五味子甲素调控有丝分裂逆转卵巢癌细胞紫杉醇耐药的机制探究

Exploring the Mechanism by Which Schisandrin A Modulates Mitosis to Reverse Taxol Resistance in Ovarian Cancer Cells

关键词:  卵巢癌  五味子甲素  紫杉醇  化疗敏感性  有丝分裂

Key words:  ovarian cancer  schizandrin A  taxol  chemosensitivity  mitosis

摘要:

目的 探讨五味子甲素增强卵巢癌紫杉醇耐药株（A2780/TAX）对紫杉醇化疗敏感性的作用及机制，为卵巢癌的减毒增敏治疗提供新思路。方法 体外培养A2780/TAX细胞，分为对照组、紫杉醇组、五味子甲素组、五味子甲素+紫杉醇组进行干预；噻唑蓝溴化四唑（MTT）法检测细胞活力；流式细胞术Annexin V-FITC/ PI双染检测细胞凋亡；流式细胞术检测细胞周期；克隆形成实验评价增殖能力；划痕实验评价迁移能力；蛋白免疫印迹检测AIF、XIAP、PARP、Cleaved PARP、Caspase 3、Cleaved Caspase 3、Caspase 7、Cleaved Caspase 7的蛋白表达水平；转录组测序分析差异基因；免疫荧光染色法进行细胞骨架和细胞核染色;RT-qPCR检测CHEK1、CHEK2、CENPA、TTK、PRC1、ANLN的mRNA水平。结果 五味子甲素与低浓度紫杉醇联用显著抑制A2780/TAX细胞活力和增殖能力；细胞凋亡率显著高于单独用药组；蛋白免疫印迹显示，药物联用显著降低AIF、XIAP、PARP、Caspase 3、Caspase 7蛋白表达量，提高Cleaved PARP、Cleaved Caspase 3和Cleaved Caspase 7蛋白水平；划痕实验表明药物联用组抑制细胞迁移；转录组测序显示正常组与联用组有1515个上调基因，1582个下调基因，GO和KEGG富集分析表明差异基因主要富集在有丝分裂细胞周期、DNA代谢等生物过程及相关信号通路。细胞周期检测显示联用将细胞阻滞在G2/M期。对富集到“有丝分裂细胞周期”生物过程的基因进行聚类分析，发现大部分基因聚类在“有丝分裂”集群。免疫荧光染色显示联用诱导细胞发生多核现象。RT-qPCR结果表明药物联用能显著降低有丝分裂相关基因mRNA水平。表明结论 五味子甲素与低浓度紫杉醇联用可抑制A2780/TAX细胞活力和增殖能力，诱导凋亡，抑制迁移，具体机制可能与调控A2780/TAX细胞有丝分裂有关。

Abstract:

OBJECTIVE To investigate the effects and mechanisms of schizandrin A in enhancing the chemosensitivity of taxol-resistant ovarian cancer cells (A2780/TAX) to taxol, providing novel insights into toxicity reduction and efficacy enhancement for ovarian cancer treatment. METHODS A2780/TAX cells were cultured in vitro and divided into four groups: control, taxol, schizandrin A, and schizandrin A+taxol. Cell viability was assessed via MTT assay. Apoptosis was detected by Annexin V-FITC/PI double staining using flow cytometry. Cell cycle distribution was analyzed by flow cytometry. The proliferative capacity was evaluated via colony formation assay and the migratory ability was tested by wound healing assay. The protein expression levels of AIF, XIAP, PARP, Cleaved PARP, Caspase 3, Cleaved Caspase 3, Caspase 7, and Cleaved Caspase 7 were measured via western blotting assay. Transcriptome sequencing was performed to identify differentially expressed genes and cytoskeletal and nuclear morphology was visualized by immunofluorescence staining. The mRNA levels of CHEK1, CHEK2, CENPA, TTK, PRC1 and ANLN were detected by RT-qPCR. RESULTS schizandrin A combined with low-dose taxol significantly inhibited A2780/TAX cell viability and proliferation. The apoptosis rate in the combination group was markedly higher than in monotherapy groups. The results of western blotting revealed that combination therapy downregulated AIF, XIAP, PARP, Caspase 3, and Caspase 7 expression while elevating Cleaved PARP, Cleaved Caspase 3, and Cleaved Caspase 7 levels. The combination group exhibited suppressed cell migration in wound healing assays. Transcriptome sequencing identified 1,515 upregulated and 1,582 downregulated DEGs between control and combination groups. GO and KEGG enrichment analyses indicated that DEGs were primarily associated with mitotic cell cycle regulation and DNA metabolic processes. Cell cycle analysis demonstrated G2/M phase arrest in the combination group. Clustering analysis of genes enriched in the biological process of “mitotic cell cycle” showed that the majority of these genes were clustered within the “mitosis” category. Immunofluorescence staining revealed multinucleation induced by the combination treatment and the results of RT-qPCR indicated that the combination of drugs can significantly reduce the mRNA levels of genes related to mitosis. CONCLUSION schizandrin A synergizes with low-dose taxol to inhibit A2780/TAX cell viability, proliferation, and migration while promoting apoptosis. The underlying mechanism may involve dysregulation of mitotic processes in A2780/TAX cells.