Experimental Research In Targeting Hypoxia-inducible Factor-1α For Pancreatic Cancer Therapy

Part I :Correlation of mRNA expression of hypoxia induciblefactor - 1 α to biological features of pancreatic cancerOBJECTIVE Hypoxia inducible factor1α (HIF-1α) has been universally detected in many types of cells and plays a key role in modulation of tumor related genes. The purpose of this study was to explore the relationship between the HIF-1a mRNA expression and biological characteristics in pancreatic cancer, such as tumor angiogenesis, tumor cell proliferation, differentiation, apoptosis and metastasis. METHODS Reverse transcriptional polymerase chain reaction was used to detect the HIF-1α mRNA expression. The expression of survivin, proliferating cell nuclear antigen (PCNA) and CD34 proteins was measured immunohistochemically. The correlation between the expression level of HIF-1α mRNA and survivin, PCNA and CD34 was analyzed. RESULTS There was very significant difference in the expression of HIF-1α between the pancreatic cancer tissue and adjacent normal tissue (P<0.01), but no significant difference was found among tumor histopathological grades (P>0.05). There was significant difference in the expression of HIF-1α mRNA between JPS stage I-II and stage III-IV (P<0.05). The expression of HIF-1α mRNA was closely correlated with the expression of survivin, PCNA and CD34 proteins. CONCLUSION The expression level of HIF-1α mRNA is surmised to have a significant correlation with tumor angiogenesis, cell proliferation, apoptosis and metastasis. Inhibition of HIF-1α may be an important and approachable therapeutic target for pancreatic cancer.Part II:Effects of Hypoxia inducible factor - 1α RNA interferance on pancreatic cancer cell proliferation, apoptosis and metastasis under hypoxia in vitroOBJECTIVE The objective of this study is to explore the effect of hypoxia on pancreatic cancer cell proliferation, apoptosis and metastasis and to explain the role of hypoxia inducible factor - 1α in the procedure and the possibility of targeting hypoxia inducible factor - 1α for pancreatic cancer therapy. METHOD A hypoxia inducible factor - 1α siRNA expression vector was constructed to transfect pancreatic cancer cells and silence their hypoxia inducible factor - 1α expression. MTT assay is used to analysis the growth curves of pancreatic cancer cells; marked pancreatic cancer cells with human Annexin V-PE kit were analysed by FCM; Transwell was used to evaluate the ability of pancreatic cancer cells to metastasis; furthermore, RT-PCR and Western blot are both used to measure hypoxia inducible factor - 1α , proliferating cell nuclear antigen (PCNA) , VEGF, NM23a and survivin expressions. RESULTS Under hypoxia, growth rate and metastatic ability of pancreatic cancer cells increased and number of apoptotic pancreatic cancer cells reduced significantly. Hypoxia inducible factor - 1α , proliferating cell nuclear antigen (PCNA) , VEGF and survivin expressions increased simutaniously, whereas angiopoietin-1 and NM23a expressions decreased. After pancreatic cancer cells was transfected with hypoxia inducible factor - 1α siRNA expression vector and hypoxia inducible factor - 1α was silenced, the ability of pancreatic cancer cells to adapt hypoxia was frustrated and the hypoxic effects dissapeared. CONCLUSION Our results suggest that Hypoxia inducible factor - 1α may be involved in not only pancreatic cancer proliferation but also apoptosis and metastasis under hypoxic conditions. Hypoxia inducible factor - 1α may be a potential target for pancreatic cancer therapy. [Keyword] Part III:In vivo influence of hypoxia inducible factor - 1α on thebiologic features of pancreatic cancerOBJECTIVE Based on the results of the second phase, the tumor models were constructed to further attest the validity of targeting hypoxia inducible factor - 1α as a therapeutic approach for pancreatic cancer. METHOD Pancreatic cancer cells were treated with hypoxia and RNA interferance. Four weeks after treated pancreatic cancer cells were respectively injected into subcutaneous part and caudal vein, we oberserved the tumor size of subcutanious tumor as well as hepatic and lung metastatic lesions in caudal vein injected mouses. Tumor, hepatic and lung samples were embeded with paraffin and tested hypoxia inducible factor - 1α, PCNA, survivin and CD34 expressions by immunohistochemistry. Tunnel assay was used to detect the apoptosis index of these samples. RESULTS Hypoxic pancreatic cancer cells without transfection produced more and larger tumor masses with high apoptotic rate and metastasis. Hypoxia inducible factor - 1α, PCNA, survivin and CD34 expressions upregulated significantly in these samples. Ability to form metastasis and tumorigenicity of pancreatic cancer cells transfected with hypoxia inducible factor - 1 α siRNA expression vector were both depressed. The apoptosis of each metastasis responded to the enviroment in vivo in a like manner. CONCLUSION After introduction of hypoxia inducible factor - 1α siRNA expression vector into pancreatic cancer cells, their tumorigenicity, proliferation and ability to form metastasis were decreased significantly. This suggest that targeting hypoxia inducible factor - 1α may be a potent therapeutic method for pancreatic cancer.