The Effects Of ARHI On Pancreatic Cancer Cell Proliferation, Apoptosis, Invasion And Metastasis In Vitro And Related Mechanisms

Pancreatic cancer is the fifth to seventh mortality in China and it is generally diagnosed at an advanced stage as one of the most aggressive human malignancies in the world.In addition the mortality rate approximating the incidence rate demonstrate the worse prognosis in pancreatic cancer.Further studies should be required to elucidate the useful molecule clues for diagnosis and treatment in pancreatic cancer.Pancreatic cancer displays a wide range of genetic and epigenetic alterations that contribute to its aggressive phenotype during which oncogenes are activated and the function of tumor suppressor genes is lost.It is known that loss or downregulation of tumor suppressor genes is thought to contribute to the pancreatic carcinogenesis.ARHI is a maternally imprinted tumor suppressor gene that belongs to the Ras superfamily. Unlike Ras,ARHI exerts opposite functions and has the characteristics of a tumor suppressor gene.ARHI is widely expressed in several normal tissues,including breast, ovary and pancreas.Downregulation of ARHI has been observed in＞60%of breast and ovarian cancers;However the expression and function of ARHI in pancreatic cancer has received relatively little attention.Role of ARHI in pancreatic cancer was studied in professor Qian laboratory since 2000.We have measured the expression of ARHI in normal pancreas and in pancreatic cancer tissue.In pancreatic cancer cell lines with low ARHI expression,the methylation status of ARHI has been explored.Pancreatic cancer cells with low ARHI expression have been transfected with the plRES2-EGFP-ARHI plasmid to examine the functional impact of ARHI re-expression on cancer cell growth in culture.The previous work showed ARHI is widely expressed in in the cytoplasm of normal pancreatic duct and acinar cells,but is dramatically down-regulated in 48.6%of pancreatic cancers.Re-expression of ARHI through plasmid transfection inhibited RAS/mitogen-activated protein kinase signaling,suppress STAT3 activity,and induce apoptosis.We proposed that ARHI gene plays an important role as a candidate tumor suppressor gene in pancreatic carcinogenesis.Additional studies will be required to elucidate further the role of ARHI on the basis of previous work.Strategies of the study:1.To observe the effects of ARHI on pancreatic cancer cell proliferation and apoptosis with stable pancreatic cancer cell line with ectopic expression of ARHI.2.To investigate the impacts of ARHI on cell cycle regulation and relevant mechanisms.3.To explore the effects of ARHI on pancreatic cancer cell invasion and metastasis as well as the regulation mechanisms.For this reason,we observe the influence of ARHI on proliferation,apoptosis, invasion and metastasis of pancreatic cancer cells in vitro.1.The tumor suppression property of ARHI on pancreatic cancer and its effect on PI-3K/AKT pathwayMethods:1) PANC-1 and MiaPaCa-2 celllines were transfected with the ARHI constructs or with an empty plasmid as a control using lipofectmine 2000,and ARHI or vector stable transfected cell line was established by G418 selection.ARHI mRNA and protein expression was analyzed by RT-PCR and western blot methods respectively.2) The growth curve and MTT methods were used to detetmine the effect of ARHI on the proliferation of stable transfected cell line.3) Flow cytometry was used to analyze the effect of ARHI on the apoptosis of stable transfected cell line.4)To observe the effects of ARHI on PI-3K/AKT signal pathway and death associated protein kinase 1,Western blot was used to examine the expression of protein T-AKT,p-AKT and DAPK1.Result:1) The ARHI stable transfected pancreatic cancer cells produced obvious expression of ARHI protein and mRNA,but failed to induce detectable ARHI expression in empty plasmid transfected cells.2) Ttansfection with ARHI markedly inhibited growth of PANC-1 pancreatic cancer cells and MiaPaCa-2 pancreatic cancer cells between 3 and 5 days after seeded.It was seen most clearly on days 5,and the cell number increased 8.5 times over 5 days in vector group,whereas the number of PANC-1 cells transfected with ARHI increased 2.8 times.The difference between ARHI transfectants vs.vector transfectants was statistically significant at 3,4,and 5 days(P＜0.05 or P＜0.01).Similar results were obtained by MTT assay.3) In ARHI group,PANC-1,MiaPaCa-2 cells apoptosis rate were 32.9%and 39.5% respectively,and were 13.8%and 15.2%in vector group.ARHI could increase the percentage of apoptosis of cell and induced apoptosis of stable transfected cell line, compared to vector.Difference between the two groups is statistically significant (P＜0.05).4) The level of phosphorylated AKT in ARHI transfectants was found down-regulated when compared with vector controls,but total AKT protein remained unchanged in ARHI or vectoe-transfected cell lines tested.5) ARHI could up-regulated expression of DAPK1 protein and mRNA,compared with vector controls.Summary:1) ARHI or vector stable transfected pancreatic cell line was established by G418 selection.2) ARHI inhibited the proliferation of PANC-1 or MiaPaCa-2 pancreatic cancer cell.3) ARHI induced apoptosis of