MicroRNA-186 regulates the invasion and metastasis of bladder cancer via vascular endothelial growth factor C.

The present study aimed to investigate the expression of microRNA (miRNA or miR)-186 in tumor tissue, blood and urine from patients with bladder cancer. The mechanism by which miR-186 regulates the invasion and metastasis of bladder cancer was also assessed. A total of 76 patients who underwent surgical resection of bladder cancer tissues between August 2012 and January 2016 were included in the present study. Blood and urine samples were also collected from the 76 patients and another 66 healthy subjects. Expression of vascular endothelial growth factor C (VEGF-C) mRNA and miR-186 was measured using reverse transcription-quantitative polymerase chain reaction. Western blot analysis was performed to assess VEGF-C protein expression in tumor tissues. The content of VEGF-C protein in blood and urine samples was measured using an enzyme-linked immunosorbent assay. To identify the direct interaction between miR-186 and VEGF-C mRNA, a dual luciferase reporter assay was performed. The present findings demonstrated that VEGF-C mRNA expression in tumor tissues, blood and urine of bladder cancer patients was upregulated. VEGF-C protein expression in bladder cancer tissues was also enhanced. VEGF-C protein content in blood and urine from bladder cancer patients was elevated, consistent with the results for VEGF-C mRNA. Expression of miR-186 was reduced in tumor tissues, blood and urine. Dual luciferase reporter assay demonstrated that miR-186 regulated the expression of VEGF-C by binding with its 3'-untranslated region. Therefore, the results of the present study indicate that the expression of VEGF-C mRNA and protein is upregulated in tumor tissues, blood and urine from patients with bladder cancer, while that of miR-186 is downregulated in these samples. miR-186 potentially regulates the invasion and metastasis of bladder cancer via VEGF-C, and may become a gene marker for bladder cancer in the future.

Exploration of the pathways and interaction network involved in bladder cancer cell line with knockdown of Opa interacting protein 5.

OBJECTIVES: In our previous study, we displayed that knockdown of Opa interacting protein 5 (OIP5) inhibited cell growth, disturbed cell cycle and increased cell apoptosis in bladder cancer (BC) cell line. Our present study aimed to explore the underlying pathways and interaction network involved in the roles of OIP5 in BC. METHODS: Microarray analysis was conducted to obtain mRNA expression profiling of OIP5 knockdown (shOIP5) and control (shCtrl) BC cell lines. Bioinformatics analyses were performed including differentially expressed mRNAs (DEGs) identification, protein-protein interaction network construction, biological functions of prediction and ingenuity pathways analysis (IPA). Western Blotting (WB) was subjected to validate the protein expression levels of candidate DEGs in shOIP5 BC cell line. RESULTS: Respective 255 up- and 184 down-regulated DEGs were identified in shOIP5 group compared with shCtrl group. In the PPI network, CAND1 and MYC had the highest connectivity with DEGs. 439 DEGs were significantly enriched in inflammatory response, regulation of cell proliferation, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and bladder cancer. In the disease and function enrichment, DEGs were obviously involved in cellular movement, cellular growth and proliferation, cancer, inflammatory response, cell death and survival. In the OIP5 regulatory network, CDH2, IRS1, IRAK3, ID1, TNF, IL6, ITGA6, MYC and SOD2 interacted with OIP5. The WB validation results were compatible with our bioinformatics analyses. CONCLUSIONS: OIP5 interaction network might function as an oncogene in BC progression based on aberrant inflammatory responses. Our study might provide valuable information for investigation of tumorigenesis mechanism in BC.

Opa interacting protein 5 acts as an oncogene in bladder cancer.

