Application Of Survivin Gene In Diagnosis And Biotherapy Of Transitional Cell Carcinama Of Bladder

Part I Clinical significance of Survivin expression in tumor tissues and urine exfoliate cells of patinets with transitional cell carcinoma of bladderBackground: Transitional cell carcinoma (TCC) of bladder is the most common malignant tumor in uropoiesis system. Up to date, there is still lack of an ideal marker for the diagnosis of TCC except CT and MRI imaging and cystoscopy. Cystoscopy is an invasive examination, which increases the possibility of urinary tract infection. Urine cytology has low sensitivity (21%~40%) in diagnosis of bladder cancer, especially for those with medium or high differentiation. Urologists are in the ongoing search for novel markers for the improvement of bladder cancer diagnosis and prognosis. Survivin is a 16.5kDa protein overexpressed in almost all malignancies but rarely detected in normal differentiated adult tissues. Functionally, Survivin has been shown to inhibit apoptosis, promote cell proliferation and enhance angiogenesis, even consistent with its role in these processes. Because of the large difference in expression between normal and malignant tissue and its causal role in cancer progression, Survivin is currently undergoing intensive investigation as a potential tumor marker. Emerging data suggests that measurement of Survivin can aid the early diagnosis of bladder cancer, determine prognosis in multiple cancer types and predict response to diverse anti-cancer therapies. These preliminary findings on the diagnostic, prognostic and predictive potential of Survivin should now be confirmed in large prospective trials. However, only a few studies have dealt with Survivin expression in TCCB with a small number of patients, and the results were controversial, moreover, the correlations between Survivin gene expression and pathological factors for bladder cancer remain unclear. Furthermore, assays for the measurement of Survivin should be simplified, standardized and evaluated in external quality assurance schemes. In the present study, both immunohistochemistry and RT-PCR were to examine Survivin expression in urine exfoliate cells and tumor tissues of patients for the purpose of comparing the differential expression of Survivin in non-TCCB, TCCB of different clinical or pathological parameters, and of determining whether expression of Survivin is associated with TCCB clinical outcomes. Objective:To investigate correlation of detections by two methods that were used to measure Survivin expression in urine exfoliate cells and tumor tissues of patients with TCCB, and further develop their clinical significances.Methods: All 48 specimens of pathologically confirmed TCCB in experimental group were collected in Wuhan Tongji Hospital during the surgical operation from 2004 Sep. to 2006 Sep.. Among those patients of 35 male and 13 female, their mean age was 56.4±2.6 years. Of 48 cases, 30 cases were first discovered and 18 cases relapsed. The pathological grade showed Grade I was 9 cases, Grade II was 30 and Grade III was 9 cases. The clinical stage was that Tis~T1 40 cases, T2~T4 8 cases. 16 specimens in control group were obtained from the patients (8 cases with glandular cystitis, 5 cases with BPH, 3 cases with cystophthisis) during bladder operations and were pathologically confirmed to be normal bladder tissue. Nested RT-PCR were respectively used to determine Survivin mRNA in urine exfoliate cells and tumor tissues of all cases in both experimental and control groups, meanwhile, immunohistochemistry was used to detect Survivin protein and identify the pathological grade and clinical stage in tumor tissues, as well as analysis of the relationship of Survivin expression and pathological parameters by statistics methods.Results:The positive expression rate of Survivin was 75.0%, and the mRNA expression detected by RT-PCR parallelly correlated with protein expression by immunohistochemistry in bladder tumor tissues from 48 TCCBs. The positive result of urine detection by RT-PCR was (34/48)70.8%, there was no significant difference of the expression rate between tissue specimen and urine specimen in TCCB patients(P>0.05). While no positive expression was found in urine and 1case of positive expression in non-neoplastic bladder tissues from 16 control cases. This indicated that the positive expression rate of Survivin in tumor tissues of TCCBs was significantly higher than that in bladder tissues of control group(P<0.05). The expression level of Survivin elevated as the grade and stage got higher in TCCBs. The positive rates in grade I, II, III were 55.6%, 76.7%, and 88.9%; in stage of Tis~T1, T2~4 72.5%, 87.5%; and primary, recurrent cases 65.5%, 89.5%, respectively. Multivariate Cox proportional hazards model analysis identified Survivin expression as an independent prognostic factor of disease-free survival (P=0.009, r=3.12). The positive expression of Survivin was strongly associated with pathological grade, clinical stage or relapse (P<0.05), but not with individual age, sex, tumor number, size, or growth mode.Conclusions:The up-regulated expression of Survivin in TCCB suggests that Survivin may play an important role in tumorigenesis and progression through inhibiting cell apoptosis. Besides, the higher the grade and stage are, the higher the expression level