PANC-1 or MiaPaCa-2 pancreatic cancer cell.4) Up-regulation of DAPK1 by ARHI might be associated with the suppression of apoptosis.5) ARHI expression blocked the activation of PI-3K/AKT signaling in pancreatic cancer cells.6) ARHI gene is a candidate tumor suppressor gene in pancreatic cancer.2.Reexpression of the tumor suppressor ARHI inhibited the growth of pancreatic cancer cells by inducing cell cycle G1 arrestMethods:This study focused on performing the following research:1) we analyzed the effects of ARHI on the cell cycle progression by flow cytometry.2) Western blot methods was used to detect target proteins,including p-AKT,MDM2,p53.p21^WAF1, p27^KIP1,cyclinA-Cdk2,cyclinE-Cdk2,cyclinD1-Cdk4.3) PI-3K inhibitor LY294002 were performed in ARHI transfectants,then detected proteins above by western blot methods.Results:1) The results indicated that reexpression of ARHI generated a markedly effects on the cellular proliferation,which lead to reduction of S phase and cellular arrest in the G₁ phase,compared with vector-transfectants.The fraction of cells in the G₁ phase increased from 56 to 75%(PANC-1) or 54 to 67%(MiaPaCa-2) compared with vector-transfectants.The difference between ARHI transfectants vs.vector transfectants was statistically significant(P＜0.05 or P＜0.01).No change in G₂-M cells was observed.2) Up-regulation of p53 and p21^WAF1 protein expression were found through inhibiting PI-3K/AKT pathway in ARHI-transfected cells,following Down-regulation of p-AKT and MDM2,compared with vector controls.PI-3K inhibitor LY294002 was able to block AKT activation,increasing p21^WAF1 expression via up-regulation of p53,but ARHI expression displayed no change.3) The p27^kip1 expression was increased in ARHI transfectants compared with vector transfectants through inhibition of PI-3K/AKT pathway.Additionally,PI-3K inhibitor LY294002 resulted in the induction of p27^kip1 expression by regulating AKT activation.4) In comparison with vector transfectants,the protein levels of cyclin A,cyclin D1, CDK2 and CDK4,but not cyclin E,were reduced by ARHI reexpression in pancreatic cancer cells.PI-3K inhibitor LY294002 had no effects on modulating expression of cell cycle regulatory proteins above.Summary:1) ARHI blocked cell cycle progression at G₁ phase in pancreatic cancer cells.2) Expression of ARHI blocked PI-3K/AKT/MDM2 survival signaling in pancreatic cancer cells.3) ARHI expression up-regulated p53,p21^WAF1 and p27^kip1 through PI-3K/AKT/MDM2 inactivation.4) ARHI inhibited proliferation of pancreatic cancer cells by modulating the expression of cell cycle regulation factors including CDK2,CDK4,and cyclins A and D1.5) ARHI plays an important role in the development of pancreatic cancer.3.The effects of ARHI on pancreatic cancer cell invasion and metastasis in vitroMethods:1) Transwell chamber motility assay and crape-motility assay were applied to study the influence of ARHI on motility of pancreatic cancer cells.2) Transwell chamber invasion assay was used to determine the effect of ARHI on invasion of pancreatic cancer cells.3) Pull-Down experimental was used to detect the changing of RhoA and Ras activation in ARHI and vector-transfected pancreatic cancer cells.Results:1) Crape-motility assay revealed a decreased motility speed in ARHI group,compared with vector group.2) In the Transwell motility experiment,PANC-1 or MiaPaCa-2 cell migration rate of ARHI-transfected group was(13.23±3.44)%or(14.02±2.08)%,respectively,and the migration rate of vector group was(19.54±3.19)%or(21.50±2.41)%.The two groups is statistically significant.(p＜0.05)3) In the Transwell invasion experiment,PANC-1 or MiaPaCa-2 cell invasion rate of ARHI-transfected group was(8.47±1.74)%or(7.78±0.70)%respectively,and the invasion rate of vector group was(11.58±1.76)%or(10.43±1.14)%.The two groups is significant difference.(p＜0.05)4)) Reexpression of ARHI decreased the expression of active Ras in PANC-1 or MiaPaCa-2 cell,when compared with the vector group.No difference in the active level of RhoA-GTP could be observed between ARHI transfectants vs.vector transfectants.Summary:1) ARHI could inhibit pancreatic cancer cell migration and metastasis in vitro.2) ARHI-transfectecd cell displayed a decrease of active Ras3) The effect of ARHI on cell motility and invasion is not associated with Ras-RhoA pathway.Conclusions:1) ARHI inhibited the cell proliferation and triggered apoptosis of PANC-1 or MiaPaCa-2 pancreatic cancer cell.2) ARHI blocked cell cycle progression at G₁ phase in pancreatic cancer cells.These results suggest that the PI-3K/AKT pathway plays a pivotal role in the pathogenesis of pancreatic cancer and ARHI exerts its growth-inhibitory effects through modulation of several key G₁ regulatory proteins,such as p21^WAF1,p27^kip1,CDK2, CDK4,and cyclins A and D1.3) ARHI could inhibit pancreatic cancer cell migration and metastasis in vitro and the effect of ARHI on cell motility and invasion is not associated with Ras-RhoA pathway.4) We concluded ARHI partially abolished malignant phenotype and firstly demonstrated ARHI gene was a candidate tumor suppressor gene in pancreatic cancer.5) ARHI may represent a modulator of cancer cell proliferation and may play an important role in the pancreatic carcinogenesis.