Polytrichum commune L.ex Hedw ethyl acetate extract-triggered perturbations in intracellular Ca²⁺ homeostasis regulates mitochondrial-dependent apoptosis.

Polytrichum commune L.ex Hedw (PCLH), a moss of Bryopsida, has been used as a traditional Chinese medicine and shown to possess anticancer activities. Previous studies have indicated its anti-leukemia effect but the potential mechanisms have not been fully explained. The present study aimed to further investigate the efficacy of PCLH ethyl acetate fraction (PC-EEF) and the associated mechanisms in human leukemia cells. Phytochemical analysis of PC-EEF was performed by spectrophotometry and HPLC. MTT analysis and trypan blue exclusion assay were adopted to examine its cytotoxicity on a panel of leukemia cells (K562, U937, HL-60 and K562/DOX cells) and non-cancerous cells (human PBMCs). Anti-proliferative effect was monitored by colony formation assay and EdU incorporation assay. Ultrastructural alterations on K562 cell membrane surface were observed by scanning electron microscopy. Changes on plasma membrane integrity, cell membrane potential, mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. Fluorescence microscope was performed to assess [Ca(2+)]i level, mitochondrial injury and cytochrome c release. Apoptosis-associated protein expression was analyzed by western blot. The role of Ca(2+) in PC-EEF-induced cell death was investigated by Ca(2+) chelating reagent BAPTA-AM. PC-EEF possessed relatively high flavonoid content (about 88.84 ± 0.89%) and showed significant cytotoxicity to human leukemia cells. PC-EEF could cause obvious cell morphological deformation, membrane integrity loss and membrane depolarization. Meanwhile, PC-EEF treatment could dramatically potentiate perturbations in cellular Ca(2+) homeostasis. Subsequently, mitochondrial membrane potential (MMP) collapse, cytochrome c release and Bcl-2/Bax down-regulation were all observed. Consistent with these results, PC-EEF treatment resulted in significant activation of caspase 3, poly (ADP-ribose) polymerase (PARP) degradation and apoptosis. Moreover, PC-EEF-caused cytotoxicity, membrane damage, mitochondrial injury and apoptosis were remarkably reversed by BAPTA-AM. PC-EEF damaged the membrane system and triggered Ca(2+)-dependent mitochondrial apoptosis, which may provide some new insights into its efficacy against human leukemia cells.