[肿瘤坏死因子-α与溴隐亭联合治疗对HepG2多药耐药亚细胞系化疗敏感性的研究]

[Study on sensitivity to chemotherapy by combination therapy with tumor necrosis factor-alpha and bromocriptine in the multidrug resistant subcell line of HepG2].

作者信息

Ding Lei, Chen Xiao-ping, Zhang Zhi-wei, Jing Kai, Cao Bin, Zhu Peng, Li Jie, Zhang De-yong

机构信息

Hepatic Surgery Center, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.

出版信息

Zhonghua Wai Ke Za Zhi. 2006 Dec 1;44(23):1644-7.

PMID:17359700

Abstract

OBJECTIVE

To investigate the change of chemosensitivity of hepatocarcinoma cell line (HepG(2)/ADM) after treated by bromocriptine (BCT) combination with human tumor necrosis factor-alpha (TNF-alpha).

METHODS

Firstly, TNF-alpha gene was transfected into HepG(2)/ADM cell line by liposome to establish a cell model expressing the TNF-alpha protein stably. All experiments were divided into four groups and named blank control group (group A), drug resistant group HepG(2)/ADM (group B), TNF-alpha gene group HepG(2)/ADM/TNF (group C) and BCT group (group D) respectively. And group D came from group C treated with BCT simultaneously. MTT assay was tested to detect the sensitivity to ADM of each group and Rhodamine 123 (Rh123) was applied to test the function of P-gp by flow cytometric analysis (FCM). MDR associated genes and proteins and PKC-alpha protein were detected by immunohistochemistry (IHC), Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) methods, respectively. The expression and the apoptosis rate of Bcl-2 in the hepatocarcinoma cells were detected by FCM.

RESULTS

There was significant difference between group C and D in the rate of reversing resistance and the intracellular Rho123 accumulation (P < 0.01). MDR1 mRNA and P-gp protein expression in group C and D were low similar to that in group A, but no difference could be found among them (P > 0.05). As we found that PKC-alpha protein expression was downregulated in group D but Bcl-2 protein expression was downregulated in group C, and there was significant difference compared to other groups. The apoptosis rate of hepatocarcinoma cells was much higher in group D than that in group C (P < 0.01) with FCM, but similar to group A (P > 0.05).

CONCLUSIONS

Synergistic effect of BCT and TNF-alpha on reversing hepatocellular carcinoma multidrug resistance could enhance the susceptibility of HepG(2)/ADM cells to cytotoxic drugs.

摘要

目的

探讨溴隐亭（BCT）联合人肿瘤坏死因子-α（TNF-α）处理后肝癌细胞系（HepG(2)/ADM）化疗敏感性的变化。

方法

首先，通过脂质体将TNF-α基因转染至HepG(2)/ADM细胞系，建立稳定表达TNF-α蛋白的细胞模型。所有实验分为四组，分别命名为空白对照组（A组）、耐药组HepG(2)/ADM（B组）、TNF-α基因组HepG(2)/ADM/TNF（C组）和BCT组（D组）。D组来自同时用BCT处理的C组。采用MTT法检测各组对阿霉素（ADM）的敏感性，应用罗丹明123（Rh123）通过流式细胞术（FCM）检测P-糖蛋白（P-gp）的功能。分别采用免疫组织化学（IHC）、蛋白质印迹法（Western blot）和逆转录聚合酶链反应（RT-PCR）方法检测多药耐药相关基因和蛋白以及蛋白激酶C-α（PKC-α）蛋白。通过FCM检测肝癌细胞中Bcl-2的表达及凋亡率。

结果

C组和D组在耐药逆转率和细胞内Rho123蓄积方面存在显著差异（P<0.01）。C组和D组中多药耐药基因1（MDR1）mRNA和P-gp蛋白表达与A组相似且较低，但三组之间无差异（P>0.05）。我们发现D组中PKC-α蛋白表达下调，而C组中Bcl-2蛋白表达下调，与其他组相比存在显著差异。FCM检测显示，D组肝癌细胞凋亡率远高于C组（P<0.01），但与A组相似（P>0.05）。