Anticancer effect of peanut hairy root culture crude extract on human cholangiocarcinoma cell lines

Abstract

Cholangiocarcinoma (CCA) is a major health problem of Thailand especially North and Northeast regions. Most of the cases of CCA are diagnosed at an incurable advanced or metastatic stage. The major limitations of current anti-CCA therapy are recurrence, chemoresistance and toxic effects resulting in poor prognosis and high mortality rate. Therefore, the discovery of novel anti-CCA agents remain an urgent challenge. Stilbenoid is a natural stilbene-base compound that possesses anti-oxidant, anti-inflammatory, cardioprotective, and anticancer properties. It has been recently reported that peanut hairy root culture crude extracts (PCE) are rich in 3 majors stilbene derivative compounds including trans-arachidin-1, -3 and resveratrol. Since, resveratrol has been extensively studied for anticancer activity. Interestingly, knowledge about anticancer effect of trans-arachidin-1 and -3 are little. In this study, we aimed to investigate the anticancer activity of PCE on CCA cells. Anti-proliferative effect of PCE in 4 CCA cell lines with different cancer potential, namely KKU-213, KKU-214, KKU-055, and KKU-213L5 were investigated by using MTT assay. The results showed that PCE significantly inhibited cell proliferation of those a CCA cells by dose and time dependent manners compared with the vehicle control. The strongest effect was detected in KKU-213L5, a highly metastatic cell line, with inhibitory concentration 50% (IC50) values of 947.64 ± 11.49, 403.65 ± 14.83 and 226.9 ± 16.31 µg/ml at 24, 48 and 72 h treatment, respectively. Then, effect of PCE on apoptotic induction was investigated in KKU-213L5 by using Annexin-V/7AAD staining, Hoechst33342 staining and Western blot. The results revealed that PCE treatment at high doses significantly induced CCA cell apoptosis characterized by increasing early and late apoptotic populations and DNA condensation. Moreover, PCE increased the expression of cleaved Poly (ADP-ribose) polymerase (PARP) but not cleaved-caspases 3 and 9 suggested that PCE may induce apoptosis in KKU-213L5 cells through caspase-independent pathway. Furthermore, anti-metastatic effects of PCE in KKU-213L5 were determined on cell migration and invasion abilities by scratch wound healing and transwell invasion assay, respectively. The results demonstrated that low doses (non-cytotoxic concentration) of PCE effectively reduced cell migration and invasion abilities in KKU-213L5. In addition, the gelatin zymography showed that the activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in PCE treated KKU-213L5 cells were not suppressed when compare to untreated KKU-213L5 cells. These results suggest that peanut hairy root culture extract can potentially inhibit CCA cell growth and metastasis suggests the therapeutic potential on anti-cancer candidates.

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