title: Polytrichum commune L.ex Hedw ethyl acetate extract-triggered perturbations in intracellular Ca²⁺ homeostasis regulates mitochondrial-dependent apoptosis.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  The present study aimed to further investigate the efficacy of PCLH ethyl acetate fraction (PC-EEF) and the associated mechanisms in human leukemia cells.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Yuan
authors.affiliation: School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an, Shaanxi, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, China.
authors.forename: Pan
authors.initials: P
authors.lastname: Wang
authors.affiliation: Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, China.
authors.forename: Yali
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, China.
authors.forename: Quanhong
authors.initials: Q
authors.lastname: Liu
authors.affiliation: School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an, Shaanxi, China. Electronic address: tangwei@snnu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.affiliation: Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, China. Electronic address: wangxiaobing@snnu.edu.cn.
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Wang
mesh_terms: D000085:Acetates / Q000737:chemistry; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D044002:Bryophyta / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018922:HL-60 Cells; D006706:Homeostasis; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy* / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008516:Medicine, Chinese Traditional; D008928:Mitochondria / Q000187:drug effects; D010936:Plant Extracts / Q000494:pharmacology*; D020298:U937 Cells
chemical_list: D000085:Acetates; D000972:Antineoplastic Agents, Phytogenic; D010936:Plant Extracts; C007650:ethyl acetate; D002118:Calcium
keywords: Apigenin (PubChem CID: 5280443); Apoptosis; Ca(2+); Flavonoids; Luteolin (PubChem CID: 5280445); Mitochondria; Polytrichum commune L.ex Hedw; Rutin (PubChem CID: 5280805)