PURPOSE: To explore the biological functions and mechanism of Opa interacting protein 5 (OIP5) in bladder cancer (BC). METHODS: We investigated the expression of OIP5 in BC through immunohistochemical staining (IHC) and its correlation with clinicopathologic features of BC patients. Moreover, knockdown of OIP5 was performed in BC cell lines and colony formation capacity, cell growth curve, cell cycle phase and cell apoptosis assay was applied for investigating the roles of OIP5 in BC. Moreover, the expression of OIP5 was validated through the Cancer Genome Atlas (TCGA) database. The diagnosis value of OIP5 was accessed by receiver operating characteristic (ROC) analysis in TCGA database. RESULTS: The expression of OIP5 in BC tissues was significantly higher than that in adjacent non-tumor tissues and bladder mucosa tissues with chronic cystitis. Higher protein expression level of OIP5 predicted shorter survival time in patients with BC, which was significantly correlated with larger tumor size, high-grade tumor and advanced T classification. The expression of OIP5 was considerably decreased after lentivirus infection both at mRNA and protein levels. Functional assay displayed that silencing of OIP5 inhibited colony formation capacity and cell growth in BC cell lines. Cell cycle assays indicated that suppressed OIP5 disturbed the balance of the cell cycle in BC cell lines, which increased the cell population of the G1 phase and decreased the cell population of the S phase. Furthermore, knockdown of OIP5 expression enhanced cell apoptosis process. The expression of OIP5 was significantly up-regulated in BC compared with adjacent non-tumor tissues based on TCGA database. OIP5 had the potential diagnostic value for BC. CONCLUSIONS: Our work demonstrated that OIP5 might function as an oncogene to promote colony formation capacity and cell growth, arrest cell cycle and suppress cell apoptosis in bladder cancer.

Application of transrectal ultrasound-guided repeat needle biopsy in the diagnosis of prostate cancer in Chinese population: A retrospective study.

BACKGROUND: Transrectal ultrasound-guided repeat needle biopsy (TUGRNB) is widely used for diagnosis of prostate cancer (PCa). However, significance of TUGRNB in Chinese population was rarely reported. A retrospective study was conducted to evaluate the significance of TUGRNB applied in prediction of PCa in Chinese population. MATERIALS AND METHODS: A total of 960 from January 2009 to December 2012 were included. Repeat needle biopsy rate and PCa positive detection rate were evaluated. Relationship between prostate specific antigen (PSA) levels and PCa positive rates was analyzed. RESULTS: PCa positive detection rate after initial needle biopsy was 28.4%, which was lower than the rate of repeat needle biopsy (40%). The rate for immediate transurethral resection (TUR), surgery after initial needle biopsy, was 27.1%, however with a low PCa positive detection rate (0.66%). The repeat needle biopsy rate was lower compared with the initial biopsy rate (P < 0.05). Meanwhile, immediate TUR rate was significantly higher than that of the repeat needle biopsy rate (P < 0.05). Among the three groups, the PCa positive detection rate in repeat needle biopsy group was the highest. In subgroups with different PSA levels, the PCa positive rate increased with the elevation of PSA level. In cases with PSA > 20 ng/ml, PCa positive rate was significantly higher than those with PSA < 20 ng/ml (P < 0.05). CONCLUSION: PCa positive detection rate following repeat needle biopsy in Chinese population was higher, although the repeated needle biopsy rate was still in a low level. TUGRNB should attract more attention in the diagnosis of PCa.

Protective effect of Rhodiola rosea polysaccharides on cryopreserved boar sperm.

Cryopreservation brings sublethal damage to sperm, resulting in reduced fertile life of sperm. Rhodiola rosea polysaccharides (RPs) have antiviral, antioxidant and antitumor activities. In the present study, the cryoprotective effect of RPs on boar sperm quality parameters after frozen-thawed process was investigated. Boar sperm was cryopreserved in the extender with RPs added at concentrations of 0 (used as control), 2, 4, 6, 8 and 10mg/L and their effects on the quality of frozen-thawed boar sperm were assessed. Addition of RPs significantly improved sperm motility, mitochondrial activity, acrosomal integrity, plasma membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased sperm malonaldehyde level (p<0.05). The results indicated that the addition of RPs to the freezing extender decreased the cryodamage to the boar sperm.

Alloantigen-specific T-cell hyporesponsiveness induced by dnIKK2 gene-transfected recipient immature dendritic cells.

