Attenuated Salmonella Typhimurium Carrying The Plasmid Co-expressed SiRNA-Stat3 And GRIM-19 For Prostate Cancer Therapy

Prostate cancer is a leading cause of cancer-related deaths in the Westerncountries, and in China, the incidence and mortality rates of prostate cancer haveincreased markedly. Men currently diagnosed at the early stages of prostatecancer can, in many cases, be effectively treated by surgery or radiation.However, in one third of the patients, the disease will recur and ultimatelydevelop into hormone-refractory prostate cancer and metastatic prostate cancersthat are essentially incurable. As a new treatment of tumor gene therapy orbiology therapy is thought highly of increasingly.Stat3 is an important member of signal transducers and activators oftranscription (STAT) family. Stat3 signaling pathway has been shown to play akey role in promoting proliferation, differentiation, antiapoptosis, and cell cycleprogression. Persistently active Stat3, and its overexpression have been detectedin human prostate cancers and have been suggested to be associated withprostate cancer progression. Aberrantly active Stat3 promotes uncontrolledgrowth and survival via dysregulation of expression of downstream targetedgenes, such as cyclin D1, c-Myc, Bcl-xL, Bcl-2, Mcl-1, and VEGF;these genesinfluence cell cycle progression or inhibit apoptosis. Constitutive Stat3 signalingrepresents one of the key molecular events in the multistep process leading tocarcinogenesis. Thus, Stat3 represents a potential molecular target for therapeuticintervention of prostate cancer.Small RNA (siRNAs) are short double-stranded RNA molecules that cantarget complementary mRNAs for degradation via a cellular process termedRNA interference (RNAi). In previous studies, we applied a DNA vector -basedStat3-specific RNAi approach to block Stat3 signaling, and this procedure byinhibition of Stat3 and its related genes suppressed growth induction of apoptosisin both prostate cancer cells in vitro and in prostate tumors implanted in nudemice. However, in mammalian cells, RNAi can not completely block geneexpression, especially for abnormally expression gene. In order to seek a bettertherapeutic regimen and to enhance the curative effect, we have developed acombined therapy involving the use of attenuated Salmonella typhimurium asa tumor-targeting anticancer agent and with the plasmid co-expressedsiRNA-Stat3 and GRIM-19 carried by this bacteria for prostate cancertherapy.GRIM-19 (gene associated with retinoid-IFN-induced mortality 19) wasoriginally isolated as a growth suppressive gene product in the IFN-β/RAinduced cell death pathway using a genetic screen. More recently, mutations inthe human GRIM-19 coding region have been found in the H?rthle cell thyroidcarcinomas. Subsequent studies showed that expression of GRIM-19 is eitherlost or severely depressed, in a number of human renal carcinoma (RCC) and insome other tumors (personal communication). Although the biological relevanceof GRIM-19 to human cancer is unclear, some reports suggest that GRIM-19 isan inhibitor of transcription factor Stat3. The resultant, down regulation ofGRIM-19 promotes tumor growth via an augmentation of Stat3 dependent geneexpression, and GRIM's tumor suppressor function appears to be modulated viainactivation of Stat3. GRIM-19 interacts specifically with the transcription factorStat3 and inhibits Stat3-dependent gene expression. The transactivation domain(TAD) of Stat3 appears to be the binding target of GRIM-19, and this bindingmay result the inhibition for Stat3 activity. It is conceivable that down regulationof GRIM-19 provides growth advantage and overexpression enhances cell death.Thus, GRIM-19 acts as a double edged sword-that-on one hand ensures cellularenergy production as part of mitochondrion, and on the other functions as agrowth suppressive protein by inhibiting Stat3.At present, the primary limitation of cancer therapy is lack of selectivity oftherapeutic agents for tumor cells. Current efforts are focused on discovering anddeveloping anticancer agents that selectively target only tumor cells and sparenormal tissue to improve the therapeutic index. It has been known that attenuatedSalmonella typhimurium