Cytotoxic effect of Ziziphus Spina-Christi extract alone and in combination with doxorubicin on breast cancer cells

E. El-Shafey, E. Elsherbiny
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Abstract

Ziziphus Spina-Christi (L.) (ZSC) is a traditional Arabian medicinal plant used to treat inflammatory symptoms, swellings and pain since long. Triple negative breast cancer (TNBC) is a form of cancer with a poor prognosis owing to the paucity of therapy alternatives. Two of the most critical pathways of TNBC development are Wnt/β-catenin signaling and autophagy. In the present study, we intended to identify the possible mechanisms of the cytotoxic effects mediated by ZSC extract on MDA-MB-231 breast cancer cells and to improve the efficacy of DOX in combination with ZSC. The MTT test was used to estimate cell viability and IC50 values. Apoptosis was detected using AnnexinV-FITC detection kit. ELISA was used to measure caspase-3 levels. Cell cycle and the level of autophagosome marker LC3-II were analysed using flow cytometry. Acidic vesicular organelle (AVOs) formation was observed by fluorescence microscopy. Real-time PCR was used to monitor changes in gene expression of β-catenin and autophagic adapter NBR1. It was shown that ZSC treatment dose-dependently inhibited MDA-MB-231 cell viability and induced apoptosis with accompanying elevation of caspase-3 level. Besides ZSC caused a significant elevation in LC3II level and downregulation of NBR1 gene expression with subsequent downregulation of β-catenin gene expression, indicating the inhibition of the oncogenic Wnt pathway. ZSC and DOX combination had synergistic cytotoxic effect by more effective suppression of Wnt pathway and induction of apoptosis and autosis. Keywords: apoptosis, autophagic adapter NBR1, autophagosome marker LC3-II, breast cancer cells, DOX, Wnt/β-catenin signaling, Ziziphus Spina-Christi
刺五加提取物单独或与多柔比星联合使用对乳腺癌细胞的细胞毒作用
Ziziphus Spina-Christi(L.)(ZSC)是一种传统的阿拉伯药用植物,长期以来用于治疗炎症、肿胀和疼痛。三阴性乳腺癌(TNBC)是一种预后较差的癌症,原因是缺乏替代疗法。Wnt/β-catenin信号传导和自噬是TNBC发展过程中最关键的两个途径。在本研究中,我们旨在确定 ZSC 提取物对 MDA-MB-231 乳腺癌细胞产生细胞毒性作用的可能机制,并提高 DOX 与 ZSC 联用的疗效。实验采用 MTT 试验估算细胞活力和 IC50 值。使用 AnnexinV-FITC 检测试剂盒检测细胞凋亡。ELISA 用于测量 Caspase-3 的水平。使用流式细胞术分析细胞周期和自噬体标记物 LC3-II 的水平。通过荧光显微镜观察酸性囊泡细胞器(AVOs)的形成。实时 PCR 被用来监测β-catenin 和自噬适配体 NBR1 基因表达的变化。结果表明,ZSC能剂量依赖性地抑制MDA-MB-231细胞的活力,并诱导细胞凋亡,同时伴有caspase-3水平的升高。此外,ZSC还能显著提高LC3II水平,下调NBR1基因表达,进而下调β-catenin基因表达,表明其抑制了致癌的Wnt通路。ZSC和DOX联用具有协同细胞毒作用,能更有效地抑制Wnt通路,诱导细胞凋亡和自噬。关键词:细胞凋亡;自噬适配体NBR1;自噬体标志物LC3-II;乳腺癌细胞;DOX;Wnt/β-catenin信号传导;酸枣仁碱
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