Background:Small-cell lung cancer (SCLC) has a poor prognosis because it is often diagnosed after it has spread and develops multi-drug resistance. Epibrassinolide (EB) is a plant steroid hormone with widespread distribution and physiological effects. In plants, EB-activated gene expression occurs via a GSK-mediated signaling pathway, similar toWnt-β-catenin signaling in animal cells that is elevated in cancer cells.Methods:This mechanistic parallel prompted investigations of the molecular interactions of EB on drug-sensitive (H69) and multi-drug-resistant (VPA) SCLC cells. Cellular and molecular investigations were performed.Results:Pharmacologic interactions between EB and theWntsignaling inhibitors IGC-011 and PRI-724 were determined by the combination index method and showed antagonism, indicating that EB acts on the same pathway as these inhibitors. Following incubation of drug-sensitive and drug-resistant SCLC cells with EB, there was a reduction in β-catenin (e.g., 3.8 to 0.7 pg/µg protein), accompanied by a reduction in β-catenin promoter activity, measured by firefly luciferase-coupled promoter element transfection. Cellular β-catenin concentration is regulated by the active form of GSK3β. InWntsignaling, active GSK3β is converted to inactive pGSK3β, thereby increasing the concentration of β-catenin. After incubation of SCLC cells with EB, there was a reduction in the inactive form (pGSK3β) and a relative increase in the active form (GSK3β). In vitro enzyme assays showed that EB did not inhibit purified GSK3β, but there was non-competitive inhibition when SCLC cell extracts were used as the source of enzyme. This indirect inhibition by EB indicates that it may act on theWntpathway by blocking the phosphorylation of GSK3β. The protein levels of three SCLC tumor markers, namely, NSE, CAV1, and MYCL1, were elevated in drug-resistant SCLC cells. EB incubation led to a significant reduction in the levels of the three markers. Two major effects of EB on SCLC cells are the promotion of apoptosis and the reversal of drug resistance. Transcriptional analyses showed that after exposure of SCLC cells to EB, there were increases in the expression of genes encoding apoptotic inducers (e.g.,BAXandFAS) and effectors (e.g.,CASP3) and reductions in the expression of genes encoding apoptosis inhibitors (e.g.,survivin). PGP1 and MRP1, two membrane efflux pumps expressed in SCLC cells, were elevated in drug-resistant cells, but EB incubation did not affect these protein levels. Cellular assays of drug efflux by PGP1 showed an increase in drug-resistant cells, but EB did not alter efflux activity. Following exposure to human liver microsomes, EB was metabolized by NADPH-dependent oxidation and UDPG-dependent glucuronidation, as evidenced by the elimination of EB cytotoxicity against SCLC cells.Conclusions:Taken together, these data indicate that EB, a steroid hormone in plants consumed in the human diet, is pharmacologically active in drug-sensitive and drug-resistant SCLC cells in theWntsignaling pathway, alters apoptotic gene expression, and is a substrate for microsomal modifications.
背景：小细胞肺癌（SCLC）预后较差，因其常在扩散后确诊并易产生多药耐药性。表油菜素内酯（EB）是一种植物甾体激素，分布广泛且具有多种生理效应。在植物中，EB通过GSK介导的信号通路激活基因表达，该通路类似于动物细胞中在癌细胞中异常活跃的Wnt-β-catenin信号通路。 方法：基于这种机制相似性，本研究探讨了EB对药物敏感型（H69）与多药耐药型（VPA）SCLC细胞的分子作用机制，并开展了细胞与分子水平实验。 结果：通过联合指数法测定EB与Wnt信号抑制剂IGC-001和PRI-724的药理相互作用显示拮抗效应，表明EB与这些抑制剂作用于同一通路。药物敏感型与耐药型SCLC细胞经EB孵育后，β-catenin含量降低（如从3.8降至0.7 pg/µg蛋白），萤火虫荧光素酶报告基因检测显示其启动子活性同步下降。细胞β-catenin浓度受GSK3β活性形式调控：在Wnt信号通路中，活性GSK3β会转化为非活性pGSK3β，从而提升β-catenin浓度。EB孵育后，SCLC细胞中非活性形式（pGSK3β）减少，活性形式（GSK3β）相对增加。体外酶学实验表明，EB对纯化的GSK3β无抑制作用，但以SCLC细胞提取物为酶源时出现非竞争性抑制。这种间接抑制作用提示EB可能通过阻断GSK3β磷酸化影响Wnt通路。耐药SCLC细胞中三种肿瘤标志物（NSE、CAV1和MYCL1）的蛋白水平升高，EB孵育可显著降低这些标志物水平。EB对SCLC细胞主要产生两种效应：促进细胞凋亡与逆转耐药性。转录分析显示，SCLC细胞暴露于EB后，促凋亡诱导基因（如BAX、FAS）与效应基因（如CASP3）表达上调，凋亡抑制基因（如survivin）表达下调。耐药细胞中两种膜外排泵PGP1与MRP1表达升高，但EB孵育未影响其蛋白水平。PGP1介导的药物外排细胞实验显示耐药细胞外排活性增强，而EB未改变该活性。经人肝微粒体代谢后，EB通过NADPH依赖性氧化和UDPG依赖性葡萄糖醛酸化被代谢，其针对SCLC细胞的细胞毒性随之消除。 结论：综上所述，这些数据表明EB——一种人类饮食摄入的植物甾体激素——在药物敏感型与耐药型SCLC细胞中通过Wnt信号通路发挥药理活性，改变凋亡相关基因表达，并能被微粒体修饰代谢。
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