Immature dendritic cells (iDCs) have been shown to be able to induce peripheral T-cell tolerance through distinct pathways. Here, we investigated the tolerogenic property of recipient iDCs whose maturation was arrested by a dominant negative mutant of inhibitor of nuclear factor kappa-B kinase 2 (dnIKK2) gene. We found that dnIKK2-iDCs presented a typical semi-mature morphology and expressed lower levels of CD80 and CD86, slightly higher MHC-II than untransfected iDCs. The expression of these molecules had no significant change even dnIKK2-iDCs were pulsed by donor antigen. In primary mixed leukocyte reaction (MLR), dnIKK2-iDCs exhibited impaired ability to stimulate allogeneic T-cells, but induced CD4(+)CD25(-) T-cell formation. In co-culture MLR, these CD4(+)CD25(-) T-cells suppressed T-cell alloreaction in an antigen-specific manner. Besides, CD4(+)CD25(-) T-cells inhibited IL-2 and IFN-γ release, whereas promoted IL-10 and TGF-ß secretion. These data suggested recipient dnIKK2-iDCs could maintain peripheral tolerance through down-regulating costimulatory molecule expressions and inducing CD4(+)CD25(-) T-cell formation.

[The value of histopathologic examination of testis tissue from testicular sperm aspiration].

OBJECTIVE: To evaluate histopathologic examination of the testis tissue from testicular sperm aspiration (TESA). METHODS: We analyzed the results of inverted microscopy and histopathologic examination of 96 samples of testis tissue from TESA, and compared the accuracy of the two methods in detecting sperm in the testis tissue. RESULTS: Among the 11 cases in which sperm was found by inverted microscopy, 9 were confirmed by histopathologic examination, and among the 57 cases in which sperm was not detected by inverted microscopy, 11 (19.3%) were found with sperm by histopathologic examination. Histopathologically, the cases in which sperm was not found by inverted microscopy included Sertoli-cell-only syndrome (n = 34), maturation arrest (n = 12) and hypospermatogenesis (n = 11). CONCLUSION: Histopathologic examination may reveal sperm in the TESA testis tissue proved to be sperm-absent by microscopy, and thus offer valuable information for a second testicular sperm retrieval.

Prolongation of rat renal allograft survival by CD4⁺CD25⁻ T cells induced by recipient dendritic cells transfected with IKK2dn.

Previous studies have demonstrated that recipient-derived immature dendritic cells transfected by recombinant adenovirus-mediated IKK2dn (Adv­IKK2dn) and loaded with donor splenocyte lysate generate CD4+CD25- T cells (Adv-IKK2dn-CD4+CD25- T cells). These cells may inhibit T cell responses in vitro. In the present study, Lewis (LW) rats were administered with an intravenous injection of naive CD4+ T cells, empty adenovirus (Adv-0)-dendritic cell-generated CD4+CD25- T cells (Adv-0-CD4+CD25- T cells), Adv-IKK2dn-CD4+CD25- T cells or an equal volume of normal saline, seven days prior to transplantation. The potency and the mechanism of action of Adv-IKK2dn-CD4+CD25- T cells was analyzed, as well as an investigation of their tolerogenic properties in vivo. Administration of Adv-IKK2dn-CD4+CD25- T cells in vivo to LW rats was observed to markedly prolong the survival of a kidney allograft from Brown Norway rats. Furthermore, the Adv-IKK2dn-CD4+CD25- T cell-treated group exhibited significantly reduced levels of interleukin (Il)-2 and interferon-γ production and increased Il-10 and transforming growth factor-ß (TGF-ß) secretion. The serum creatinine levels remained at low levels in the Adv-IKK2dn-CD4+CD25- T cell-treated group. Their ability to induce allogeneic T cell proliferation was markedly reduced compared with the other groups. These observations indicated that Adv-IKK2dn-CD4+CD25- T cells induce prolongation of kidney allograft survival in vivo, which is hypothesized to be due to the high expression levels of Il-10 and TGF-ß.

Prostate cancer in the senior men from rural areas in east district of China: contemporary management and 5-year outcomes at multi-institutional collaboration.