of Survivin is. Survivin is correlated with recurrence of TCCB. And detecting the Survivin expression in urine may be a new method for screening and diagnosis in early stage or monitoring recurrence after operation. High and specific expression of Survivin in tumor tissue would contribute it to be a new targeted gene in biotherapy of TCCB.Part II Antisense Oligodeoxynucleotide Targeting Survivin Expression Induces and Sensitizes Bladder Cancer Cells to ChemotherapyBackground: Diminished apoptosis plays a critical role in tumor initiation, progression, and drug resistance. Several IAPs that inhibit apoptosis have been identified. Survivin, a novel gene encoding a structurally unique apoptosis inhibitor IAP, is abundantly expressed during fetal development, and almost undetectable in terminally differentiated adult tissues. However, Survivin is prominently expressed in transformed cell lines and in all the most common human cancers in vivo, including transitional cell carcinoma of bladder (TCCB). It is also a bifunctional protein that acts as a cell division regulator and an apoptosis suppressor. To evaluate the abilities of inhibiting Survivin expression and enhancing sensibility to chemotherapy, A Survivin-ASODN eukaryotic vector pcDNA3-SVVas was prepared in previous study.Objective: To evaluate the effects of antisense oligodeoxynucleotide(ASODN) of Survivin gene on MMC-induced apoptosis in bladder cancer cell line T24.Methods: A Survivin-ASODN eukaryotic vector pcDNA3-SVVas prepared in previous study was delivered into T24 mediated by liposomal reagent. The mRNA expression of Survivin measured by RT-PCR. Cell survival fraction was determined using the trypan blue dye exclusion assay. Cell counting method and MTT colorimetry. were tested to investigate the sensibility of transfected cells to MMC. Induced Apoptosis was detected by DNA gel electrophoresis and nuclear staining,as well as apoptosis rate by FCM.Results: We obtained two positive cell clone T24-SVVas and T24-neo by liPofectin. Compared to T24 and T24-neo cells, the mRNA expression of Survivin in T24-SVVas cells decreased, as well as significantly inhibited in T24-SVVas cell growth (p<0.05). The sensibility of T24-SVVas cells to MMC increased significantly. Agarose gel electrophoresis of genomic DNA from T24 SVVas displayed typical DNA ladder, but DNA from control groups did not. Nuclear staining found cellular nuclei become condense in T24-SVVas cells. FCM showed that cell apoptosis rate of T24-SVVas with MMC is significantly higher than that of other groups.Conclusion: As the human Survivin-ASODN could decrease the expression of Surviving to enhance MMC-induced apoptosis in bladder cancer cell line T24 in vitro and sensitize T24 cells to chemotherapy, this may lay an experimental foundation for further research of biotherapy in bladder cancer, it also deserves further investigation as a novel approach to selective tumor therapy. Part III Survivin-targeted siRNA induces apoptosis in human bladder cancer T24 cell linesBackground: Bladder cancer has been known as the most common type of urinary system tumours in China. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin is recognized as a general target in cancer therapy because of its selective overexpression in the majority of tumors. In bladder cancer (BCa), its expression correlates with tumor grade, recurrence risk and survival. By inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. RNA interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression, which would cause strong and stable degradation of the specific mRNA and decreased protein expression. For these reasons, we studied the influence of small interfering RNA (siRNA) targeting Survivin on the growth and apoptosis of bladder cancer cells.Objective: This study was aimed to investigate the effect of T24 cell growth, apoptosis, and sensitivity to chemotheraputic drug MMC by small interferencing RNA silencing Survivin gene in human bladder transitional cell carcinoma (BTCC).Methods: The eukaryotic vector pGenesil-1 was designed to contain 3 Survivin-targeted siRNA, promoter U6, and marked protein EGFP. Liposomal transfection by LipofectamineTM2000 was respectively performed for pGenesil-1-SVVsi and a blank vector pGenesil-1 delivered into T24, positively transfected cells (T24-SVVsi and T24-neo) were harvested to continue next tests after transfection for 48h, as well as no transfection T24 as control. Survivin protein detected by using Westernbloting and mRNA expression was quantified by real time RT-PCR; The viability of 3 groups of treated cell was examined by using MTT and Colony formation assays were performed to evaluate the long-term survival of treated cells; Morphological change was observed with transmission electron microscope, Cell cycle analysis and apoptosis dectection of treated cells was carried out by FACS after double staining cells with propidium iodide (PI) and FITC-labeled Annexin V; MTT assay and cell counting method was performed to evaluate the sensibility of T24 cells to chemotherapeutics MMC.Results: Abnormal cell morphological change was found in T24-SVVsi, no apoptotic evidences were observed in 2 control groups; In T24-SVVsi group the mRNA and protein expression quantified by real time RT-PCR and Westernbloting was reduced by at least 50%; T24-SVVsi showed a significant reduction (up