The Effects And The Mechanism Of Pak1 On Gastric Cancer Cell Proliferation,Invasion And Metastasis

PurposeAlthough the overall incidence of gastric cancer is decreasing in almost every country,it is still the fourth most common malignancy and the second leading cause of cancer-related death worldwide.Despite the advances in diagnosis and treatment,the prognosis for patients with advanced gastric cancer remains dismal.In 2000,about 880 000 people were diagnosed with gastric cancer and approximately 650 000 died of the disease.Over the past several years,it has been clearly demonstrated that multiple genetic alterations are responsible for the development and progression of gastric cancer.Changes in the expression of specific genes in gastric cancer play important roles in diverse cellular functions,such as cell adhesion,signal transduction,cell differentiation,development,metastasis,DNA repair,and glycosylation changes.A better understanding of molecular changes during stomach carcinogenesis may lead to new paradigms and possible improvements in cancer diagnosis,treatment,and prevention.However,the precise mechanisms of molecular genetic changes that contribute to malignant phenotypes of gastric carcinoma remain largely unknown.p21-activated kinase 1(Pak1),a serine/threonine kinase,is an effector of the small Rho GTPases Cdc42 and Rac1.Once Pakl has been activated by one of these GTPases it mediates downstream signalling events involved in cytoskeletal reorganization,cell motility,apoptosis and transformation.Other signaling molecules can also affect Pak1 activity,including JAK2,PI3K and PKA.Activation of Pak1 by diverse signals leads to autophosphorylation of several sites,including Thr423,which lies within the auto-inhibitory loop of the kinase.Previous studies have also revealed that increased expression of Racl is related to higher TNM stages of gastric carcinoma,and down-regulated RhoA expression could inhibit the proliferation of gastric cancer cells. Despite these reports,there has been little work on the role of Pak1 in human gastric cancer.Mehtods1.The effects of Pak1 on gastric cancer cell proliferation,invasion and metastasis a.The expression of Pak1 in gastric cancer tissue was detected by western blot and immunohistochemistry.b.BGC823,MGC803,SGC7901,MKN1,MKN45 cells were analyzed with Pak1 kinase assay,and examed the ability of migration and invasion.c.chemically synthesized Pak1 siRNA were transfected into BGC823 cells.After transfection, the cells were processed for western blot analysis,MTT proliferation assay,migration and invasion assay.d.We construct the stable Pak1 knockdown cell lines and analysis the proliferation ability by growth curve and anchorage-independent growth assay,and then test the migration and invasion ability.e.Wild type BGC823 cells,control stable cell lines,and Pak1 knockdown cells(clone17) were injected into three separate groups of nude mice by means of subcutaneous injection,celiac injection and tail vein injection,the proliferation and metastasis ability of Pak1 knockdown cell lines were detected in vivo.2.The mechanism of Pak1 on gastric cancer cell proliferation,invasion and metastasisa.Pak1 mediate the expression of Cyclin B1 in BGC823 cells.(1)we examined Cyclin B1 expresssion in the implant tumors from the control and clone 17 nude mice mentioned above by immunohistochemistry.(2)The expression of Cyclin B1 in stable Pak1 knockdown cell lines was tested by western blot.(3)Catalytically active T423E Pak1 plasmids were transiently transfected into BGC823 cells.Western blot analysis cyclin B1 protein expression.(4)To confirm whether Pak1 affected the transcription of cyclin B1,we next examined the mRNA level of cyclin B1in four cell lines by northern blot analysis.b.Pak1 modulates cyclin B1 promoter activity.(1)We transiently transfected down-regulated Pak1 cells with luciferase reporter constructs containing the human cyclin B1 promoter and measured luciferase activity.(2)We transiently transfected BGC823 cells with GST-PID and measured luciferase activity.(3)we next examined the effects of wild-type(WT) Pak1 or active T423E Pak1 on the transcriptional regulation of cyclin B1.c.AO/EB staining to detect apoptosis with peritoneal mesothelial cells induced by stable Pak1 knockdown cell lines.d.The expression of miRNA-100 in 17 clone was measured by Q-PCR.e.We transiently transfected miRNA-100 into control cells,inhibitor of miRNA-100 into 17 clone.After transfection,the ability of proliferation,migration, invasion were measured.Results1.The effects of Pakl on gastric cancer cell proliferation,invasion and metastasisa.Increased expression of Pak1 presents inâ…¥stages of gastric carcinoma,and gastric cancer metastasis to lymph nodes.b.higher migration and invasion ability exists in gastric cell with more actived Pak1.c.Transient knockdown of Pak1 expression inhibits the proliferation migration and invasion of BGC823 cells.d.Stable knockdown of Pak1 expression inhibits the proliferation,migration and invasion of BGC823 cells.e.Downregulation of Pak1 inhibits the tumorigenicity and metastasis of BGC823 cells in vivo.2.The mechanism of Pak1 on gastric cancer cell proliferation,invasion and metastasisa.Pak1 can mediate the expression of Cyclin B1 in BGC823 cells.b.Pak1 can effect the Cyclin B1 promoter activity in BGC823 cells.c.Pak1 promots BGC823 cells to induce peritoneal mesothelial cells appearing apoptosis.d.Pak1 can mediate the expression of miRNA-100 in BGC823 cells. e.miRNA-100 can effect the proliferation,migration and invasion of BGC823 cells.Conclusions1.Pak1 can effect the proliferation,migration and invasion of BGC823 cells in vitro and in vivo.2.Pak1 can mediate the expression of Cyclin B1 in BGC823 cells.3.Pak1 can mediate the expression of miRNA-100 in BGC823 cells.4.miRNA-100 can effect the proliferation,migration and invasion of BGC823 cells.