| Bladder cancer is one of the most common urological malignant diseases in the world. Bladder cancer was the fourth in men and the eighth in women most common cancer respectively. In 2006, in Europe, an estimated 104,400 incident cases of bladder cancer were diagnosed, of which 82,800 were diagnosed in men and 21,600 in women. This represents 6.6% of the total cancers in men and 2.1% in women, with an estimated male-to-female ratio of 3.8:1. Bladder cancer resulted in 4.1% of total deaths for cancer in men and 1.8% of total deaths in women. At the initial diagnosis of bladder cancer, 70% of cases are diagnosed as non-muscle-invasive disease and 30% as muscle-invasive disease. The most important performances of urothelial cancers of the bladder are metachronous or synchronous multifocal occurrence with high recurrence .Operation especially radical cystectomy is the standard treatment for muscle invasive bladder cancer in most countries in the world. However when patients , with an initially non-insive and invasive bladder cancer, presenting with pT0 or T1 status at first resection are selected for transurethral resection of bladder tumour(TURBT)alone, about 60-99% of them will recurs and 15-20% of them progressed during organ-preserving treatment. Organ-preserving treatment such as transurethral resection of bladder tumour, followed by neoadjuvant chemotherapy, radiotherapy and immune revulsive, was identified as a prognostically important variable. However these therapeutic means we use can't stop the progression and recurrence of bladder cancer and at the same time the side effects of chemotherapy, radiotherapy and drugs fast in patients remain the tough bladder-preserving strategy, the bladder remains a potential source of recurrence. Therefore many scholars is still exploring new effective treatments of bladder cancer. Gene therapy was identified as that functional genes are transfected into the patients'body to correct the defective gene or block the overexpressed gene for treatment and prevention strategies of disease. For more than twenty years research gene therapy has gradually become mature and is accepting as extremely promising approach to treating cancer after regular treatment such as operation, chemotherapy and radiotherapy. Human being pay close attention to targeted therapy today. Inhibing the expression of activated oncogene in cancer cells has gradually been one trend of gene therapy.The discovery that RNAi works in mammalian cell function, its use as a tool to understand or screen for genes functioning in cellular pathways in normal and diseased cells and animals, and its potential for therapeutic gene silencing.Signal transducers and activators of transcription( Stat)proteins comprise a family of transcription factors latent in cytoplasm that consists of seven different members. They were discovered in the course of studies of signaling specificity from IFN receptors.The Stat transcription factors family are activated by a series of extracellular signaling proteins such as cytokine, growth factors, and Hormones that bind to specific cell-surfacer eceptors. The resulting signal transduction pathways permit them to play different roles in normal physiological cell processes, such as differentiation, proliferation, apoptosis and angiogenesis. However, aberrant activation of Stat Signaling gives rise to different pathological events, for example, cell transformation and oncogenesis. In addition, constitutively activated Stat3 induces cellular transformation in vitro and tumor formation in nude mice.Thus, Stat3 may be an important molecular target for tumor therapy. Stat3, a member of the Stat family, is a key signal transduction protein that mediates signaling by numerous cytokines, peptide growth factors, and oncoproteins. Accumulating evidence demonstrates that Stat3 activation plays important roles in cell diferentiation, proliferation, development and apoptosis. Elevated activity of Stat3 has been detected frequently in a lot of human tumors, including carcinoma of rectum, hematologic malignancies, head and neck, breast and prostate cancer. Cell strains from multiple myelomas that have become growth factor independent require constitutively active Stat3 to protect against apoptosis. In addition, constitutively activated Stat3 induces cellular transformation in vitro. Stat3 participates in oncogenesis through up-regulation of genes encoding apoptosis inhibitors (Bcl-xL, Mcl-1 and survivin), cell-cycle regulators (cyclin D1 and c-Myc) and inducers of angiogenesis vascular endothelial growth factor (VEGF).Stat3 is activated by phosphorylation at tyrosine residue 705 (Y705), which leads to dimer formation, its nuclear translocation, DNA binding and target