Rhinacanthin-C Extracted from Rhinacanthus nasutus (L.) Inhibits Cholangiocarcinoma Cell Migration and Invasion by Decreasing MMP-2, uPA, FAK and MAPK Pathways

Parichart Boueroy,Sakawrat Kanthawong,Chariya Hahnvajanawong,Pongpun Siripong,Sunitta Saensa‑Ard

doi:10.31557/apjcp.2018.19.12.3605

Parichart Boueroy, Sakawrat Kanthawong + Show 3 more

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https://doi.org/10.31557/apjcp.2018.19.12.3605

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Abstract

Cholangiocarcinoma is a malignant tumor with high metastatic and mortality rates. We investigated the effects of rhinacanthin-C on cell proliferation, migration, invasion and the expression of proteins regulating cancer cell invasion-regulated proteins in a cholangiocarcinoma (KKU-M156) cell line. Cytotoxicity of rhinacanthin-C was determined by the SRB assay. Using wound-migration, chamber-migration and chamber-invasion assays, we assessed the effects of rhinacanthin-C against KKU-M156 cells. The activities of matrix metalloproteinases 2 and 9 (MMP-2, MMP-9) and urokinase-type plasminogen activator (uPA) were determined using gelatinase and uPA zymography assays. The expression of invasion-regulated proteins was investigated using western-blot analysis. After treatment with rhinacanthin-C, KKU-M156 cells exhibited antiproliferative effects in a dose-dependent manner with greater efficacy than in Vero cells: IC50 values were 1.50 and 2.37 µM, respectively. Rhinacanthin-C significantly inhibited cell migration and invasion of KKU-M156 cells in a dose-dependent manner. Consistent with this observation, treatment with rhinacanthin-C was associated with a decrease in the expression levels of FAK, p-FAK, MMP-2, and a decrease in the levels of p38-, JNK1/2- and ERK1/2-MAPK pathways as well as inhibiting NF-κB/p65 expression and translocation of NF-κB/p65 to the nucleus. We have shown for the first time that the anti-metastatic effects of rhinacanthin-C on KKU-M156 cells are mediated via inhibition of the expression of invasion-regulated proteins. Rhinacanthin-C may deserve consideration as a potential agent for the treatment of cholangiocarcinoma.

Highlights

Cholangiocarcinoma is the second-most prevalent primary hepatic tumor worldwide and is associated with a high mortality rate
We investigated the effects of rhinacanthin-C on cell proliferation, migration, invasion and the expression of proteins regulating cancer cell invasion-regulated proteins in a cholangiocarcinoma (KKU-M156) cell line
And 3C, rhinacanthin-C caused a significant delay in migration of KKU-M156 cells into the wound area compared with the dimethyl sulfoxide (DMSO)-treated group, whereas the migration of Vero cells was only slightly affected (Figure 3A and 3C). This was confirmed by the chamber migration assay, which is semi-quantitative and showed that rhinacanthin-C significantly reduced the migration of KKU-M156 cells in a dose-dependent as shown in Figure 4A and 4B

Summary

Introduction

Cholangiocarcinoma is the second-most prevalent primary hepatic tumor worldwide and is associated with a high mortality rate. This cancer is highly metastatic, leading to unresponsiveness to chemotherapeutic drugs. Rhinacanthone has been reported to inhibit proliferation, cell cycle arrest and induce apoptosis in human cervical carcinoma HeLa cells in dose- and time-dependent manners (Siripong et al, 2009). The same researcher reported that rhinacanthins (C, N and Q) exhibit anti-proliferative effects and induce apoptosis in human cervical carcinoma (HeLaS3) cells mediated through G2/M cell-cycle arrest and by the activation of caspase-3 (Siripong et al, 2006a)

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