Plasmid-based HPV E6-specific SiRAN And Co-expression Of Wild-type P53Suppresses The Growth Of Cervical Cancer Delivered By Attenuated Salmonella Typhimurium

Purpose:Co-expression plasmid pSi-E6-P53through reduced oncogenes E6and increased tumor suppressor gene P53plays on antitumor activity ofhuman cervical cancer, explore the oncogene and tumor suppressor geneimbalances possible mechanisms leading to cancer.Methods:The co-expression plasmid pSi-E6-P53was constructed usingrecombinant DNA technology. Cervical cancer SiHa cells weretransfected in vitro, using Lipofectamine, with plasmid expressing eitherpSi-E6or pP53alone, or the combined therapeutic plasmid pSi-E6-P53.The mRNA and protein expression levels of E6, P53and related geneswere detected by RT-PCR and Western Blot analysis. Cell apoptosis wasdetected by TUNEL analysis. A nude mouse, cervical cancer subcutaneous xenograft model was successfully established in vivo.Attenuated Salmonella Typhimurium carrying one of the aboveconstructed plasmids was injected via tail vein into mice containing atransplanted cervical tumor to observe its impact on tumor growth. ThemRNA and protein expression levels of E6, P53and related genes wereagain studied by RT-PCR and Western Blot analysis. The effects ofattenuated Salmonella Typhimurium carrying the different plasmids onhealthy heart, liver, spleen, lung, and kidney tissues of nude mice, as wellas on the implanted tumor tissue was assessed by using H&E staining andimmunohistochemical staining for PCNA protein expression.Results:The single expression plasmids and the co-expression plasmidpSi-E6-P53were constructed successfully and verified by restrictionendonuclease digestion and DNA sequencing analysis. The studies using plasmid-transfected SiHa cells showed that plasmids expressingsiRNA-E6reduced E6expression and plasmids expressing P53enhancedP53mRNA and protein expression. The combination therapeutic plasmid,as compared to either single treatment alone, demonstrated statisticallysignificant alterations of both E6and P53levels, and was also associatedwith up-regulation of Bax,cleaved-Caspase3,Caspase8andcleaved-Caspase9, and the down-regulation of Bcl-2expression (P<0.05).Further, SiHa cells transfected with pSi-E6-P53underwent significantapoptosis compared with the control groups, as assessed by TUNEL assay(P<0.05). Nude mice implanted with SiHa cells and treated with theco-expression plasmid showed significantly reduced average tumorweights and volumes, compared with those observed after treatment withthe pSi-E6or pP53plasmids alone (P<0.05). There were virtually nohistological changes, observed following H&E staining, in healthy heart, liver, spleen, lung, kidney or in tumor tissues of mice treated withattenuated Salmonella Typhimurium carrying the various treatmentplasmids. Treatment plasmid pSi-E6-P53was observed to reduce PCNAexpression (P<0.01).Conclusions:1. Co-expression plasmid pSi-E6-P53through reduced oncogenesE6and increased tumor suppressor gene P53, play the inhibitory effect onhuman cervical cancer, the mechanism may promote tumor cellapoptosis-related.These studies suggest that the combination ofsiRNA-E6and P53plays a pro-apoptotic role by inducing the expressionof caspase8, and initiates a caspase activation cascade via the extrinsic (ordeath receptor) pathway or by repressing Bcl-2. Further, this combinationtherapy enhanced the expression of interrelated proteins Bax, Cyto-c, andcleaved-caspase9, plus activated Caspase3via the intrinsic or mitochondrial pathway and, most importantly, exhibited a significanttherapeutic effect on cervical cancer.2. The combination of siRNA-E6and P53by down-E6, reduce thedegradation of P53, and P53tumor suppressor gene expression increasedto overcome alone SiRNA E6and P53alone shortcomings, from theregulator E6oncogene and tumor suppressor gene P53balancedperspective treatment for cervical cancer provides new clues.