Induction Of Therapeutic Apoptosis By Activating Caspase-3 Gene Expression With Probasin-based Promoters In Prostate Cancer

Prostate cancer is presently the most commonly diagnosed non-skin cancer in men and is the second leading cause of cancer-death in men in the west countries. The incidence rate has been increasing year by year with the change of average life span and dietary structure in China. While frequently curable in its early stage, many patients present with advanced or metastatic disease with concomitantly fewer curative treatment options and with a median life expectancy of 3 years.Gene therapy holds the promise of providing asystemic method for repairing, replacing, or deleting agenetic defect. Hence, the feasibility of using a prostate specific promoter to express exogenous therapeutic genes in prostate cancers offers an attractive way of potentially targeting locally advanced and metastatic tumors for eradication or control.Probasin is a nuclear basic protein which was first described in the rat dorsolateral lobes of the prostate. Amino acid sequence analysis confirmed that PB was a member of the outlier lipocalins. Immunohistochemical analysis confirmed that PB was found both in the secretions and in the nucleus.The expression of PB is regulated by androgen and prostate specific.So people pay more attention to probasin promoter after PSA to the therapy of prostate cancer.The activation of caspase is the common downstream event of apoptosis pathway of different cells.Caspase-3 is one of the most major effective caspases. It exists as latent zymogen in the cells, each zymogen contains an N-terminal prodomain, a large subunit containing the active site cysteine and a C-terminal small subunit. An aspartate cleavage site separates the prodomain from the large subunit, and an interdomain linker containing one or two aspartate cleavage sites separates the large and small subunits and then form the activated caspase-3 which can lead to cell apoptosis.In our study , genomic DNA was extracted from benign prostate tissues of rat and the sequence(-426,+28)of probasin promoter fragment and ARR2PB promoter were obtained by polymerase-chain-reaction(PCR) and over-lap PCR.To measure the activity and tissue specificity of two promoters, the correct PB and ARR2PB promoters were inserted to the pPRIME vector which containing GFP gene and pGL3-Basic luciferase vector ,and then the lipofection-mediated gene transfers were performed with the recombinant vectors: pPRIME,PB-pPRIME and ARR2PB-pPRIME in different human prostate cancer cell lines:LNCaP,PC3 ,DU145,PC3m,non-prostate cancer cell lines:HeLa,HepG2,A549 and normal cell lines:Chang liver .The GFP fluorescence expressed by the recombinant vectors showed that the activity of ARR2PB promoter was much higher than that of PB promoter,but it was not as high as CMV.And the fluorescence in the LNCaP was the most.Then fluorescence expression was tested by flow cytometry.The result confirmed what we observed in fluorescence microscope. After the recombinant luciferase vectors:PB-PGL3,ARR2PB-PGL3 and P3.1-LUC were transferred into LNCaP,PC3,HeLa and Chang liver cells, luciferase assay also showed the tissue specificity of the probasin promoters in the prostate tissue and that the order of activity of the promoters was CMV>ARR2PB>PB.Next the GFP gene in pPRIME was replaced by the caspase-3 gene, and then three recombinant expression vectors: PB-pPRIME-caspase-3, ARR2PB-pPRIME-caspase-3,pPRIME-caspase-3 were constructed successfully . And then we transfected recombinant vectors into prostate cancer cells LNCaP and PC-3. After the two cells were transfected with recombinant vectors, the apoptosis indexs of them were detected by flow cytometry. Apoptosis of cells was also tested by Immunofluorescence, Hoechst33258 staining and transmission electron microscope(TEM). Immunofluorescence showed that the cleaved caspase-3 was expressed in the prostate cells. Hoechst33258 staining also showed cell apoptosis affected by the recombinant vectors. The apoptosis index of LNCaP cells which transfected with pPRIME-caspase-3 ,PB-pPRIME-caspase-3 and ARR2PB-pPRIME-caspase-3 were 10.55﹪, 7.74﹪and 10.5﹪, and the apoptosis index of PC3 cells were 71.71﹪,57.56﹪and 61.82﹪. The result confirmed apoptosis index increased compared with control groups.In summary, the study provided some constructs for study the expression quantity and tissue specificity of probasin promoters and indicated the probasin maybe a good candidate of gene therapy for prostate cancer.