Research background. From ancient times plants have been used for medicinal purposes against various ailments. In the modern era, plants are a major source of drugs and are an appealing drug candidate for the anticancer therapeutics against various molecular targets. Here we tested Tamarix articulata methanolic extract of dry leaves for anticancer activity against a panel of hepatocellular carcinoma cells.
Experimental approach. Cell viability of hepatocellular carcinoma cells was determined by MTT assay after dose-dependent treatment with extract of Tamarix articulata. Phase-contrast microscopy and DAPI staining were performed to analyze cellular and nuclear morphology. Immunoblotting was performed to determine the expression of proteins associated with autophagy, apoptosis, and cell cycle. However, flow cytometry was used for the quantification of apoptotic cells and the analysis of cells in different phases of the cell cycle after treatment with varying doses of Tamarix articulata. Additionally, acridine orange staining and DCFHDA dye were used to analyze the quantification of autophagosomes and reactive oxygen species.
Results and conclusion. Our results demonstrate that Tamarix articulata methanolic extract exhibits promising antiproliferative activity with IC50 values (271±4.38), (298±7.08) and (336±6.11) µg/mL against HepG2, Huh7D12, and Hep3B cell lines, respectively. Mechanistically, we found Tamarix articulata methanolic extract induces cell death by activating apoptosis and autophagy pathways. First, Tamarix articulata methanolic extract promotes autophagy which was confirmed by acridine orange staining. The immunoblotting analysis further confirms that Tamarix articulata methanolic extract consistently induces the conversion of, LC3I to LC3II form with a gradual decrease in expression of autophagy substrate protein p62 at higher doses. Second, Tamarix articulata methanolic extract promotes reactive oxygen species production in hepatocellular carcinoma cells and activates reactive oxygen species-mediated apoptosis. Flow cytometry and immunoblotting analysis showed that Tamarix articulata methanolic extract induces dose-dependent apoptosis and activates proapoptotic proteins caspase-3 and PARP1. Additionally, Tamarix articulata methanolic extract triggers the arrest of the G0/G1 phase of the cell cycle and upregulates the protein expression of p27/Kip, and p21/Cip, with a decrease in cyclin-D1 expression in hepatocellular carcinoma cells.
Novelty and scientific contribution. The current study demonstrates that Tamarix articulata methanolic extract exhibits promising anticancer potential to kill tumor cells by programmed-cell-death type I and II mechanisms and could be explored for potential drug candidate molecules to curtail cancer in the future.
Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells
