Sadava David, Kane Susan E
Department of Cancer Biology, Beckman Research Institute of the City of Hope, 1500 East Duarte Rd., Duarte, CA 91010, USA.
Department of Cancer Biology, Beckman Research Institute of the City of Hope, 1500 East Duarte Rd., Duarte, CA 91010, USA.
Biochem Biophys Res Commun. 2017 Nov 4;493(1):783-787. doi: 10.1016/j.bbrc.2017.08.094. Epub 2017 Aug 25.
Small-cell lung carcinoma (SCLC) has a dismal prognosis in part because of multidrug resistance (MDR). Epibrassinolide (EB) is a steroid hormone in plants, with many physiological effects. It acts via a membrane receptor and GSK3 pathway, resulting in stabilization of a transcription factor. The parallels to the Wnt signaling pathway, which is activated in SCLC and results in increased β-catenin, prompted investigations of the effects of EB on drug-resistant (VPA17) and drug-sensitive (H69) SCLC cells. EB was cytotoxic to both cell lines (IC = 2 μM), indicating a lack of cross-resistance in the VPA17 cell line. EB was pro-apoptotic after 24 h as measured by ELISA of BUdR-labeled DNA fragments and caspase-3 specific activity (2.5 enzyme units/mg protein vs. 0.01 units/mg protein for untreated controls). Matrigel assays showed that EB reduced the SCLC cell invasion phenotype by 80%. Pre-incubation of VPA17 cells in 1 μM EB for 96 h reversed resistance to etoposide (IC = 6.0 μM, reduced to 1.8 μM with EB) and doxorubicin (IC = 0.37 μM, reduced to 0.09 μM). Synergism between EB and chemotherapy drugs was investigated by exposure of VPA17 cells to 1:1 ratios at the respective IC values, with serial dilutions at 0.25 to 2.0 × IC and determination of the combination index (CI). EB and etoposide showed synergism (CI = 0.80 at ED50); EB and doxorubicin also showed synergism (CI = 0.65 at ED50). Incubation of SCLC cells in EB led to a time- and dose dependent reduction of β-catenin (maximum 80% reduction). Gene expression analyses of SCLC cells showed EB incubation resulted in significant reduction in expression of β-catenin-dependent genes that are anti-apoptotic (e.g., c-Jun, survivin), cell division-related (e.g., CCND1 cyclin, sox9), and metastasis-related (e.g., MMP7, uPAR). WIKI4, a known inhibitor of Wnt signaling, was cytotoxic to SCLC cells (IC = 0.02 μM). Synergism between EB and WIKI4 was determined by the CI method and showed antagonism (CI = 1.09 at ED50), suggesting that EB and WIKI4 act on the same pathway. Taken together, these data indicate that EB, a natural product with widespread occurrence in plants, is pharmacologically active in both drug-sensitive and drug-resistant SCLC cells and acts through the Wnt signaling pathway.
小细胞肺癌（SCLC）预后不佳，部分原因是多药耐药（MDR）。表油菜素内酯（EB）是植物中的一种类固醇激素，具有多种生理效应。它通过膜受体和GSK3途径发挥作用，导致转录因子稳定。Wnt信号通路在SCLC中被激活并导致β-连环蛋白增加，这促使人们研究EB对耐药（VPA17）和药物敏感（H69）SCLC细胞的影响。EB对两种细胞系均具有细胞毒性（IC = 2 μM），表明VPA17细胞系不存在交叉耐药性。通过ELISA检测BUdR标记的DNA片段和caspase-3特异性活性（2.5酶单位/毫克蛋白，而未处理对照为0.01单位/毫克蛋白），发现EB在24小时后具有促凋亡作用。基质胶试验表明，EB使SCLC细胞侵袭表型降低了80%。将VPA17细胞在1 μM EB中预孵育96小时可逆转对依托泊苷（IC = 6.0 μM，经EB处理后降至1.8 μM）和阿霉素（IC = 0.37 μM，经EB处理后降至0.09 μM）的耐药性。通过将VPA17细胞暴露于各自IC值的1:1比例，并以0.25至2.0×IC进行系列稀释，然后测定联合指数（CI），研究了EB与化疗药物之间的协同作用。EB和依托泊苷显示出协同作用（ED50时CI = 0.80）；EB和阿霉素也显示出协同作用（ED50时CI = 0.65）。将SCLC细胞在EB中孵育导致β-连环蛋白呈时间和剂量依赖性降低（最大降低80%）。SCLC细胞的基因表达分析表明，EB孵育导致抗凋亡（如c-Jun、生存素）、细胞分裂相关（如CCND1细胞周期蛋白、sox9）和转移相关（如MMP7、uPAR）的β-连环蛋白依赖性基因的表达显著降低。WIKI4是一种已知的Wnt信号抑制剂，对SCLC细胞具有细胞毒性（IC = 0.02 μM）。通过CI方法确定了EB和WIKI4之间的协同作用，结果显示为拮抗作用（ED50时CI = 1.09），表明EB和WIKI4作用于同一路径。综上所述，这些数据表明，EB这种在植物中广泛存在的天然产物，在药物敏感和耐药的SCLC细胞中均具有药理活性，并通过Wnt信号通路发挥作用。
