title: Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.
abstract: BACKGROUND
  Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells.
  
  
  METHODS
  Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes.
  
  
  RESULTS
  In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation.
  
  
  CONCLUSIONS
  In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an increase in [Ca2+]i, P38 activation, and ROS generation via NADPH oxidase and mitochondria.
authors.forename: Tae Hwan
authors.initials: TH
authors.lastname: Kim
authors.forename: Ju Sung
authors.initials: JS
authors.lastname: Kim
authors.forename: Zoo Haye
authors.initials: ZH
authors.lastname: Kim
authors.forename: Ren Bin
authors.initials: RB
authors.lastname: Huang
authors.forename: Young Lye
authors.initials: YL
authors.lastname: Chae
authors.affiliation: Department of Radiotherapy, The First Affiliated Hospital, Guangxi Medical University, Nanning, China. 13807806008@163.com.
authors.forename: Ren Sheng
authors.initials: RS
authors.lastname: Wang
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D062610:Fungal Polysaccharides / Q000494:pharmacology*; D006801:Humans; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D055492:Polyporus / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020940:Reishi / Q000737:chemistry; D015398:Signal Transduction / Q000187:drug effects; D014020:Tissue Extracts / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D062610:Fungal Polysaccharides; C579322:Khz extract; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014020:Tissue Extracts; D019255:NADPH Oxidases; D048051:p38 Mitogen-Activated Protein Kinases; D002118:Calcium