细胞外腺苷诱导人肝癌 HepG2 细胞凋亡过程中 NF-κB 的激活作用。

Involvement of NF-kappaB activation in the apoptosis induced by extracellular adenosine in human hepatocellular carcinoma HepG2 cells.

机构信息

Department of Gastroenterology, Second Affiliated Hospital, Shantou University Medical College, Shantou, China.

出版信息

Biochem Cell Biol. 2010 Aug;88(4):705-14. doi: 10.1139/O10-008.

PMID:20651843

Abstract

Adenosine can exhibit cytotoxic activity in vivo and in vitro, though its mechanisms are still uncertain. In this study, we investigated the adenosine-mediated apoptotic signaling pathway and the role of NF-kappaB in human hepatocellular carcinoma HepG2 cells. HepG2 cells were treated with different concentrations of adenosine for 12-48 h, and the effect of adenosine on cell proliferation was evaluated by MTT assay. The cytotoxicity of adenosine alone or in combination with an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was also evaluated by MTT assay and the mode of cell death was detected by Hoechst 33342 staining. Cell cycle progress was performed by flow cytometry with PI staining. The protein expressions of Bcl-2, p53, NF-kappaB subunit p65, and caspase-3 were assayed by Western blot. Caspase-3 activity was measured by spectrophotomteric assay. The results showed that adenosine significantly reduced the viability of HepG2 cells in a dose- and time-dependent manner, with IC 50 (24 and 48 h) of 2.52 and 1.89 mmol x L(-1), respectively. The apoptotic index (percentage of sub-G1 phase) of HepG2 cells in adenosine treatment alone for 12 and 24 h or in combination with PDTC were 8.30%, 22.32% and 20.18%, 30.89%, respectively. All of them were higher than that in the control group (0.81%, p < 0.01). The characteristic changes of cell apoptosis (chromatin condensation and sub-G1 peak) were observed under fluorescent microscopy and flow cytometry. We also found that the apoptotic process triggered by adenosine was involved in G0-G1 cell-cycle arrest, enhanced the activity of caspase-3, upregulated p53 and NF-kappaB p65 expression, and downregulated Bcl-2 expression. Inhibition of NF-kappaB by PDTC decreased NF-kappaB p65 expression, enhanced cell apoptosis ratio, and increased caspase-3 activity. NF-kappaB may play an anti-apoptosis role in adenosine-induced HepG2 cytotoxicity.

摘要

腺嘌呤在体内和体外都能表现出细胞毒性活性，但其机制尚不清楚。在这项研究中，我们研究了腺嘌呤介导的凋亡信号通路以及 NF-κB 在人肝癌 HepG2 细胞中的作用。用不同浓度的腺嘌呤处理 HepG2 细胞 12-48 小时，通过 MTT 法评估腺嘌呤对细胞增殖的影响。还通过 MTT 法评估了腺嘌呤单独或与 NF-κB 抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）联合使用的细胞毒性，并用 Hoechst 33342 染色检测细胞死亡方式。用碘化丙啶（PI）染色通过流式细胞术检测细胞周期进程。用 Western blot 法测定 Bcl-2、p53、NF-κB 亚基 p65 和 caspase-3 的蛋白表达。用分光光度计法测定 caspase-3 活性。结果表明，腺嘌呤以剂量和时间依赖的方式显著降低 HepG2 细胞的活力，IC 50（24 和 48 小时）分别为 2.52 和 1.89 mmol x L(-1)。腺嘌呤单独处理 12 和 24 小时或与 PDTC 联合处理时 HepG2 细胞的凋亡指数（亚 G1 期的百分比）分别为 8.30%、22.32%和 20.18%、30.89%，均高于对照组（0.81%，p < 0.01）。荧光显微镜和流式细胞术观察到细胞凋亡的特征变化（染色质浓缩和亚 G1 峰）。我们还发现，腺嘌呤引发的凋亡过程涉及 G0-G1 细胞周期停滞，增强了 caspase-3 的活性，上调了 p53 和 NF-κB p65 的表达，下调了 Bcl-2 的表达。PDTC 抑制 NF-κB 降低了 NF-κB p65 的表达，增强了细胞凋亡比例，增加了 caspase-3 的活性。NF-κB 可能在腺嘌呤诱导的 HepG2 细胞毒性中发挥抗凋亡作用。