Effects of an ethyl acetate extract of Daphne altaica stem bark on the cell cycle, apoptosis and expression of PPARy in Eca-109 human esophageal carcinoma cells

Abstract

Daphne altaica Pall. (D. altaica; Thymelaeaceae) has long been used in traditional Kazakh medicine for the treatment of cancer and respiratory diseases. Previous studies have demonstrated the in vitro anticancer effects of D. altaica extract and its constituents in certain cancer cell lines; however, the underlying molecular mechanisms are not completely understooD. The present study aimed to investigate the molecular mechanisms underlying the activity of an ethyl acetate extract of D. altaica (Da-Ea) by assessing its effects on cell morphology, cell apoptosis, cell cycle progression and the expression levels of peroxisome proliferator-activated receptor y (PPARy) in Eca-109 cells. Cell morphology was observed under a phase contrast microscope. Cell apoptosis and cell cycle progression were assessed by flow cytometry following Annexin V/propidium iodide (PI) double staining and PI single staining, respectively. The mRNA and protein expression levels of PPARy were deter¬ mined by reverse transcription-quantitative PCR and western blotting, respectively. Compared with the control group, the percentage of apoptotic cells, cell cycle arrest at S phase and apoptotic morphological cell characteristics were increased in da-Ea-treated Eca-109 cells. Furthermore, da-Ea treat¬ ment upregulated the mRNA and protein expression levels of PPARy compared with the control cells. High-performance liquid chromatography with diode-array detection indicated that daphnetin-7-o-p-d-glucoside, daphnetin, demethyldaph noretin-7-o-p-d-glucopyranoside and genkwanol A were the main constituents of da-Ea. Collectively, the results suggested that da-Ea displayed antiproliferative activities in Eca-109 cells by inducing apoptosis and S phase cell cycle arrest, as well as upregulating PPARy expression levels.