Study On The Expression Of Hepatoma-derived Growth Factor In Gastric Cancer And The Mechanism Of Its Influence On Gastric Carcinoma AGS Cells

Gastric cancer is one of the most common malignant tumors in the world, and its mortality ranks the second in China. With the improvement of surgical technique in recent years, the 5-year survival rate is higher than 90% in patients with early gastric cancer, but it is still ranges between 5%-15% in the advanced gastric cancer patients. The early gastric cancer diagnosis as well as new and more effective therapies are therefore necessary for improving gastric cancer survival and decreasing its mortality. Exploring the molecular mechanism of gastric cancer helps to screen specific molecular targets also useful for the prevention and reduction incidence of gastric cancer.Most gastric cancers arise from precancerous lesions, but the de novo cancer develops directly from normal mucosa. The carcigenesis process of stomach mucosa typically involves several stages and multiple factors and steps, with interior and exterior factors acting on normal stomach stem cells. The research on the molecular alterations at different levels of precancerous lesions and cancer tissues reveals that during the multi-stage tumorigenesis of the gastric epithelial cells, different molecular alterations take place, accompanied by the imitative activation of oncogenes or the LOH of oncogene suppressors. The progress from precancerous lesions to gastric cancer is also accompanied by the mutation of DNA repair genes and other epigeneticchanges. The abnormal proliferation of the cells is the initial steps in the development of tumor.HDGF, or the hepatoma-derived growth factor, is a heparin-binding growth factor, which was separated from the supernatant of the serum-free culture of human hepatoma HuH-7 cells in 1994. HDGF was confirmed to have been involved in the growth and development of multiple embryonic tissues such as the liver, kidney, lung, and blood vessels. It is high-expressed in these organisms and tissues, and the expression decreases after birth. HDGF also possesses a mitogen role, and after entering and anchoring on the nucleus, it promotes the proliferation of the fetal liver cell, hepatoma cell, fibroblast cell, endothelium and vascular smooth muscle cell. HDGF is also a paracrine and autocrine growth factor, which facilitates the proliferation of itself and the cells in the vicinity. HDGF has also been found to be a neurotrophic factor, which can promote the survival of the neurocytes. Recent studies have revealed a close relationship between HDGF and the genesis and development of tumor. In the process of spontaneous development from hepatocyte to hepatoma in mice, the expression of HDGF increases dramatically after tumorigenesis. It happens in hepatoma, NSCLC, pancreatic cancer, gastric cancer and so on, and its over-expression is correlated with prognosis. However, the molecular mechanism of the participation of HDGF in the genesis and development of tumor is still not clearly understood.With immunohistochemical staining, gastric tumor and adjacent mucosa samples were examined, evaluates the relationship between the level of HDGF and the clinical pathological characteristics and the progression of gastric cancer as well the significance of this relationship. By further constructing the eukaryotic expression vector containing HDGF to transfect AGS cells, the present study evaluates the effects of the over-expression of HDGF on the biological behaviors in AGS cells and on the key molecular activities in signal pathways. Finally, it analyzes the influences on the biological behavior of the AGS cells and the changes of the key molecules after HDGF is silenced by specific siRNA. The aim of this study is to explain the molecular mechanism of HDGF involved in gastric carcinogenesis, which may be useful for the diagnosis and therapy of gastric cancer.Results1. HDGF is involved in the genesis and development of human gastric cancer.The expression of HDGF in gastric tumor and adjacent mucosa samples in 205 cases were detected by EnVision's two-step Immunohistochemical method. The results showed that the HDGF level of cell nucleus gradually elevates in the carcinogenesis process of gastric mucosa. In benign gastric diseases like superficial gastritis, atrophic gastritis, the HDGF of gastric mucosa cell nucleus was not expressed or had a very low expression. The expression began to increase in the stage of intestinal metaplasia and atypical hyperplasia (dysplasia) of gastric precancerous lesions compared with that in normal mucosa(p<0.01). It became even higher in early gastric cancer, with early gastric cancer higher than in intestinal metaplasia(p<0.01). There is no difference between early gastric cancer and atypical