accumulate and replicate preferentially in tumors to alevel that is 1,000-fold greater than found in cells from normal tissues. S.typhimurium is a facultative anaerobic bacterium, which is capable of replicatingpreferentially in tumor cells and inhibition of growth, is associated with lysis oftumors with a necrotic/hypoxic centre. These observations suggest that the tumortargeting bacteria in combination with other antitumor agents, represents apromising strategy for the treatment of primary and metastatic tumors.Objective:Evaluation of our new conception i.e: using co-expressed plasmidscontaining siRNA-Stat3 and GRIM-19 and those plasmids will be carried byattenuated Salmonella bacteria and were induced to primary tumor andmetastatic tumors for developing more effective treatment for prostate cancer.Methods:(1) Construction of a recombinant plasmid co-expressing siRNA-Stat3 andGRIM-19:Using human Stat3 gene sequences from GenBank, selected suitable targetsite, synthesized oligonucleotides as DNA template encoding siRNA-Stat3,annealed and ligated into pSilencerTMneo 3.1-H1 siRNA expression vector toconstruct plasmid pSH1Si-Stat3;amplified H1 promoter and the sequence ofSiRNA-Stat3 by using pSH1Si-Stat3 plasmid as template by PCR, then clonedinto plasmid pcDNA3.1(+) eukaryotic expression vector, designed plasmidpH1Si-Stat3. Amplified the sequence of GRIM-19 gene by using pCXN2mycAGRIM-19 plasmid as template by PCR, then cloned into pH1Si-Stat3 vector toconstruct a plasmid pGRIM-19-Si-Stat3 that co-expressed both siRNA-Stat3 andGRIM-19.(2) Studies in vitro:The prostate cancer cell lines, PC-3M and RM-1, were transfected withplasmids pH1Si-Stat3, pGIM-19, or pGRIM-19-Si-Stat3. To determine theexpression levels of the Stat3 siRNA and GRIM-19 after transfection, thesemi-quantitative RT-PCR analysis, immunocytochemical detection and Westernblot analyses with the samples extracted from transfected and control cells wereperformed. The cells were also analyzed for cell cycle phase distribution andapoptosis rate by flow cytometry, and stained by Annexin V-CY3 apoptosisdetection kit to detect the early apoptosis. A stable cell line which could maintainpGC-Si-Stat3 plasmid in transfected cells was established. Gelatin zymographywas used to study the effects of the activities of MMP-2 and Transwell chambermigration assay was used to study the migration ability of cells, thoseexperiments are relevance to metastasis of cancer cells.(3) Studies in vivo:To study the effects of pGIM-19, pH1Si-Stat3 and pGRIM-19-Si-Stat3 onprostate tumor growth in vivo, we developed a nude mouse tumor xenograftmodel. Mice were transplanted via.s.c. with PC-3M cells into the right flank.On day 12, palpable tumors had developed, and the pGRIM-19, pH1Si-Stat3,pGRIM-19-Si-Stat3 plasmids and scramble control, mock control buffer wereinjected via.i.t. The effects of the different constructs on suppression of tumorswere analysed by various procedures. For example, evaluation of the role ofcombination therapy with tumor-targeted bacteria carrying plasmid containingantitumor molecules, the primary tumor and spontaneous metastasis micemodels were established by surgical orthotopic implantation in c57BL6 mice.Attenuated S. typhimurium carried pGC-si-Stat3 or pGC-scramble plasmids wereintroduced via i.v. or i.t. into the mice. The TUNEL assay was used to detect theapoptosis of tumor cells. Northern blot, RT-PCR and Western blot analysis wereused to detect the expression levels of related genes and proteins,Immunohistochemistries for Stat3,GRIM-19 and Ki-67 were performed todetermine the distribution of Stat3 and GRIM-19 expression. Gelatinzymography was used to detect MMP-2 expression in tumor tissue.Results:(1) The plasmids pH1Si-Stat3 containing siRNA-Stat3 and pGRIM-19-Si-Stat3 which was designed for co-expression of siRNA-Stat3 and GRIM-19were constructed, and confirmed by restriction enzyme digest and sequenceanalysis.(2) Stat3 is overexpressed in prostate cancer tissues and GRIM-19 isseverely depressed in prostate tumor tissues.We examined 29 samples of normal prostate tissues and 38 samples fromprostate cancer tissues. The results of immunohistochemical analyses showedthat GRIM-19 expression in normal prostate tissue is abundant and in prostatecancer tissue is severely depressed. In contrast, there is overexpression of Stat3in prostate cancer tissues compared with normal prostate tissues.