OBJECTIVE: Prostate cancer is an underreported and emerging problem in China. Here we summarize the data for Chinese patients with prostate cancer (PCa), describe available treatment options, and report 5-year outcomes at multiple tertiary care institutions. PATIENTS AND METHODS: A series of 1611 patients (mean age 76.51 years) diagnosed with PCa were enrolled. Survival rates for patients were analyzed using the Kaplan-Meier method. Prognostic factors for disease-specific survival were analyzed using the log-rank test and Cox proportional hazards model. RESULTS: Seven hundreds and thirty-two patients with a prostate tumor clinical stage of III or IV and 879 with a tumor clinical stage of I or II were diagnosed. The disease-specific survival rates at 1, 3 and 5 years were 94.6%, 81.3% and 72.6%, respectively. Five-year disease-specific survival rates were 99.2% for patients with low clinical stage PCa who underwent radical prostatectomy, 76.5% for those who underwent transurethral resection of the prostate plus hormone therapy, 38% for those who received hormone therapy plus radiation therapy and 29% for those that received hormone therapy alone. CONCLUSIONS: In keeping with a lack of prostate-specific antigen (PSA)-based screening, Chinese men present later in life and course of their disease, with over 27% men dying of PCa at five years. Debulking of tumors by surgery and radiation therapy for high grade tumor may provide some survival benefit in the senior men but further study is required to validate these findings. It is important of the annual use of PSA test for men over 50 years old to detect the PCa in the early stage in this nation.

[Expressions of interleukin-15 and osteopontin mRNA in early stage of acute rejection of renal allograft in rats].

OBJECTIVE: To study the expressions of interleukin-15 (IL-15), osteopontin (OPN), granzyme B (GraB) and perforin (PFP) mRNA in early stage of acute rejection (AR) of renal allograft in rats. METHODS: The rat renal transplantation model was established. Male Brown Norway and Lewis rats were used as donors and recipients. Four groups were designated: CsA group (BN-->LEW, n = 10, recipients were treated with CsA i.p.); AB group (BN-->LEW, n = 10, recipients were treated with anti-IL-15 neutralizing antibody i.p.); AR group (BN-->LEW, n = 10, recipients were treated with normal saline i.p.) and control group (LEW-->LEW, n = 6, recipients were treated with normal saline i.p.). The blood samples of recipients were drawn at Days 1, 3, 5 and 7 post-transplantation. The serum expressions of IL-15, OPN, PFP and GraB mRNA of recipients were detected by real-time PCR. Allograft tissues were analyzed by pathological assays. RESULTS: In comparison with other groups, the expressions of OPN, IL-15, PFP and GraB mRNA in AR group were gradually up-regulated and peaked at Day 5. The expressions of IL-15 mRNA in CsA and AB groups were 9685 +/- 1440 and 4346 +/- 741 respectively at Day 5 post-operation. It was significantly lower than that in AR group (17 022 +/- 2153, P < 0.01). The expression of IL-15 mRNA in AB group was significantly lower than that in CsA group (P < 0.01). The expressions of OPN mRNA in CsA and AB groups (13 226 +/- 1565 vs 19 112 +/- 2908) were both significantly lower than that in AR group (24 663 +/- 2449, P < 0.01). But the expression of OPN mRNA in AB group was higher than that in CsA group (P < 0.01). At Day 5 post-transplantation, both the expressions of PFP and GraB mRNA in AB and CsA groups was lower than that in AR group (P < 0.01). The pathological results showed that severe AR occurred at Day 7 post-transplantation in AR group and whereas the extent of rejection sign relieved in AB group. CONCLUSION: In early stage of AR of renal allograft in rats, the expressions of OPN, IL-15, PFP and GraB mRNA are up-regulated. Blocking IL-15/IL-15R pathway in early stage of AR can down-regulate the expressions of PFP and GraB mRNA.

Comparative analysis of upper ureteral stones (> 15 mm) treated with retroperitoneoscopic ureterolithotomy and ureteroscopic pneumatic lithotripsy.

OBJECTIVE: To investigate an appropriate treatment for patients with upper ureteral stones, > 15 mm in size, by comparing the therapeutic outcomes for those undergoing retroperitoneoscopic ureterolithotomy (RPUL) and rigid ureteroscopic pneumatic lithotripsy (URSPL) retrospectively. PATIENTS AND METHODS: During the study period, 81 patients with a large upper ureteral stone (> 15 mm) were divided into two groups. RPUL was performed with retroperitoneal approach, and the stone was removed in group A. URSPL was conducted using a rigid ureteroscope, and pneumatic probe was used for lithotripsy in group B. The patient characteristics, success rate, stone-free rate, operation time, and complications were analyzed prospectively in the two groups. RESULTS: The success rates of operation were 94.5% (34/36) in group A and 88.8% (40/45) in group B, but there were no significant differences between two groups (P > 0.05). After 4 weeks of follow-up, the stone-free rate after RPUL (100%, 34/34) and URSPL (77.5%, 31/40) groups were statistically different (P = 0.006). Furthermore, simultaneous ureterolithotomy and ureteroplasty by retroperitoneal laparoscopic surgery were performed on four patients combined with ureteral stricture. However, the mean operation time and hospital staying time after surgery in group A were longer than that in group B, and the differences were statistically significant (P < 0.05). The complication rate after RPUL (17.6%, 6/34) was lower than that after URSPL (20%, 8/40), but the differences were not statistically significant (P > 0.05). CONCLUSION: RPUL is a safe and effective treatment technique for large, impacted, upper ureteral stones >15 mm in size when first-line treatments have failed or are unlikely to be effective. It can handle with combined pathologies simultaneously.

