白花丹素对肝癌索拉菲尼耐药细胞HepG2R增殖和凋亡的影响及其机制

doi:10.13481/j.1671-587x.20180620

摘要/Abstract

摘要： 目的：探讨白花丹素（PLB）对肝癌索拉菲尼耐药细胞HepG2R增殖和凋亡的影响，阐明其可能机制。方法：体外培养肝癌HepG2细胞，建立索拉菲尼耐药细胞模型HepG2R，MTT法鉴定耐药倍数和筛选药物作用浓度及时间，依据其结果选择索拉菲尼和PLB作用浓度分别为5μmol·L^-1和2μmol·L ^-1，各组药物作用时间为48 h。将HepG2R细胞随机分为对照组、索拉菲尼（5μmol·L^-1）组、PLB （2μmol·L ^-1）组和索拉菲尼（5μmol·L^-1）联合PLB （2μmol·L ^-1）组（联合组）。MTT法检测各组细胞活力，克隆形成实验检测各组细胞克隆形成率，Hoechst33342实验和细胞流式术检测各组细胞凋亡率，Western blotting法检测各组细胞中cleaved Caspase-3、Bax和Bcl-2蛋白表达水平，计算Bax/Bcl-2比值。采用活性氧（ROS）检测试剂盒检测细胞中ROS水平。结果：随着索拉菲尼浓度的升高，HepG2和HepG2R细胞活力逐渐降低，耐药细胞HepG2R对索拉菲尼的半数抑制浓度（IC₅₀）值是HepG2的4.5倍（P<0.05）。随着PLB浓度升高和作用时间延长，耐药细胞对索拉菲尼敏感性升高。与对照组、索拉菲尼组和PLB组比较，联合组耐药细胞克隆形成率降低（P<0.05或P<0.01）。Hoechst33342实验，对照组细胞核淡染，索拉菲尼组和PLB组细胞核少部分浓染、明亮；联合组细胞核大部分浓染，细胞核染色质固缩、明亮。流式细胞术和Western blotting法检测，与对照组、索拉菲尼组和PLB组比较，联合组细胞凋亡率明显升高（P<0.05或P<0.01），细胞中cleaved Caspase-3蛋白表达水平和Bax/Bcl-2比值明显升高（P<0.05或P<0.01）。联合组细胞中ROS水平明显高于其他各组（P<0.05或P<0.01）。结论：PLB能改善肝癌对索拉菲尼的耐药情况，其机制可能与升高耐药细胞中ROS水平有关。

关键词: 白花丹素, 肝肿瘤, 索拉菲尼, HepG2细胞, 细胞增殖, 细胞凋亡

Abstract: Objective: To explore the effects of plumbagin (PLB) on the proliferation and apoptosis of hepatocellular carcinoma hepG2R cells resistant to sorafenib,and to clarify its mechanism.Methods: The hepatocellular carcinoma cells hepG2 were cultured in vitro and the models of sorafenib-resistant HepG2R cells were set up. MTT assay was used to identify the resistance factor and screen the concentration and time of drug. According to the results, the concentrations of sorafenib and PLB were confirmed as 5 μmol·L^-1 and 2 μmol·L ^-1. The action time in various groups was 48 h. The HepG2R cells were randomly divided into control group, sorafenib (5 μmol·L^-1) group, PLB(2 μmol·L^-1) group, sorafenib (5 μmol·L^-1) combined PLB(2 μmol·L^-1) group (combination group). MTT assay was used to detect the cell vitality in various groups. The clone formation rates of cells in various groups were detected by clone formation assay. The apoptotic rates of cells in various groups were determined with Hoechst33342 assay and flow cytometry(FCM). The expression levels of cleaved Caspase-3,Bax and Bcl-2 proteins in the cells in various groups were examined by Western blotting method,and the Bax/Bcl-2 ratio was calculated. The reactive oxygen (ROS) levels in the cells in various groups were detected by ROS detector kit.Results: As the increasing of concentrations of sorafenib, the cell vitalities of HepG2 and HepG2R cells were gradually decreased;the IC₅₀ of sorafenib-resistant HepG2R cells was 4.5 times as much as HepG2 cells (P<0.05). The sensitivities of sorafenib resistant HepG2R cells were increased with the increasing of PLB concentrations and prolongation of time. Compared with control group, sorafenib group and PLB group, the clone formation rate of the cells in combination group was decreased(P<0.05 or P<0.01). The Hoechst 33342 assay results showed the nuclei were lightly stained; a few of the nuclei in sorafenib group and PLB group were strongly stained and bright; while in combination group, the nuclei were mostly stained and chromatin was condensed and bright.The FCM results showed that compared with control group, sorafenib group and PLB group, the apoptotic rate of cells in combination group was significantly increased (P< 0.05 or P< 0.01). The Western blotting results showed that the expression level of cleaved Caspase-3 in the cells and the Bax/Bcl-2 ratio in combination group were increased significantly (P<0.05 or P<0.01). The ROS level in the cells in combination group was higher than those in the other groups(P< 0.05 or P<0.01).Conclusion: PLB can improve the resistance of hepartocellular carcinoma to sorafenib and the mechanism may be related to increasing the ROS level.

Key words: plumbagin, liver neoplasms, sorafenib, HepG2, cell proliferation, apoptosis

中图分类号:

R735.7

朱德强, 陈学军. 白花丹素对肝癌索拉菲尼耐药细胞HepG2R增殖和凋亡的影响及其机制[J]. 吉林大学学报(医学版), 2018, 44(06): 1223-1229.

