Abstract 3709: Molecular targets of total methanolic and ethyl acetate fraction of triphala in colon cancer

Abstract

Abstract We previously demonstrated that the methanolic extract of triphala, a herbal preparation of a mixture of three fruits, namely, Terminalia chebula, Emblica officinalis and Terminalia bellerica was effective against colon cancer cells. In order to identify its mechanism of action, HCT-116 cells (1×106 cells) were seeded in 25mm2 petri dishes and treated with varying concentration of total extract. Cells stained using Alexa Fluor 488 Annexin V/Dead Cell Apoptosis assay revealed a dose dependent increase in early apoptosis which plateaued with 200 µg/ml, and a dose dependent increase in late apoptosis upto 400 µg/ml. This and previous finding of down-regulation of cyclin D1 suggested that the inhibitory action of triphala is facilitated both by antiproliferation and induction of apoptosis. In addition, significant downregulation of select upstream and downstream molecules of NFκB signaling was observed. In order to identify the bioactive components of the mixture, the total methanolic extract was subfractionated with various organic solvents and tested for antiproliferative activity using HCT-116 cells. The efficacy was determined to be in the following order: ethyl acetate fraction>total methonolic fraction>chloroform fraction>butanol fraction>hexane fraction>residual aqueous fraction. When normal colon cells CRL-1459 (5×103 cells) were treated with either the total methanolic or ethyl acetate fraction, the IC50 was found to be 7 fold higher for both the fractions compared to colon cancer cells, suggesting that the antiproliferative activity is specific to malignant cells. FTICR-MS analysis of the effective fractions revealed enrichment of most of the phenolics such as gallic acid, ellagic acid, and several other tannins into the ethyl acetate fraction. In order to identify the molecular targets of the ethyl acetate fraction, HCT-116 (1×106 cells) were seeded in 6 well plates and treated with LPS, 100 or 200 µg/ml ethyl acetate extract of triphala or both for 6 hours. qRT-PCR analyses revealed that the ethyl acetate fraction is more effective in down-regulating both constitutive and induced expression of TNFα and COX2 compared to the total methanolic extract. In addition, down-regulation of LPS-induced NFκB expression was more pronounced with ethyl acetate extract compared to methanolic extract. Ethyl acetate extract was also effective in downregulating the cell cycle regulatory molecule cyclin D1. However, the total extract was more effective in modulating the expression of IL-6 and IFNγ, suggesting that the various fractions of triphala may have distinct targets of colon carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3709. doi:10.1158/1538-7445.AM2011-3709