A Study On The Role Of Heme Oxygenase-1 In The Apoptotic Process Of Eca-9706 Cell Line Induced By Sodium Arsenite

Esophageal Squamous carcinoma is one of the common cancers in the digestive system with high malignancy ,which has poor prognosis and many difficult in therapy. Investigating a new and more effective way of killing the cancer cells is what medical researchers are taking effort on. Recently,it is found that heme oxygenase-1(HO-1) is a inducible enzyme which exists widely in mammal cells. It has many important effects including anti-inflammation and anti-apoptosis and so on,which has great relationship with the effluence and development of tumor. So this study is to find whether or not it would assist killing tumor cells with chemotherapeutics via changing the expression of HO-1,in hope of finding a new effective way of managing tumors.Part one: construction and expression of eukaryotic expression vector of hHO-1 gene.Objective: To construct eukaryotic expression vector pcDNA3.1-hHO-1 of homo sapiens HO-1 gene and detect its expression.Methods: We get the hHO-1 gene by digesting the amplification vector pGEM-TEasy-hHO-1 with HindШ+BamHI. Then the hHO-1 gene is inserted into eukaryotic expression vector pcDNA3.1. After it show that the insertion is successful by digesting with HindШ+BamHI, we transfect the Eca-9706 cell line with this reorganized vector. Then we take the general RNA and protein 24 hours after cell transfection. We detect the expression of HO-1 in cells transfected with reorganized vector using RT-PCR and western blotting methods to decide whether or not the reorganized eukaryotic expression vector is successfully constructed. Results: Digesting with HindШ+BamHI,it proves that the HO-1 gene has been cloned into eukaryotic expression vector pcDNA3.1. 24 hours after transfected with the reorganized vector, RT-PCR and western blotting results suggest that the reorganized vector has worked well in the cells and expressed great quantity of HO-1 product.Conclusion: we have construct a eukaryotic expression vector pcDNA3.1-hHO-l of homo sapiens HO-1 gene successfully.Part two: The apoptosis of Eca-9706 cells induced by NaAsO₂ and the expression of HO-1.Objective: To study the apoptotic-inducible effect of NaAsO₂ on Eca-9706 cell line and the change of HO-1 expression during this apoptotic process.Methods: Treating Eca-9706 cells with different concentration of NaAsO₂, we use MTT method to detect the influence of NaAsO₂ on Eca-9706 cells'growth;we observe the alternation-of cell figure appearances at different time points under microscope; Using DNA ladder and FCM to detect the apoptotic effects. At the same time,we detect the change of HO-1 mRNA expression using RT-PCR method.Results: Low concentration of NaAsO₂ (under 2.5μmol/L) has no inhibitory action but weak growth facility effect on Eca-9706 cells and has no apoptotic effect. The concentration above 15μmol/L NaAsO₂ has inhibitory effect on Eca-9706 cells which increases parallel with the incease of drug concentration. The MTT and FCM results show that treating the cells with a NaAsO₂ concentration above 15μmol/L can have apoptotic effects and the apoptotic rate rises parallel with the drug concentration. RT-PCR result shows that after treating cells with 15μmol/L NaAsO₂,the rise of HO-1 mRNA expression can be detected at 4 hours. it rises to the highest level at 8 hours and after that ,it begin to decrease rapidely to the normal level.Conclusion :Above 15μmol/L NaAsO₂ can lead Eca-9706 cells to apoptosis. The transient rise of HO-1 expression may have a protective role agaist the apoptotic effect.Part three: The influence of HO-1 expression on the cell apoptosis induced by NaAsO₂.Objective: To study whether or not changing HO-1 expression can alter the cell apoptotic effect induced by NaAsO₂ so as to augment the tumor-killing effect of chemotherapeutics by changing HO-1 expression .Methods: Transfecting Eca-9706 cells with pcDNA3.1-hHO-1 as one group, inducing or inhibiting HO-1 expression with 40μmol/L heme and 20mol/L SnPPIX respectively as another two groups. MTT method , taking DNA fragments and FCM methods are used to detect the alteration of cytotoxic effect and apoptotic effect of NaAsO₂ on Eca-9706 cells.Results: Transfecting cells with pcDNA3.1-hHO-1 gene or treating with 40μmol/L heme can attenuate the cytotoxic effect of NaAsO₂ and attenuate the apoptotic rate when treated with 15μmol/L NaAsO₂.As a result,it need higher concentration of NaAsO₂ to get DNA ladder. By contrast, 20μmol/L SnPPIX augments the cytotoxic effect and apoptotic effect of NaAsO₂ in every way.Conclusion: Inhibiting HO-1 expression with 20μmol/L SnPPIX can augment the inhibitory and apoptotic effect of NaAsO₂ on Eca-9706 cell line.So,inhibiting the intracellular HO-1 expression of Eca-9706 cells may augment its sensitivity to the apoptotic effect induced by chemotherapeutics. This offer a new way to kill Esophageal Squamous carcinoma.