The effect of brassinolide, a plant steroid hormone, on drug resistant small-cell lung carcinoma cells

Abstract

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 (IC₅₀ = 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 (IC₅₀ = 6.0 μM, reduced to 1.8 μM with EB) and doxorubicin (IC₅₀ = 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 IC₅₀ values, with serial dilutions at 0.25 to 2.0 × IC₅₀ 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 (IC₅₀ = 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.

Keywords: Apoptosis; Brassinolide; Lung carcinoma; Multidrug resistance; Plant hormone; Sensitization; Wnt signaling.