gene transcription. Phosphorylation of Stat3 at tyrosine 705 (Y705) is critically its dimerization and nuclear translocation; both prerequisites of Stat3 activation. Thus, we considered that inhibiting this phosphorylation at residue 705 might reduce VEGF expression in tumor cells.Thus, the Stat3 signaling pathway may represent a potential molecular target for novel therapeutic approaches for bladder cancer. As outlined below, Stat3 can be targeted at one or more steps. Strategies to target Stat3 include decreasing Stat3 levels, reducing tyrosine phosphorylation of Stat3 and receptor complexes, reducing Stat3 recruitment to receptor complexes and its dimerization, and reducing Stat3 binding to promoters. Studies have shown that using a variety of approaches, such as tyrosine kinase inhibitors such as tyrphostin AG490, Antisense oligonucleotides, decoy oligonucleotides, and dominant-nagative Stat3 proteins can inhibit Stat3 expression in cancer cells to surpress proliferation and induce apoptosis.Small interfering RNA (siRNAs)are short doubles tranded RNA (dsRNA)Molecules that can target complementary mRNAs for degradation via a Cellular process termed RNA interference (RNAi). RNAi is usually activated by introducing long double-stranded RNA molecules into cells which are cleaved into 21-nt to 23-nt RNAs referred to as siRNAs by an endonuclease named Dicer in animal cells. The siRNA molecules then serve as a guide for sequence-specific degradation of homologous mRNAs. siRNA has been used for functional analysis of genes in many species including invertebrates, plants, and mammalian cells. Recently, siRNA has emerged as powerful RNAi reagents for gene silencing and inhibition of viral propagation. The potential of using siRNA for silencing specific genes has been demonstrated in treatment of viral diseases and cancer.At present, the primary limitation of cancer therapy is lack of selectivity of therapeutic agents for tumor cells. Current efforts are focused on discovering and developing anticancer agents that selectively target only tumor cells and spare normal tissue to improve the therapeutic index. It has been known that attenuated Salmonella typhimurium accumulate and replicate preferentially in tumors to a level that is 1,000-fold greater than found in cells from normal tissues. S. typhimurium is a facultative anaerobic bacterium, which is capable of replicating preferentially in tumor cells and inhibition of growth, is associated with lysis of tumors with a necrotic/hypoxic centre. These observations suggest that the tumor targeting bacteria in combination with other antitumor agents, represents a promising strategy for the treatment of primary and metastatic tumors.The present study was designed to investigate the potentiall use of siRNA to block Stat3 expression, its efect on growth and apoptosis of human bladder cancer cells, and effect on tumor formation in Balb/c nude mice.We construct pSHlSi-Stat3 and transfected it in bladder cancer cells to observe its inhibition effect on Stat3 expression and its effect on growth, proliferation and apoptosis of bladder cancer cells T24. We further use attenuated S. typhimurium carrying Si-Stat3 to observe its therapeutic effect on nude mice transplantation tumor. Our results showed that targeting Stat3 signaling using siRNA technique may serve as a novel therapeutic Strategy for treatment of bladder cancer.However, to our knowledge, no reports have been published to date concerning the effect of siRNA against the Stat3 gene in bladder cancers cells. In our studies we have used T24 bladder cancer cells, which have shown to be of value for the study of bladder cancer.Study objectives:(1) to determine if Stat3 siRNA can inhibit the expression of Stat3 gene in bladder cancer cells in vivo and in vitro.(2) to explore that the attenuated S. typhimurium with siRNA-Stat3 plasmids were induced to primary tumor and subcutaneous transplantation tumor model for developing more effective treatment for bladder cancer.Methods:(1) Study on the Expression of Stat3, Survivin, VEGF in tissue of Bladder Transitional cell carcinoma and their Clinical Significances(2) Construction of a recombinant plasmid expressing Stat3 siRNAUsing Stat3 gene sequences from GenBank, selected suitable target site,synthesized oligonucleotides as DNA template encoding Stat3 siRNA, annealed and ligated into pSilencerTM neo3.1-H1 expression vector to construct plasmid pSilencerTM neo3.1-H1-Stat3(pH1Si-Stat3, Si-Stat3).(3) Studies in vitro:The bladder cancer cell lines were transfected with plasmids pH1Si-Stat3. To determine the expression of the Stat3 at 72h after transfection, semi -quantitative RT-PCR analysis , Western blot and immunocytochemical detection analyses with the samples extracted from transfected and control cells were performed. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry, and stained by acridine orange to detect the early apoptosis.MTT assay showed that the growth-inhibition rate of the Stat3 siRNA groups was significantly higher than that of the vehicle group or plasmid group.(4) Studies in vivo:To study the effects of pHlSi-Stat3 on bladder tumor growth in vivo, we developed a nude mouse tumor xenograft model.First of all, mice were transplanted via.s.c. with T24 cells into the left hip for tumor-bearing nude mice.Then the primary tumor and spontaneous metastasis mice models were established by surgical orthotopic implantation in nude mice. On day 14 and 21, palpable tumors had developed in the spontaneous metastasis mice models, and Attenuated S. typhimurmm carried Si-Stat3 or scramble plasmids were introduced via intratumor injection into the mice. On day 10 and 17, the primary metastasis bladder tumors had developed in the primary bladder cancer metastasis models of nude mice, and Attenuated S. typhimurmm carried Si-Stat3 or scramble plasmids were introduced via intraperitoneal injection into the mice.The HE staning was used to detect the level of the tumor tissues.The TUNEL assay was used to detect the apoptosis of tumor cells. RT-PCR and Western blot analysis were used to detect the expression levels of related genes and proteins. Immunohistochemistries for Stat3, VEGF, Survivin, Bcl-2 and Ki-67 were performed to determine the distribution of their related proteins.Results:(1) Stat3, Survivin,VEGF protein showed positive expression of 64%, 56% and 68% respectively in bladder cancer tissue than in normal bladder mucosa with statistical significience(p<0.01).The expression rate of Stat3, Survivin,VEGF were strongly correlated whit pathological grade and clinical statge of the TCC (P<0.01) . And these three factors were showed hightly negative correlation with prognosis of these patients(with p<0.01;p<0.01; p<0.05, respectively) .(2) The plasmids pHlSi-Stat3 containing siRNA-Stat3 were constructed, and confirmed by restriction enzyme digest and sequence analysis.(3) Transfection with pHlSi-Stat3 inhibited the growth of T24 cells and induced apoptosis effect for suppression of tumors. The results from semi-quantitative RT-PCR analysis, immunocytochemical detection and Western blot analyses for the samples from T24 cells after transfection with Si-Stat3, demonstrated that the plasmids containing Si-Stat3, could specifically reduce Stat3 expression. The Si-Stat3 transfected groups showed: (i) that the total Stat3 expression levels were not reduced, (ii) that the expression levels of Bcl-2, cyclin D1, survivin and VEGF were significantly depressed. The results of MTT, FCM and acridine orange detection assay demonstrated that the Si-Stat3 group showed remarkable apoptosis effects compared to untreated and scramble groups. Examination of subcutaneous tumors in nude mice showed that in the group transfected with Si-Stat3, both the average weights and volumes of the tumors were lower than those of other groups. The results indicated the effect for suppression of tumor growth by the siRNA-Stat3.(4) Systemic administration of attenuated S. typhimurium carrying Si- Stat3 for bladder cancer therapy.An animal model of bladder cancer demonstrating primary bladder tumor and subcutaneous metastasis by surgical orthotopic implantation in nude mice has been obtained. In primary bladder tumor model and subcutaneous metastasis bladder tumor model ,the group treated with attenuated S. typhimurium carrying Si-Stat3 and Si-scramble plasmids introduced via. intratumor injection or via intraperitoneal injection showed selectively growth suppression of tumors compared with untreated and Si-Scramble groups, respectively. Average tumor weights and volumes in the mice treated with Si-Stat3 carried by Salmonella were significantly lower than the untreated and scramble groups. Average tumor weights and volumes in the scramble groups notable showed lower than that of the untreated. The tumor tissue cells developed apoptosis.Conclusions:(1) we successfully constructed pH1Si-Stat3 plasmid.(2) In this studies, we show for the first time that a bladder cancer therapy with SiRNA-Stat3 showed a remarkable effect with respect to suppression of tumors in vitro and in a nude mice model, and Si-Stat3 which was carried by attenuated S. typhimurium showed significant suppression effect for primary transplanted tumor growth of bladder cancer. RNAi technique may be a promising tool for cancer therapy.(3) Stat3 signaling pathway is an ideal molecule target to gene therapy of bladder cancer. |
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