Abstract
BackgroundKhz-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.MethodsKhz-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.ResultsIn 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.ConclusionsIn 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.
Highlights
Khz crude polysaccharides (Khz-cp) is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz)
Extraction of Khz-cp First, 1 kg of powder was added to 8.5 L of clean water, heated to 115°C, and extraction was performed for 60 min under pressure
The induction of preferential apoptosis by Khz-cp in transformed cells was evaluated in a series of cell lines that comprise an in vivo lung epithelial carcinogenesis model
Summary
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. Cancer develops because of abnormal cellular proliferation or defective apoptosis that leads to uncontrolled growth [1]. Defective signaling during the regulation of cell death can result in the abnormal proliferation of cells and can cause cancer. Repairing defective cell death mechanisms or developing drugs or food components that induce cell differentiation may be a promising approach for the generation of anticancer agents [2,3]. Polyporus umbellatus induces G2/M cell cycle arrest and apoptosis in HepG2 cells, thereby causing growth suppression [11]
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