P22^phox And The Effects And Mechanisms Of Reactive Oxygen Species On Tumor Formation And New Angiogenesis

With the aging of the human population increases,increasing environmental pollution and other factors,cancer has become the major risk fact to human.Prostate cancer is one of the most common malignant tumors for men.In the world,the morbidity and death rate of prostate cancer is the second and sixth,respectively,the incidence of prostate cancer in our country arises.No obvious symptoms in early stage of prostate cancer render it very difficult to diagnose at early stage.When prostate cancers diagnosed,are often in the advanced stages,even metastatic to born.The principle therapeutic method is surgery.Even though the surgical techniques have been developed rapidly,the five-year survival rate hasn't been improved yet.Thus,a better understanding of the mechanisms leading to the initiation and development of prostate cancer is required to develop preventive and therapeutic strategies.Reactive oxygen species(ROS)include superoxide(O2·-),hydrogen peroxide(H2O2),hydroxyl radicals(·OH)and a variety of their reaction products.There is a growing interest in ROS biology and cancer.Numerous studies have supported the role of ROS in promoting tumor development.In the context of prostate cancer,we previously found that prostate cancer cells spontaneously produce excessive ROS.However,the direct role of ROS in prostate cancer development is not known.Endogenous ROS production sites include:the mitochondrial respiratory chain,NADPH oxidase complex,endoplasmic reticulum of electron transfer and so on.The NADPH oxides consist of the catalytic subunit gp91phox,together with the regulatory subunits p22phox,p47pphox,p40phox,p67phox and the small GTPase RAC.The enzyme activity of gp91phox is regulated by the assembly of these regulatory subunits with gp91phox to form an active complex.NOX catalytic and regulatory subunits have been implicated in one or more of the most common cancer types.Increased mRNA and/or protein expression of NOX1,NOX2,NOX4 and NOX5 or their regulatory components has been detected at higher levels in various cultured cancer cell lines or human tumours.However,the expression and role of NADPH oxides subunit p22phox in regulating tumorigenesis and angiogenesis remain to be elucidated.We detected ROS levels in 12 pairs of prostate cancer specimens and corresponding adjacent normal tissues.In prostate cancer,ROS level is upregulated in tumor tissues compared with controls.Catalase and DPI can obviously decreased endogenous ROS production in prostate cancer cells.These results indicated ROS production is important for prostate cancer development.We assay the mRNA expression levels of NOX1?NOX2?NOX3?NOX4?NOX5 and p22phox in prostate cancer cells.These results showed that NOX1 and p22phox obviously over expression.Next,we detected p22phox expression levels in 5 human normal prostate tissues and 33 human prostate cancer tissues which were paraffin-embedded by Immunohistochemical staining,and combined with clinical features for further analysis.In prostate cancer,p22phox is upregulated in tumor tissues compared withcontrols.Higher expression levels of p22phox are positively correlated with grades.ROS production is usually overexpressed in tumor,and regulating many signaling cascades including phosphatidylinositol-3-kinase(PI3K)/protein kinase B(AKT)and mitogen-activated protein kinases(MAPK)/extracelluar regulated protein kinase(ERK)pathways.Knockdown of p22ph0x with siRNAs decreased ROS levels using intracellular CM-H2DCFDA staining by flow cytometry,indicating that p22phoxis required for ROS generation.In order to investigate roles of p22phox in prostate cancer tumor growth and angiogenesis,we established a stable cell line that downexpressed p22phox which was originally with high levels of p22phox.Knock down of p22phox deactivated AKT and ERK 1/2 signaling pathway,leading to inhibition of hypoxia-inducible factor la(HIF-la)and vascular endothelial growth factor(VEGF),which were proved to be vital inducers in angiogenesis and tumorigenesis.Next,we explored the functions of p22phox in prostate cancer in vitro and vivo.Downregulation of p22phox in prostate cancer cells inhibited cell proliferation,migration and colony formation in vitro.Next,angiogenesis experiments in nude male mice shownd that knockdown of p22phox inhibited angiogenesis in vivo.Furthermore,xenograft tumor models in nude mice indicated that forced inhibition of p22phox decreased tumor growth,and slowed down the progression of prostate cancer.Studies have supported ROS production is overexpressed in ovarian cancer cells compared with immortalized ovarian surface epithelial cells.Next,we explored the role of p22phox in ovarian cancer development.We analyzed p22phox levels in 14 human ovarian cancer specimens and 7 human normal ovarian specimens.In ovarian cancer,p22phox level is upregulated in tumor tissues compared with controls.Stable ovarian cancer cell line that knockdown of p22phox shown inhibition of cell proliferation and cell cycle.We also found that knockdown of p22phox inactivation AKT and ERK1/2 signaling pathway and decreased p53 expression.In this study,we identified human prostate tumors and cells showed increased levels of endogenous ROS and expression of p22phox,endogenous p22phox was correlates with increased ROS levels,p22phox modulated tumor growth and angiogenesis through HIF-1 and VEGF expression via AKT and ERK1/2 signaling pathways.Collectively,these findings reveal the origin source of endogenous ROS in prostate cancer and ovarian cancer,suggest that p22phox may be an important factor in tumor growth,and provide new information for developing p22phox-based therapeutic strategies for tumor treatment in the future.