to 47%) of viability; Cell cycle analysis and quantitation of apoptosis revealed both a specific G2/M arrest and an induction of apoptosis, as well as the occurrence of multinucleated cells. only T24-SVVsi group exhibited a prolonged duplication time up to 1.4-fold at 48 h after treatment. Furthermore, compared to control groups, the half inhibition concentration (IC50) of MMC to bladder cancer cells in T24-SVVsi group was significantly reduced by MTT assay, the difference was significant by statistical analysis.Conclusion: Survivin-siRNA may silence the Survivin expression to induce T24 cells apoptosis, inhibit cell proliferation and sensitize T24 cells to chemotheraputic drug (MMC). This suggested that the anti-Survivin siRNA treatment might represent a suitable therapeutic approach to selectively inhibit growth of BCa cells in addition to commonly applied therapy schemesPart IV Effect of Survivin-targeted complex system on transitional cell carcinoma of the bladderBackground: Bladder cancer is the most common cancer of urinary deseases in China. Survivin, a member of inhibitor of apoptosis protein (IAP) gene family, is abundantly expressed in a wide range of malignancies, including carcinoma of the bladder urothelium. Our previous study demonstrated Survivin as a novel intervention target to suppress the growth and induce apoptosis in cancer cells by antisense oligodeoxynucleotide (ASODN) or small interfering RNA(siRNA). But the efficiency of their inducing gene knockdown is hampered by low transfection efficiency; also several gene-transected approaches limited in vivo application due to their toxicity or damage. Furthermore, the mechanism by which Survivin plays a role in the regulation of cell death is still unknown. In the present study, basing the previous foundation that TfR is overexpressed on the surface of TCC and TfR-ScFv is a ideal tumor-targeted molecule by carrying expression vector to intra-cellular internalization,we established the Survivin-targeted complex system to investigate its biological effect on bladder cancer cell.Objective: By mixing double expression vector and target molecule TfRscFv-GAL4 at a adopted ratio in vitro experiment, we first established the Survivin-targeted complex system(TfRScFv-GAL4-GAL4rec-SurvivinsiRNA–pGes) to incubate with T24 cell line and investigate its biological effectMethods:①4.5% native polyacrylamide gel electrophoresis was used to identify the conjugated target system(TfRScFv-GAL4-GAL4rec-SurvivinsiRNA–pGes) of renatured fusion protein TfRScFv-GAL4 and recombinant vector GAL4rec-SurvivinSiRNA-pGes at molar ratio of 1:2.5;②The expression rate of green fluorescent protein(GFP) in T24 was determined by FCM;③The targeted complex intake mediated by the fusion protein TfRScFv-GAL4 and effector expression were observed through inversted fluophot.④Survivin protein measured by Western-blot and mRNA expression was quantified by real time RT-PCR;⑤Growth inhibition of T24 cells was determined by use of the colorimetric MTT⑥It was observed using Hochest staining kit that tumor cell apoptosis induced by the targeted complex system TfRScFv-GAL4- GAL4rec-SurvivinSiRNA-pGes.⑦Cell cycle analysis and apoptosis dectection of treated cells was carried out by FACS with propidium iodide (PI) and FITC-labeled Annexin V Results:①The only one protein band could be seen in the TfRScFv group with different concentration of GAL4rec-SurvivinSiRNA-pGes in 4.5 % native polyacrylamide gel electrophoresis, but the moved protein band was dose-dependent with addition of vector GAL4rec- SurvivinSiRNA-pGes in GAL4 and TfRScFv-GAL4 group, these results showed fusion protein TfRScFv-GAL4 could specially bind to GAL4rec-SurvivinSiRNA-pGes by GAL4 fragment, and the molar rate is 1:2.5.②The expression rate of GFP is 44.30%.③Of all groups, two groups of the targeted complex system and TfRScFv-GAL4-GAL4rec-pGes were co-cultured with TCCB T24 cell lines for 48h, it was observed the green fluorescin in the tumor cell endochylema but not in other control groups through fluorescent microscope. Also microscope could find that cytoplasm condensed in some adherent cells and cell bodies appeared rotund like the apoptosis cell morphous in the targeted complex system group, whereas the adherent cells grow well in the control groups.④In targeted complex system group the mRNA and protein expression was reduced by at least 53%;⑤It showed a significant reduction of viability and strong inhibition in cells of targeted complex system group (P<0.05);⑥in the cell nucleus staining test, it was found that numerous apoptosis cell as well as the cells with condensed cytoplasm and rotund bodies by rebuilding their pictures in the targeted complex system group.⑦Cell cycle analysis and quantitation of apoptosis revealed both a specific G2/M arrest and an induction of apoptosis by 48%. Conclusion: It firstly demonstrated the complex system TfRscFv-GAL4-GAL4rec-SurvivinsiRNA-pGes could be intaken into endochylema to express the GFP, and then siRNA induce malignant cells apoptosis. This suggested that TfRScFv-GAL4 has the tumor-targeted characteristic and could induce the expression of eukaryotic bifuction vector in the tumor cells. The investigation also suggested the targeted complex system against Survivin could remarkably inhibit proliferation of T24 and induce apoptosis. But not affect the normal cells.