双硫仑联合二价铜离子诱导铜死亡抑制肝癌细胞Hep3B增殖、迁移和侵袭

doi:10.3969/j.issn.1674-5671.2024.04.06

摘要/Abstract

摘要： 目的探讨双硫仑（disulfiram，DSF）联合二价铜离子（Cu²⁺）对肝癌细胞增殖、迁移和侵袭的影响及其作用机制。方法体外培养肝癌Hep3B细胞，分别采用DSF（30 nmol/L）溶液、Cu²⁺（1 μmol/L）溶液和铜螯合剂四硫代钼酸铵（ammonium tetrathiomolybdate Ⅵ，ATTM）（30 nmol/L）溶液单独或联合干预，以二甲基亚砜（dimethyl sulfoxide，DMSO）（30 nmol/L）作用的细胞为对照组。分别采用CCK⁃8实验、划痕实验和Transwell小室实验检测细胞的增殖能力、迁移能力和侵袭能力；采用免疫荧光实验检测细胞中铜死亡相关蛋白二氢硫辛酰胺⁃S⁃乙酰转移酶（dihydrolipoamide S⁃acetyltransferase，DLAT）和铁氧还蛋白1（ferredoxin 1，FDX1）的表达。结果与对照组相比，DSF、Cu²⁺单独或联合干预后Hep3B细胞的增殖、迁移和侵袭能力均下降（均P<0.05），其中DSF+Cu²⁺联合干预的细胞增殖、迁移和侵袭能力下降更加显著（均P<0.0001）。在DSF联合Cu2+的基础上加入ATTM后逆转了DSF联合Cu²⁺对Hep3B细胞增殖、迁移和侵袭的抑制作用，其增殖、迁移以及侵袭能力较DSF+Cu²⁺组增强（均P<0.05）。与对照组相比，DSF、Cu²⁺单独干预后铜死亡相关蛋白DLAT和FDX1的荧光强度改变并不明显，但Cu²⁺联合DSF后DLAT蛋白的荧光强度增加而FDX1蛋白的荧光强度减弱，加入ATTM后则逆转了DLAT和FDX1蛋白的表达趋势。结论 DSF联合Cu2+能抑制肝癌细胞Hep3B增殖、迁移和侵袭能力，其作用机制可能是通过诱导铜死亡的发生实现。

关键词: 肝癌, Hep3B细胞, 双硫仑, 铜死亡, 增殖, 迁移, 侵袭

Abstract: Objective To investigate the effects of disulfiram (DSF) combined with Cu²⁺ on the proliferation, migration and invasion of hepatoma Hep3B cells and the underlying mechanism. Methods Hepatoma Hep3B cells were cultured in vitro. DSF (30 nmol/L) solution, Cu²⁺ (1 μmol/L) solution and copper chelating agent ammonium tetrathiomolybdate Ⅵ (ATTM) (30 nmol/L) solution were used individually or combinedly for intervention. The cells treated with dimethyl sulfoxide (DMSO) (30 nmol/L) were used as the control group. CCK⁃8, scratch test and Transwell cell test were used to detect the proliferation, migration and invasion ability of cells. The expression of copper death related proteins dihydrolipoamide S⁃acetyltransferase (DLAT) and ferredoxin 1 (FDX1) were detected by immunofluorescence assay. Results Compared with the control group, the proliferation, migration and invasion ability of Hep3B cells were significantly decreased after DSF or Cu²⁺ or DSF+Cu²⁺ combined intervention (all P<0.05), and the decrease was more significant in DSF+Cu²⁺ combined intervention (all P<0.0001). After addition of DSF combined with Cu²⁺ to ATTM, the inhibitory effects of DSF combined with Cu²⁺ on the proliferation, migration and invasion of Hep3B cells were reversed, and the proliferation, migration and invasion ability cells were enhanced compared with DSF+Cu²⁺group (all P<0.05). Compared with the control group, the fluorescence intensity of copper death related proteins DLAT and FDX1 showed no significant increase after DSF or Cu²⁺ intervention, but the fluorescence intensity of DLAT protein increased while that of FDX1 protein decreased after DSF+Cu²⁺ combined intervention, and the expression trend of DLAT and FDX1 proteins was reversed after the addition of ATTM. Conclusions DSF combined with Cu²⁺ can inhibit the proliferation, migration and invasion of Hep3B cells, probably through the induction of cuproptosis.

Key words: Hepatoma, Hep3B cells, Disulfiram, Cuproptosis, Proliferation, Migration, Invasion

中图分类号:

R735.7

唐敏, 黄娟婵, 韦杏雯, 罗雪文, 赵伟. 双硫仑联合二价铜离子诱导铜死亡抑制肝癌细胞Hep3B增殖、迁移和侵袭[J]. 中国癌症防治杂志, 2024, 16(4): 417-423.

TANG Min, HUANG Juanchan, WEI Xingwen, LUO Xuewen, ZHAO Wei . Effects of disulfiram combined with Cu²⁺ induced cuproptosis on proli-feration,migration and invasion of hepatoma Hep3B cells [J]. Chinese Journal of Oncology Prevention and Treatment, 2024, 16(4): 417-423.

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