Effect Of Arterial Embolization On HIF-1α Pathway Of Transplanted Hepatoma: Correlation With Pulmonary Metastasis

Part I : Relationship between chemical hypoxia-reoxygenation culture and hypoxia inducible factor-la and vascular endothelial growth factor expression in Walker-256 cells Objective: To investigate hypoxia inducible factor-1α(HIF-1α) and vascular endothelial growth factor (VEGF) expression in Walker-256 cells and to study the possible relationship between HIF-1 and VEGF during chemical hypoxia and reoxygenation caused by cobalt chloride (CoCl₂) . Methods: Walker-256 cells were maintained in RPMI —1640 supplemented with 10% (v/v) fetal calf serum(FCS). Cell proliferation inhibition rate was analysis by 3- [4, 5-dimethy lthiazol-2-yl] -2, 5-diphenyl tetrazolium(MTT)in different concentration CoCl₂. Based on the dose-effect relationship between CoCl₂ and cell proliferation, 100μmol/L was chosen as working concentration. Chemical hypoxia and reoxygenation were simulated by the addition and the removal of respectively in Walker-256 cells. Cell morphological changes wereobserved by microscope, and HIF-1α VEGF mRNA and their protein expression were explored by reverse transcription polymerase chain reaction(RT-PCR) and Western blot upon different periods of chemical hypoxia and reoxygenation. Results: In Walker-256 cells treated with CoCl₂, The cell structure changes correlated with cell injury was increased gradually since chemical hypoxia. Treatment with CoCl₂ was found to apparently elevate HIF-1α and VEGF. The HIF-1α mRNA and protein expression were peaked differently at 4h and at 8h, while the VEGF mRNA and protein expression were both peaked at 8h. In Walker-256 cells treated with the removal of CoCl₂, cell injury were increased significantly at 8 h after chemical reoxygenation in contrast to those in cells at 24h after reoxygenation. HIF-1α and VEGF mRNA and protein were dramatically declined at 8h after reoxygenation, HIF-1α and VEGF mRNA and protein expression was similar with normal culture condition at 24h after reoxygenation. The VEGF mRNA of Walker-256 cells was consistent with the results in HIF-1α protein. Conclusion: Hypoxia plays an important role in over-expression of VEGF. Hypoxia can up-regulate the expression of HIF-1 protein, which activates the transcription of VEGF. HIF-1 could be the key role in the protection against tumor cell injury during chemical hypoxia- reoxygenation.Part II: Effect of Hepatic arterial Blockage on HIF-1α and VEGF expression of transplanted hepatoma in ratsObjective: To investigate the molecular biologic effect of transcatheter arterial embolization (TAE) on recurrence and metastasis of hepatic tumor. Methods: Twenty-four Wistar rats were randomly divided into two groups: the HAL group and the control group, after Walker-256 carcinoma was implanted in the left medial lobes of thelivers. Eight days later, hepatic artery ligation(HAL group) and abdominal incision (control group)was performed . Each three rats were sacrificed 1, 3, 7, 12d after treatment and tumor tissues were excised. Expression of hypoxia-inducible factor-1α (HIF-1α ) and vascular endothelial growth factor (VEGF) mRNA was checked by reverse transcription polymerase chain reaction (RT-PCR) and expression of HIF-1α and VEGF protein of tumors was examined using immunohistochemistry. The relative intensity of HIF-1α and VEGF was evaluated with a computer-assisted image-analyzer. The results were analyzed with SPSS software. Results: 1, 3d after treatment, the relative level of HIF-1α and VEGF was significantly higher in the HAL group compared with the control group (P <0.05 ) . 7, 12d after treatment, expression of HIF-1α and VEGF protein was no significant difference in the TAE group compared with the control group (P>0.05) . VEGF was well correlated to HIF-1α expression in the HAL group (P<0.05). Conclusion: HAL induces hypoxic result of tumor and promotes the expression of HIF-1 and VEGF protein. Part III: Effect of hepatic arterial embolization on HIF-1α expression of transplanted hepatoma in rats: Correlation with pulmonary metastasisObjective: To investigate the effect of transcatheter arterial embolization (TAE) on metastasis of hepatic tumor and the feasible mechanism. Methods: Walker 256 carcinosarcoma was transplanted into rat liver to establish a liver cancer model. Thirty-nine bearing tumor rats were randomly divided into three groups. Hepatic artery ligation(HAL group: 15), hepatic artery embolization (HAE group: 9) or abdominal incision (control group: 15) was performed. All three groups were sacrificed were sacrificed 12d after treatment, tumor tissues and lungs were excised. Changs in tumor volume and tumor growth inhibiting rate after treatment were evaluated, and the degreeof necrosis in tumor were observed, pulmonary metastasis was evaluated using macroscopic and microscopic method. The expression of hypoxia-inducible factor-1α (HIF-1α ) protein was detected by immunohistochemistry. Results: The tumor volumes in HAL group and HAE group were smaller than the volume in the control group, P < 0.01. Compared with HAL group, the tumor volume in HAE group decreased further, P <0.05. Compared with the control group, the degree of tumor necrosis increased in HAL and HAE group, P < 0.05. Pulmonary metastasis lesions were observed in three rats in HAL group, two rats in HAE group in comparison with one rat in control group. There was no significant difference in metastasis rate among three groups, P > 0.05. However, expression of HIF-1α was significantly higher in the HAE group compared with control group or HAL group, P < 0.05. Conclusion: TAE for transplanted cancer in rat' s liver can reduce tumor volume, and promote tumor necrosis. It has no significant influence on inhibiting lung metastasis rate. The remaining tumor cells adapt to hypoxic microenvironment through HIF pathway, which maybe stimulate metastasis.