 cells, indicated that it had cross resistance to these drugs. The doubling time of HepG2 and HepG2/erlotinib was (20.7±3.3)h and (26.7±1.2)h, respectively. In the HepG2/erlotinib resistant cell line, the cell numbers of S phase increased (P<0.05) while that of G₀/G₁ phase decreased (P<0.01). HepG2/erlotinib cells expressed higher levels of BCRP mRNA and BCRP protein compared with parental cells (P<0.05). CONCLUSION An erlotinib-resistant human hepatocellular carcinoma cell line HepG2/erlotinib is established. It shows a typical resistant phenotype and biological characteristics. The high BCRP expression may contribute to its resistance to erlotinib.