The Role Of HDGF In The Pathogenesis And Chemotherapy Resistance In Colorectal Cancer

Background:Colorectal cancer is a common prevalent malignant tumor and its incidence is increasing year by year. Currently, the major treatment for colorectal cancer is still surgery. The failure of chemotherapy after surgery is the main reason for tumor metastasis and recurrence. So it is important for us to seek effective therapeutic targets which can avoid resistance to chemotherapy.Established studies indicate that a lot of growth factors such as the epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) which play an important role in development of cancer.Recently a novel growth factor named "hepatoma-derived growth factor (HDGF) " gradually attract more and more researchers for its important role in the process of physiological or pathology. And what’s more, higher expression levels of HDGF were found in many malignant tumors such as lung cancer、gastric cancer and hepatoma than in their corresponding normal tissues. These studies implied that HDGF has a close relation with formation and growth of these malignant tumors.Aims:In order to discern the role of HDGF in the carcinogenesis and chemotherapy resistance of colorectal cancer, we make some researches as follows. In the present study, we first analyzed the expression of HDGF in normal colorectal mucosa, colitis, colonic adenoma and colorectal cancer by using immunohistochemistry staining, and discussed the relationship among HDGF, Survivin and clinical parameters. Secondly we observe the effect caused by down-regulation of HDGF on apoptosis of colon cancer cells. We knock down the HDGF Gene by a target siRNA.Thirdly we examined the cell’s sensitivity to nordihydroguaiaretic acid (NDGA) in different cells including HDGF-overexpressor cells and control cells. The HDGF-overexpressor cells was established by transfecting a restructuring plasmid which containing HDGF cDNA into cells.Methods:1. The expression of HDGF in different colorectal tissues (adenocarcinoma, adenoma, colitis and normal tissue) colitis and Survivin in adenocarcinoma are detected by immunohistochemistry. The results are analyzed by statistical analysis.2. First we knock down the HDGF gene by RNA interference. And then we observe the changes of cell’s proliferation and apoptotsis rate by MTT assay, DNA Ladder assay, Hoechst 33258 staining, and flow cytometry. at Last, we using western blot to detect the expression changes of apoptosis proteins.3. First we construct an eukaryotic expression plasmid which contains the whole HDGF cDNA fragment using genetic engineering technology. Then we established a over-expression lines LoVo/HDGF with G418 resistance. Then we detect the growth and apoptosis of different group cells during different concentrations (time) by MTT assay, and flow cytometry.Results:1. The expression level of HDGF protein in four different stages of colorectal disease (normal mucosa, inflammation, adenoma and adenocarcinoma) gradually incrased. The HDGF staining of 59.2% colorectal cancer patients is positive and the Survivin staining of 59.2% colorectal cancer patients is positive. With survivn HDGF in colorectal cancer tissue of the positive rate of expression for 59.2% and 60.8%. Both of their expression didn’t relate with the patient’s age, gender, tumor size, tumor distribution and infiltration depth (p<0.05). However, the high expression of HDGF is correlated with clinical pathologic extent, lymph node metastasis and Dukes stage. And the high expression of Survivin are correlated with lymph node metastasis and Dukes stage.in addition, Spearman analysis indicated that a close correlation between the two genes.2. Comparing to the two control group, the HDGF siRNA-treatment group cells has a significantly inhibition in proliferation and a marked increase in apoptosis. in addition, the siRNA-treatment group Cells, pERKl/2, PCNA, Survivin and Bcl-2 are obviously down-regulated and Bax obviously up-regulated. And what’s more, cyt-c was released from mitochondria into cytoplasm; Caspase-9 and caspase-3 are also activated.3. All of the three groups of cells are inhibited by NDGA treatment in a dose dependent manner. But compared to the LoVo and mock cells, LoVo/HDGF group cells exhibit an excellent protection for cells from the hurting by NDGA. The resistant to NDGA of LoVo/HDGF cells was also confirmed in vitroConclusion:1 HDGF plays an important role in the carcinogenesis in colorectal cancer. Our study also demonstrated that HDGF has a close relation to Survivin. They may interact with each other and constitute the molecular basis of malignant colorectal cancer together. Thus HDGF is an ideal colon cancer marker for prognosis2 HDGF-siRNA showed an excellent ability in inhibiting tumor cell growth and inducing apoptosis. So HDGF may be a potent good molecular target for treatment with colorectal cancer.3 HDGF-overexpression could confer the cell to resist the toxic effect from NDGA in colorectal cancer. The discovery may contribute to get clear the mechanism of chemotherapy resistance.