ZHU Deqiang, CHEN Xuejun. Effects of plumbagin on proliferation and apoptosis of hepatocellular carcinoma HepG2R cells resistant to sorafenib and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2018, 44(06): 1223-1229.

参考文献

[1] Chen X, Ma Z, Huang Y, et al. Multiparametric MR diffusion-weighted imaging for monitoring the ultra-early treatment effect of sorafenib in human hepatocellular carcinoma xenografts[J].J Magn Reson Imaging,2017,46(1):248-256.
[2] 史蕤, 李然, 赵冰清. 索拉菲尼血药浓度与疗效和不良反应的关系以及在肝细胞癌治疗应用[J]. 中国临床药理学杂志,2015,31(17):1793-1795.
[3] 刘正端, 赵智伟, 陈亚娟. 白花丹素下调FoxM1对食管鳞癌细胞增殖、凋亡的影响[J]. 药学学报, 2017,52(4):563-568.
[4] 刘方方, 谢军平, 历风元. 白花丹素体内外抗Lewis肺癌的效果及机制研究[J]. 中国药学杂志,2017, 52(7):581-586.
[5] Na S, Zhang J, Zhou X, et al. Plumbagin-mediating GLUT1 suppresses the growth of human tongue squamous cell carcinoma[J]. Oral Dis, 2018,24(6):920-929.
[6] Pan M, Li W, Yang J, et al. Plumbagin-loaded aptamer-targeted poly D,L-lactic-co-glycolic acid-b-polyethylene glycol nanoparticles for prostate cancer therapy[J]. Medicine (Baltimore), 2017, 96(30):e7405.
[7] Checker R, Gambhir L, Sharma D, et al. Plumbagin induces apoptosis in lymphoma cells via oxidative stress mediated glutathionylation and inhibition of mitogen-activated protein kinase phosphatases (MKP1/2)[J]. Cancer Lett,2015,357(1):265-278.
[8] Chao CC, Hou SM, Huang CC, et al. Plumbagin induces apoptosis in human osteosarcoma through ROS generation, endoplasmic reticulum stress and mitochondrial apoptosis pathway[J]. Mol Med Rep, 2017,16(4):5480-5488.
[9] 张晓媛, 黄鹏, 李燕京. 晚期原发性肝癌治疗的研究进展[J]. 现代肿瘤医学, 2017, 25(10):1655-1659.
[10] Pan ST, Qin Y, Zhou ZW, et al. Plumbagin induces G2/M arrest, apoptosis, and autophagy via p38 MAPK-and PI3K/Akt/mTOR-mediated pathways in human tongue squamous cell carcinoma cells[J]. Drug Des Devel Ther, 2015, 9:1601-1626.
[11] Khaw AK, Sameni S, Venkatesan S, et al. Plumbagin alters telomere dynamics, induces DNA damage and cell death in human brain tumour cells[J]. Mutat Res Genet Toxicol Environ Mutagen, 2015, 793:86-95.
[12] Zhang W, Cheng L, Hou Y, et al. Plumbagin protects against spinal cord injury-induced oxidative stress and inflammation in wistar rats through Nrf-2 upregulation[J]. Drug Res (Stuttg), 2015, 65(9):49-59.
[13] Casadei Gardini A, Faloppi L, Aprile G, et al. Multicenter prospective study of angiogenesis polymorphism validation in HCC patients treated with sorafenib. An innovate study protocol[J]. Tumori, 2017, 15(6):32-41.
[14] Zhang N, Liu L, Dou Y, et al. Glycogen synthase kinase-3β antagonizes ROS-induced hepatocellular carcinoma cell death through suppression of the apoptosis signal-regulating kinase 1[J]. Med Oncol, 2016, 33(7):60.
[15] Seol HS, Lee SE, Song JS, et al. Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production[J]. Cancer Lett, 2016, 382(2):157-165.
[16] Liu J, Wei X, Wu Y, et al. Giganteaside D induces ROS-mediated apoptosis in human hepatocellular carcinoma cells through the MAPK pathway[J]. Cell Oncol (Dordr), 2016, 39(4):333-342.
[17] Zheng R, You Z, Jia J, et al. Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells[J]. Mol Med Rep, 2016, 13(2):1570-1576.
[18] Simo A. Hepatocellular carcinoma:portuguese data in chronic hepatitis B patients on antiviral therapy and treatment results with sorafenib[J]. GE Port J Gastroenterol, 2016, 23(5):231-232.
[19] Jia D, Lu W, Zhang X, et al. Calf spleen extractive injection (CSEI), a small peptides enriched extraction, induces human hepatocellular carcinoma cell apoptosis via ROS/MAPKs dependent mitochondrial pathway[J]. J Pharmacol Sci, 2016, 132(2):122-130.
[20] Jian KL, Zhang C, Shang ZC, et al. Eucalrobusone C suppresses cell proliferation and induces ROS-dependent mitochondrial apoptosis via the p38 MAPK pathway in hepatocellular carcinoma cells[J]. Phytomedicine, 2017, 25:71-82.
[21] Hu Z, Dong N, Lu D, et al. A positive feedback loop between ROS and Mxi1-0 promotes hypoxia-induced VEGF expression in human hepatocellular carcinoma cells[J]. Cell Signal, 2017, 31(9):79-86.
[22] 王帅,尚志梅,黄利敏,等.表没食子儿茶素没食子酸酯对肝癌细胞增殖的抑制作用研究[J].解放军医学杂志,2016,41(3):199-203.
[23] 刘亮,刘江惠,陈乐彤等.青蒿琥酯对肝癌细胞生长的抑制作用及其机制[J].解放军医学杂志,2018,43(7):594-599.