(3) Transfection with pGRIM-19, pH1Si-Stat3, pGRIM-19-Si-Stat3inhibited the growth of PC-3M cells and induced apoptosis, and transfectionof co-expression plasmid pGRIM-19-Si-Stat3 showed a synergistic effect forsuppression of tumors.The results from semi-quantitative RT-PCR analysis, immunocytochemicaldetection and Western blot analyses for the samples from PC-3M cells aftertransfection with pH1Si-Stat3, pGRIM-19, and pGRIM-19-Si-Stat3demonstrated that all three plasmids containing siRNA-Stat3, GRIM-19 orsi-Stat3-GRIM-19 respectively, could specifically reduce Stat3 expression. ThepGRIM-19, and pGRIM-19-Si-Stat3 transfected groups showed: (i) that the totalStat3 and p-Stat3 expression levels were not reduced, (ii) that GRIM-19expression was obviously enhanced, and (iii) that the expression levels of Bcl-2,cyclin D1, c-Myc and VEGF were significantly depressed. The results of MTT,FCM and Annexin V-CY3 apoptosis detection assay demonstrated that thepGRIM-19-Si-Stat3 group showed remarkable apoptosis effects compared topH1Si-Stat3 and pGRIM-19 groups. Examination of subcutaneous tumors innude mice showed that in the group transfected with pGRIM-19-Si-Stat3, boththe average weights and volumes of the tumors were lower than those of othergroups. The results indicated a synergistic effect for suppression of tumor growthby the GRIM-19 and siRNA-Stat3 combination. The tumor tissue cellsdeveloped apoptosis;MMP-2 activity was suppressed, and so tumor's metastasiswas inhibited.(4) Establishment of stable cell line transfected with pGC-Si-Stat3 plasmidsand lost the ability to induce the tumor.pGC-Si-Stat3 plasmid containing siRNA-Stat3 and reporter GFP gene wasconstructed, which can express siRNA to inhibit Stat3 expression and enhancedgreen fluorescent protein too. The plasmid pGC-Si-Stat3 was transfected intoRM-1 cell lines, and a stable cell line has been selected, named RMpGC-Si-Stat3.The results from semi-quantitative RT-PCR analysis and Western blot analysesprovided strong evidence that siRNA-Stat3 can significantly suppress Stat3expression. The growth and survival of RM-1 cells were inhibited and apoptosisand G1 arrest of RM-1 cells in vitro were induced. The results of Western blotanalyses showed that Bcl-2, cyclin D1, c-Myc, VEGF expression was depressedin RMpGC-Si-Stat3 transfected cell line. MMP-2 activity was significantlysuppressed, and the invasive ability of these cells was obviously decreased.When mice were transplanted with stable cell line RMpGC-Si-Stat3 via s.c. atright flank, no tumors were induced during 60 days observation.(5) Systemic administration of attenuated S. typhimurium carrying pGC-Si-Stat3 for prostate cancer therapy.An animal model of prostate cancer demonstrating primary prostate tumorand spontaneous metastasis by surgical orthotopic implantation in c57BL6 micehas been obtained. By day 8, mice which were transplanted via. i.v. with prostatecancer cell line RM-1 developed tumors at the sites of transplantation and hadmetastasized to various organs. The group treated with attenuated S.typhimurium carrying pGC-si-Stat3 and pGC-scramble control plasmidsintroduced via.i.v. showed selectively growth suppression of tumors comparedwith mock and pGC-si-Scramble groups. In tumor-bearing mice, S. typhimuriumreplicated preferentially in tumor tissues over at least 1,000-fold in normaltissues. Average tumor weights and volumes in the mice treated withpGCSi-Stat3 carried by Salmonella were significantly lower than the controlgroups. The tumor cell metastasis was inhibited and the tumor tissue cellsdeveloped apoptosis.Conclusions:In this studies, we show for the first time that a combined-gene therapy withco-expression of SiRNA-Stat3 and GRIM-19 showed a synergistic effect withrespect to suppression of tumors in vitro and in a nude mice model, andpCGSi-Stat3 which was carried by attenuated S. typhimurium showed significantsuppression effect not only for primary transplanted tumor but also forprevention and inhibition of metastasis of prostate cancer.