Adenovirus-mediated Small Hairpin RNA Targeting Bcl-XL As Therapy For Colon Cancer

Backgroud:Apoptosis can be initiated by a receptor(extrinsic)pathway,a mitochondria(intrinsic)pathway or both.Death signals from death receptors may be transduced to mitochondria by Bcl-2 family proteins,which subsequently trigger the release of cytochrome c and initiate the apoptosome amplification loop.Thus,Bcl-2 family members are critical for apoptosis induction.Unbalanced expression of Bcl-2 family will impair apoptotic signaling pathways.For example,over-expression of anti-apoptotic proteins such as Bcl-XL leads to the development of a multiple drug-resistant phenotype.On the other hand,enforced expression of the pro-apoptotic Bax protein could induce apoptosis in various cancer cells.A growing body of evidence showed that the ratio of Bcl-2 to Bax was an important indicator of resistance to apoptosis.However,recent studies showed that expression of Bcl-2 was low whereas the expression of Bcl-XL was high in colon cancer. Furthermore,our recent data showed that down-regulation of Bcl-XL by small interfering RNA(siRNA)could induce apoptosis in 5-fluorouracil resistant colon cells and overcome the acquired resistance toward tumor necrosis factor-related apoptotic inducing ligand(TRAIL)protein in DLD1 cells, suggesting that Bcl-XL can be a new therapeutic target for colon cancer.The effect of RNA interference therapy was determined by stability of siRNA, tissue specificity and efficiency of siRNA transduction.Recent studies showed that using the DNA plasmid to express double-strand RNA could increase the stability of siRNA.However,the efficiency of in vivo delivery of plasmid remains low.Therefore,it was urgent to find a more efficient approach to deliver siRNA in vivo.As we known,adenovectors could efficiently transduce a broad range of cell types and have been used extensively in studies for gene delivery applications.So we would use a recombinant adenovirus to express small hairpin RNA(shRNA)targeting BcI-XL driven by U6 promoter in this study and determined whether it could induce RNA interference in vivo as well as its therapeutic effects on colon cancer.Method:Firstly,we would construct a recombinant adenovirus to express a shRNA targeting Bcl-XL driven by U6 promoter named Ad/Bcl-XL shRNA. Then its effects on down-regulation of Bcl-XL expression as well as apoptosis-induction in colon cancer in vitro and in vivo were asscessed by a series of methods including RT-PCR,MTT assay,cell clonogenic assay and animal experiments.Results:A recombinant adenovirus named Ad/Bcl-XL shRNA was constructed and purified.Results showed that Ad/Bcl-XL shRNA could effectively knock-down the expression of Bcl-XL on both levels of mRNA and protein.It could also decrease the cell viability in colon cancer DLD1 and Lovo cells in a dose-dependent as well as time-dependent manner(p＜0.05), while control vector Ad/GFP alone had almost no effects at all at the same time point.Treatment with Ad/Bcl-XL shRNA could dramatically suppress the colony formation in a dose-dependent manner(p＜0.05).Treatment with Ad/Bcl-XL shRNA but not a control vector led to a dramatic cleavage of caspase-9,caspase-3,and PARP.Ad/Bcl-XL shRNA also significantly suppressed tumor growth in subcutaneous tumor model derived from DLD1 cells.Furthermore,treatment with Ad/Bcl-XL shRNA did not cause obvious damage to the normal tissues or normal human fibroblast.Finally,we established a primary tumor model by inoculating the patient's colon cancer tissue into inguinal area of nude mice.Totally,we inoculated 22 samples of colon cancer and there were only three samples with tumorogenesis. Ad/Bcl-XL shRNA also significantly suppressed the growth of these tumors and prolonged the survival of tumor-burden mice.Conclusions:1)A recombinant adenovirus named Ad/Bcl-XL shRNA was constructed;2)Ad/Bcl-XL shRNA could effectively knock-down the expression of Bcl-XL;3)Ad/Bcl-XL shRNA could effectively decrease the cell viability in colon cancer cells by activation of apoptotic signaling;4)A primary tumor model was established by inoculating the patient's colon cancer tissue into inguinal area of nude mice;5)Ad/Bcl-XL shRNA also significantly suppressed tumor growth in subcutaneous tumor model derived from DLD1 cells as well as primary tumor model derived from the patient's colon cancer tissue.