Experimental Study Of Targeting Gene Therapy For Prostate Cancer Using Polyamidoamine Dendrimers As Nonviral Vector

The incidence of prostate cancer have been rising gradually in the past decades inChina. In the initial phase of growth prostate cancer is androgen-dependent andhormone withdrawal is often used to control the growth of the tumor. However,eventually all prostate cancers become hormone refractory by mechanisms that arenot yet fully characterized and at this point there is no available curative treatment.Evidently there is a great need for novel treatments for prostate cancer. Gene therapyhas opened new avenues in prostate cancer therapy.Gene therapy is a promising approach for the treatment of prostate cancer. Themain obstacle for the clinical application of prostate cancer gene therapy is the lack ofgene transfer vectors that are safe, efficacious, and tumor-selective., it is a challengeto develop safe and efficient gene carriers. In general, gene delivery systems includeviral and nonviral carriers. Viral vectors, while efficient in gene transfer, pose a safetyconcern unlikely to be abated soon, searching for nonviral vectors which are safe andease of synthesis are attractive alternatives. Among existing nonviral vectors, cationicpolymers such as generation 5 polyamidoamine dendrimers(G5-PAMAM-D)extensively studied for gene delivery. In this study, first we used G5-PAMAM-D asgene vector, transfected plasmids which express HSV-tk and shRNA into prostatecancer cells.second we synthesized G5-PAMAM-D-Tf by conjugating transferrin onG5-PAMAM-D dendrimer surface to acquire tumor-targeted gene carriers.Furthermore G5-PAMAM-D-Tf was used to tissue-specific RNAi.Our aim is findingsafe, efficacious, and tumor-selective gene vector for prostate cancer gene therapy. This study was divided into three parts:PARTⅠPolyamidoamine dendrimers-Mediated Experimental HSV-tk Gene Therapy inMurine prostate cancer modelsObjective:To identify possibility of Generation 5 Polyamidoamine dendrimers(G5-PAMAM-D) as gene vecter for HSV-tk suicide gene therapy in prostate cancer.Methods: Prosrtate cancer cells PC-3,22Rv1 were transfected by plasmidpEGFP-C1 coated with G5-PAMAM-D, Furthermore, G5-PAMAM-D were usedas gene vecter to deliver plasmid pcDNA3-tk which expresses HSV-tk ineukaryocytes into PC-3 and 22Rv1. 48 hours after transfection,the substrateganciclovir(GCV) was added to the transfected PC-3, 22Rvl cells at differentconcerntration. 24 hour later, MTT was used to evaluate the effect of GCV on theproliferation of two prostate cancer ceils. BALB/C mice subcutaneous tumor modelwere made with PC-3,Polyplex of G5-PAMAM-D and pcDNA3-tk was injectedintratumorally, 48 hours after injection, GCV was injected intraperitoneally,Thetumor volume and mice survival time was observed. Results: G5-PAMAM-D cantransfer plasmid pEGFP-C1 into two prostate cancer, cells, and EGFP wassuccessfully expressed.The number of live cells in G5-PAMAM-D nanoparticlesgroup with HSV-tk is much less than those contral group,significantdifference was found between them.The tumor volume of BALB/C mice wassignificantly smaller in PAMAM-D/pcDNA3-tk group than in Naked plasimd groupand PAMAM-D group(P＜0.01) after treatment 40 days.Survival time ofPAMAM-D/pcDNA3-tk group was longer than two contral groups. Conclusions:G5-PAMAM-D nanoparticles can deliver HSV-tk gene to prostate cancer cellseffectively;it is promising gene vecter for suicide gene therapy in prostate cancer.PARTⅡ Inhibitory Effect of Silencing STAT3 Gene with Short Hairpin RNA Mediatedby Polyamidoamine dendrimers in Growth of ProstareObjective: To investigate the possibility of G5-PAMAM-D as gene vecter foreukaryotic expression plasmid of siRNA in prostate carcinoma in vitro and vivo.Methods Firstly we construct eukaryotic expression vector of siRNApSilencing4.1-EGFP-shRNA specific for enhance green fluorescent protein(EGFP),pSilencing4.1-STAT3-shRNA for Signal Transducers and Activators ofTranscription 3 (STAT3). pEGFP-C1 and pSilencing4.1-EGFP-shRNA werecotransfected into into prostate cancer cells PC-3 and 22Rvl with G5-PAPAM-D asvector, and observed silencing of EGFP. Secondly pSilencing4.1-STAT3-shRNA wastransfected into PC-3 and 22Rvl by G5-PAPAM-D,westem blotting and apoptosisstraining wasused to detect silencing of STAT3 and growth-inhibiting.ThirdlyBALB/C mice subcutaneous tumor model were made with PC-3,Polyplex ofG5-PAMAM-D and pSilencing4.1- STAT3-shRNA was injected intratumorally, Thetumor volume was observed. Results Fluorescent detecting and Western blottinganalyses demonstrated that G5-PAMAM-D could deliverySilencing4.1-EGFP-shRNA and pSilencing4.1-STAT3-shRNA into 2 prostate cancercells,shRNA was expressed to silence EGFP and STAT3. MTT results showed thatproliferation of prostate cancer cells were suppressed by G5-PAMAM-D/pSilencing4.1-STAT3-shRNA and induce apoptosis of PC-3 cells in vitro.The humanprostate cancer in BABL/C mice was formed successfully by inoculated PC-3 cellinto male BABL/C mice. In G5-PAMAM-D/pSilencing4.1-STAT3-shRNA treatedgroup, volume of tumor was shrank remarkably after 9 days'treatment and tumorgrowth were retarded compared with the contral groups. Conclusion G5-PAMAM-Dnanoparticles can deliver plasmid vector which express shRNA into prostate cancercells effectively in vitro and vivo;it is promising gene vecter for RNA inferencetherapy in prostate cancer. PARTⅢProstate cancer siRNA therapy by tumor selective delivery with transferrin-targeted PAMAM-D nanoparticleObjectives: RNA interference (RNAi) has been proposed as a potential treatment forprostate cancer, but the lack of cellular targets limits its application. In the presentstudy we attempt to develop cancer-specific RNAi delivery system usingtransferrin-targeted PAMAM-D nanoparticle, G5-PAMAM-D-Tf for targeted genedelivery shRNA to human prostate cancer LNCaP and PC-3 cells. Methods:pSilencing4.1-EGFP-shRNA and pSilencing4.1-STAT3-shRNA were transfected intoprsotate cell and other cell using G5-PAMAM-D-Tf as vector. Reversetranscription-polymerase chain reaction (RT-PCR), fluorescence microscopy andWestern blotting were used to measure EGFP expression. Inhibition of STAT3 wasevaluated by RT-PCR and Western blotting. Cell proliferation and viability weremeasured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay. Results: We showed that with G5-PAMAM-D-Tf mediatedshRNA targeted delivery, expression of the exogenous reporters. EGFP in prostatecancer cells, but not normal cells, was specifically inhibited in vitro.G5-PAMAM-D-Tf mediated targeted delivery shRNA also depressed the expressionof STAT3. Inhibition of STAT3 affect proliferation of PC-3 and LNCap cells.Conclusion: The present study describes an efficient RNAi targeting delivery systemfor gene silencing that is specific to prostate cancer cells using the G5-PAMAM-D-Tf.This system may be useful for RNAi therapy.