hyperplasia, p=0.247. The HDGF level is even higher at the advanced stage than in early gastric cancer (p<0.01). Moreover, the expression of HDGF in the nucleus of gastric cancer cells correlateed to the state of lymphoid node metastasis, the invasion depth and the UICC staging(P<0.05). All the above suggests that over-expression of HDGF is an early event in the genesis and progression of gastric cancer and is relevant to its development. The HDGF experssion level was shown to be a prognostic factor for gastric carcinoma.2. The over-expression of HDGF promotes the proliferation of gastric cancer AGS cells in vitro through the activation of Erk1/2 pathway.To explore the biological behavior changes of the gastric cells after over-expression of HDGF, we established a cell line (HDGF-AGS) with stable expression of HDGF. The MTS/PES assay showed that HDGF-AGS cells still maintained the cell proliferation in the absence of serum stimulation compared with their parent cells and the sham cells (pcDNA-AGS)(P<0.05). However, the difference among the three was not significant when supplemented with 15% serum(P>0.05). Interestingly, the HDGF-AGS cells were more resistant to cis-DDP with half-lethal concentration, an apoptosis-inducing agent with p53-independent. Further more, we found the expression of total Erk1/2 and phosphorylated Erk1/2 increased in AGS cells transient transfected with HDGF, but not in p38 and JNK/SAPK in the MAPKpathway. The total Akt in transfected cells was increased whereas the phosphorylated Akt activity was decreased, as showed by Western blot. ELISA results found that there were no significant differences in p65, p52, p50, Rel-B and Rel-C among HDGF-AGS, pcDNA-AGS and their parent AGS cells(P<0.05). HDGF over-expression did not affect NF-kappa B activity. Western blot also showed that activated Erk1/2 by HDGF did not stimulate the Bcl-2/Bcl-xl/Bad pathway, an important apoptosis-resistance pathway. But Erk1/2 activation up-regulated the level of transcription factors including T-box 18 and b-LHL scanned by global genchip analysis.To determine whether the AGS cells with stable HDGF expression possess greater metastasis potential, we performed the experiments of Transwell soft-agar cloning formation and Transwell cell invasion in vitro. The results demonstrated that the cloning number of HDGF-AGS cells had no significant difference compared with controls, but larger cell clones were formed in soft agar cloning test in vitro. Experiments of Transwell cell invasion in vitro found that the number of cells infiltrated Matrigel had no significant differences from the controls, whether in the cases of no serum or 15% serum in cell invasion test in vitro(P>0.05). 3. The silence of HDGF inhibited the apoptosis of the gastric cancer AGS cells through the increase of phosphorylated Bcl-2 and TRAF-2/c-IAP inhibitory complex.To evaluate the biological effect of the gastric cells with HDGF silence, we designed a specific siRNA to knockdown the HDGF in AGS cells and subsequently evaluated the biological effect of the gastric cells with HDGF silence by soft agar cloning test in vitro and FCM. The results showed that the cloning formation of the AGS cells with HDGF silence in the soft agar was reduced. FCM analysis found that the knockdown of HDGF did not increase the apoptosis; however, HDGF knock -down cells showed apparent apoptosis inhibition under the condition of 0.06125ug/ml cis-DDP.Western blot further showed that HDGF knock-down AGS cells enhanced the expression of the phosphorylated Bcl-2 and total Bad, while the phosphorylation level of Bad Serl12 and Ser136 had no significant differences with the blank control and the sham transfected cells. There were no significant differences between the HDGFknockdown cells and the controls in the expression of other Bcl-2 molecules. In the immuno-coprecipitation assay with TRAF2 antibody, there was more c-IAP1 that can be precipitated by TRAF2 antibodies in HDGF knockdown cells, compared to the blank control and the sham transfected cells, while the TRAF2, c-IAP1 and XIAP showed no significant changes among these groups. Conclusions:1. The over-expression of HDGF is an early event in the genesis and progression of gastric cancer and is relevant to its development. The HDGF experssion level was shown to be a prognostic factor for gastric carcinoma.2. The over-expression of HDGF promotes the proliferation of gastric cancer AGS cells through the activation of Erk1/2 pathway, without increasing the invasive potential of the AGS cells in vitro.3. The anchorage-independent growth of AGS cells with HDGF silence in the soft agar is reduced. Silence of HDGF inhibites the cisplatin-induced apoptosis of the gastric cancer AGS cells through the increase of phosphorylated, Bcl-2 and TRAF-2/c-IAP inhibitory complex.