Abstract 183: Brassinolide, a plant steroid hormone, reverses drug resistance in human small-cell lung carcinoma cells

Abstract

Abstract Small-cell lung carcinoma (SCLC) has a dismal prognosis in part because of multidrug resistance (MDR). Epibrassinolide (EB) is a steroid hormone present in higher plants, where it has numerous physiological effects and acts via a LRR-RLK membrane receptor and GSK3/SHAGGY 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 SCLC cells, particularly showing MDR. SCLC cells were NCI-H69 and a derived line, VPA17, showing resistance to etoposide (9-fold), doxorubicin (20-fold) and vincristine (10-fold). EB was cytotoxic to both cell lines (IC50 = 2 uM), indicating a lack of cross-resistance. EB was pro-apoptotic after 24 h as measured by ELISA of BUdR-labeled DNA fragments. Apoptosis was also indicated by an increase in caspase-3 specific activity (to 2.5 enzyme units/mg protein vs. 0.01 for untreated controls). Matrigel assays showed that EB reduced the SCLC cell invasion phenotype by 80% in a time-dependent fashion. Pre-incubation of VPA17 cells in 1 µM EB for 96 h reduced the IC50 for etoposide (6.0 uM to 1.8 µM) and doxorubicin (0.37 µM to 0.09 µM). Synergism between EB and chemotherapy drugs was determined by exposure of VPA17 cells to 1:1 ratios at the appropriate IC50 values of EB: etoposide or EB: doxorubicin. After 4 days, the combination index (CI) was determined using CalcuSyn software. EB and etoposide showed modest synergism (CI = 0.80 at ED50 and CI = 0.90 at ED95); EB and doxorubicin also showed synergism (CI = 0.65 at ED50 and CI = 0.90 at ED95). The Wnt - β-catenin signaling pathway was investigated as a possible explanation for the pharmacological effects of EB. In the presence of EB, SCLC cells showed a time- and dose dependent reduction of β-catenin (maximum 80% reduction). Cell fractionation studies showed that the reduction was primarily in nuclear β-catenin. Transcription analyses of SCLC cells showed EB led to 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). Down-regulation of the β-catenin promoter was confirmed by transfection of SCLC cells with a construct containing the promoter coupled to a luciferase reporter (reduced by 85%). WIKI4, a known inhibitor of Wnt signaling, was cytotoxic to SCLC cells (IC50 = 0.02 uM). Synergism between EB and WIKI4 was determined by exposure of VPA17 cells to 1:1 ratios at the appropriate IC50 values of EB: WIKI4, as above. EB and WIKI4 showed antagonism (CI = 1.09 at ED50 and CI = 1.35 at ED95), 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. Citation Format: David Sadava, Susan E. Kane. Brassinolide, a plant steroid hormone, reverses drug resistance in human small-cell lung carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 183. doi:10.1158/1538-7445.AM2017-183