Expression Of Gadd45α/γ Protein In Human Hepatocellular Carcinoma And The Related Signal Transduction Pathways In Gadd45α-induced Growth Suppression Of Hepatoma Cells

Primary hepatoma is the most common and lethal cancer around the world which is very harmful to human health.In china,it is the second most frequent cancer.Hepatocellular carcinoma(HCC)is the most common histological type among primary hepatoma.The identification of the abnormal gene expression profile in HCC will help us to clarify the mechanisms of tumorigenesis and development of HCC and develop new targets for prevention,diagnosis and therapy.The Gadd45 family of proteins,which includesα,βandγisoforms, has recently been demonstrated to play critical role in tumor research, development and the process of turnover.Recently it was documented that Gadd45 could play critical roles in negative growth control. Nevertheless,up to now the mechanism by how Gadd45 exerts its function of negative growth control is not fully understood in hepatoma HepG2 cell lines.In this study,we described the expression of Gadd45α/γin HCC and the related signal transduction pathways that mediated cell cycle G2-M arrest by Gadd45αin HepG2 cells. PartⅠExpression and significance of Gadd45α/γprotein in human hepatocellular carcinomaObjective:To investigate the expression of Gadd45α/γprotein in human hepatocellular carcinoma(HCC)and observe cell cycle arrest inducing by Gadd45αand Gadd45γin hepatocellular carcinoma.Methods:Immunohistochemical method was used to detect the expression of Gadd45α/γfrom the hepatocellular carcinoma tissues and surrounding non-neoplastic liver tissues in 50 cases;then vectors encoding the Gadd45αor Gadd45γgenes were tranisently transfected to HepG2 hepatoma cells and analysed for the cell cycle phase using flow cytometry.Results:(1)The positive expression rates of Gadd45αin the HCC tissues and surrounding non-neoplastic liver tissues in 50 cases was 58.0%(29/50) and 62.0%(31/50)respectively.The rates were no significantly different from each other(p＞0.05).The positive expression rates of Gadd45γin the HCC tissues and surrounding non-neoplastic liver tissues in 50 cases were 30.0%(15/50)and 86.0%(43/50)respectively.The rates were significantly different from each other(p＜0.01).(2)The both expression of Gadd45αand Gadd45γwere close correlatedwith the differentiation degree(P＜0.05):the positive rate of Gadd45α/γin well differentiation carcinoma(84.2%,57.9%)was noticeable higher than poor differentiation carcinoma(41.9%,12.9%).But the expression of Gadd45αand Gadd45γwere not significantly correlated with age,gender and lymphnode metastasis(P＞0.05).(3)After transient transfection of HepG2 cells,the proteins of them were capable of arresting HepG2 cells at the G2/M phase of the cell cycle. Compare to Gadd45γ-induced G2/M arrest,Gadd45α-induced G2/M arrest was more obviously.Conclusions:The Gadd45α/γcould play critical roles in pathological type,development and the process of turnover of hepatocellular carcinoma;The Gadd45αcould play more important role in treatment of hepatocellular carcinoma.PartⅡRelated signal transduction pathways in Gadd45α-induced cell cycle G2-M arrest in hepatoma cellsObjective:To explore the related signal transduction pathways throw Gadd45α-induced cell cycle G2-M arrest in HepG2 cells and provide the primary indications of growth inhibition in hepatoma.Methods:Firstly,HepG2 cells were transfected with pCMV-tag2BGadd45αand pCMV-tag2B respectively and the changes in protein levels of Gadd45α,P38,p-P38,JNK and p-JNK were detected by Western blot. Then,the inhibitors of SB203580 and SP600125 were employed in HepG2 cells to block up the phosphorylation of P38 and JNK.Finally,the cell cycles of HepG2 cells were assayed by flow cytometry.Results: (1)In HepG2 cells transfected with pCMV-tag2B-Gadd45αexpression vector,Gadd45αprotein expression reached the peak level(0.68±0.02)at 48h and the phosphorylation levels of P38(0.94±0.01)and JNK(0.77±0.04)apparently increased.(2)The degree of G2-M arrest was obviously attenuated after applying with the inhibitors in HepG2 cells.Conclusion:Gadd45αand Gadd45γ,utilize P38 and JNK signaling pathways to induce cell cycle G2/M arrest in HepG2 Hepatoma cells...