摘要
目的:本研究旨在通过黑色素瘤耐药细胞株的构建以探究ATP结合盒B亚家族1转运蛋白(ABCB1)基因高表达所驱动的黑色素瘤A375细胞中达卡巴嗪耐药性及其靶向逆转策略。方法:通过逐步提高达卡巴嗪浓度并对黑色素瘤A375细胞进行长期诱导以构建A375黑色素瘤达卡巴嗪耐药细胞株(A375-DacR)。利用CCK-8法和流式细胞凋亡实验评估耐药细胞株耐药水平。通过qPCR及Western Blot检测A375细胞及A375耐药细胞(A375-DacR)中ABCB1基因及其编码蛋白P-gp的表达水平。通过流式细胞术检测细胞周期分布。利用ABCB1靶向抑制剂处理达卡巴嗪耐药细胞株,评估耐药性逆转效果。结果:成功构建黑色素瘤A375达卡巴嗪耐药细胞株(A375-DacR),耐药指数>5;A375-DacR细胞中ABCB1基因的mRNA水平及蛋白表达显著上调;相较于A375亲本细胞,A375-DacR细胞出现了一定程度的G_(0)/G_(1)期阻滞;靶向抑制ABCB1后,其编码蛋白P-gp表达降低,A375-DacR细胞在达卡巴嗪作用下存活率及克隆形成率下降。结论:经长期诱导后A375-DacR细胞群体出现了向静止期(G0期)的转变以适应达卡巴嗪治疗的压力。此外,ABCB1基因及其编码蛋白P-gp高表达是导致黑色素瘤A375耐药细胞耐药性形成的核心驱动因素,抑制ABCB1后A375-DacR细胞对达卡巴嗪敏感性恢复。本研究表明靶向ABCB1可能是治疗黑色素瘤达卡巴嗪耐药的有效途径。
Objective The aim of this study was to explore the resistance of dacarbazine in melanoma A375 cells driven by the high expression of ABCB1 gene and its targeted reversal strategy through the construction of melanoma drug-resistant cell lines.Methods A375 melanoma dacarbazine resistant cell line(A375-DacR)was constructed by gradually increasing Dacarbazine concentration and long-term induction of melanoma A375 cells.CCK-8 assay and flow cytometry were used to evaluate the drug resistance level of drug-resistant cell lines.The expression levels of ABCB1 gene and its coding protein P-gp in A375 cells and A375-DACR cells were detected by qPCR and Western Blot.The cell cycle distribution was detected by flow cytometry.Dacarbazine resistant cell lines were treated with ABCB1-targeted inhibitors to evaluate the effect of resistance reversal.Results The dacarbazine-resistant A375 melanoma cell line(A375-DacR)was successfully constructed with a resistance index greater than 5.The mRNA and protein expression levels of ABCB1 in A375-DacR cells were significantly upregulated,highlighting its pivotal role in the development of drug resistance.Compared to parental A375 cells,A375-DacR cells exhibited a notable G_(0)/G_(1) phase arrest.Following targeted inhibition of ABCB1,the expression of its encoded protein P-gp was reduced,leading to a decreased survival rate and clonogenic capacity of A375-DacR cells upon dacarbazine treatment.Conclusion Following long-term induction,the A375-DacR cell population exhibited a transition into the quiescent phase(G0 phase)as an adaptive response to the selective pressure of dacarbazine treatment.Moreover,the high expression of the ABCB1 gene and its encoded protein P-gp was identified as a key driver of dacarbazine resistance in melanoma A375-resistant cells.Inhibition of ABCB1 effectively restored the sensitivity of A375-DacR cells to dacarbazine.This study suggests that targeting ABCB1 may serve as an effective strategy for overcoming dacarbazine resistance in melanoma therapy.
作者
廖羿龙
邓远雄
LIAO Yilong;DENG Yuanxiong(School of Pharmaceutical Sciences,Hunan Normal University,Changsha 410013)
出处
《晓庄学院学报(医学版)》
2025年第3期8-14,共7页
Journal of Hunan Normal University(Medical Sciences)
基金
湖南省自然科学基金“基于SCFA-GPR41/43功能轴研究白背三七多糖治疗2型糖尿病的靶点和机制”(2020JJ4437)
