Hydroalcoholic root extracts of Houttuynia cordata (Thunb.) standardized by UPLC-Q-TOF-MS/MS promotes apoptosis in human hepatocarcinoma cell HepG2 via GSK-38/8-catenin/PDL-1 axis

Houttuynia cordata (Thunb.), an important medicinal plant of Northeast India, Korea, and China, is used to treat various ailments and for anticancer research. Knowing its traditional practices, we are interested in the mode-of-action of HCT on HepG2 to co-relate the traditional practice with modern drug therapeutics. UPLC-Q-ToF-Ms analysis of HCT reveals identification of 14 metabolites. Network pharmacology analysis of the 14 compounds showed interaction with 232 different targets with their potential involvement in hepatocellular carcinoma. Whole extracts impart cytotoxicity on variety of cell lines including HepG2. There was a significant morphological alteration in treated HepG2 cells due to impairment of cytoskeletal components like beta and gamma- tubulin. Arrest at G(1)-S checkpoint was clearly indicated downregulation of Cyclin D1. The root extracts actuated apoptosis in HepG2 as evident from altered mitochondrial membrane potential, Annexin V- FITC, BrdU-PI, AO/EtBr assays, and modulations of apoptotic protein expression but without ROS generation. Whole extracts caused abrogation of epithelial to mesenchymal transition with repression of Snail, N-Cadherin, Vimentin, MMP-9, and upregulation of Pan-Cadherin. Pathway analysis found GSK-3 beta in Wnt/beta-Catenin signaling cascade to be involved through Hepatocellular carcinoma (hsa05225) pathway. The GSK-3 beta/beta-Catenin/PDL-1 signaling was found to be inhibited with the downregulation of pathway components. This was further confirmed by application of EGF, an inducer of the GSK-3 beta/beta-Catenin pathway that neutralized the effect of Houttuynia cordata (Thunb.) root extract on the said pathway. Network pharmacology analysis also confirms the synergy network with botanical-bioactive-target-disease which showed Kaempferol to have the highest degree of association with the said pathway.