[Expressions of survivin and the splice variants survivin-2B and survivin-DeltaEx3 in bladder cancer and their clinical significance].

BACKGROUND AND OBJECTIVE: Studies on survivin over the past 5 years have shown that survivin participates in the genesis of several human cancers, including bladder cancer. Recent studies have indicated that survivin splice variants appeared to have unique subcellular localizations and functions as well. This study was to explore the roles of survivin and its two splice variants survivin-2B and survivin-DeltaEx3 in transitional cell carcinoma of bladder (BTCC). METHODS: The relative amount of survivin, survivin-2B, and survivin-DeltaEx3 mRNA of fresh carcinoma tissues from 60 patients with BTCC and 12 non-cancerous bladder tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), and the relationships of their expression levels in different pathologic grades to clinical stages of bladder cancer were analyzed. The time of follow-up was 4-24 months. RESULTS: Survivin, survivin-2B, and survivin-DeltaEx3 mRNA were detected in all BTCC tissues, and their relative expressions were 0.333+/-0.163, 0.056+/-0.017, and 0.124+/-0.096, respectively. In the control group,three and four samples expressed survivin and survivin-DeltaEx3 mRNA respectively, and all samples expressed survivin-2B mRNA. The expressions of survivin and survivin-DeltaEx3 mRNA were positively correlated with the pathologic grades and clinical stages (0 < r 's < 1,P < 0.05), however, survivin-2B mRNA was negatively correlated with those (-1 < r 's < 0, P < 0.05). CONCLUSION: Detecting the expression levels of survivin and its two splice variants survivin-2B and survivin-DeltaEx3 mRNA in BTCC by real-time PCR could have potential values to evaluate tumor progression and recurrence rate.

[Expression of survivin mRNA in urine exfoliated cells of patients with bladder transitional cell carcinoma detected by real-time PCR].

BACKGROUND AND OBJECTIVE: Bladder cancer is the most malignant cancer of the urinary system. However a noninvasive and sensitive method of the early diagnosis for bladder cancer has not been developed. This study was to explore the expression of survivin mRNA in urine exfoliated cells of preoperative and postoperative patients with bladder transitional cell carcinoma (BTCC), and to analyze its value in early diagnosis and postoperative monitoring. METHODS: Urine of 30 patients with initially diagnosed BTCC was collected before operation and one week, one month, six months and 15 months after operation. Urine of 10 healthy volunteers and 15 patients with cystitis was used as control. Expression of survivin mRNA in urine exfoliated cells was detected by real-time fluorescent quantitative polymerase chain reaction (real-time PCR). RESULTS: The relative copy number of survivin mRNA in the patients was (96.01+/-42.33) before operation, which was significantly higher than that of healthy volunteers and cystitis patients (P <0.05). The level of survivin mRNA was apparently declined one week after operation (25.30+/-1.51) compared with its preoperative level (P <0.05); and the level became as low as the control group after one month (13.20+/-1.49) and six months (13.90+/-1.36) (P>0.05). Patients were followed up for 15 months, and three patients recurred, whose survivin mRNA level (97.83+/-27.47) was significantly higher than that at six months after operation (P <0.05). CONCLUSION: Detecting survivin mRNA in urine exfoliated cells is sensitive for the diagnosis of BTCC. Detection survivin mRNA after operation can monitor recurrence.

Rapid membrane effect of testosterone in LNCaP cells.

Testosterone signaling is widely considered to be mediated through the transcription-regulating intracellular androgen receptor (iAR). In the human prostate cancer cell line LNCaP we demonstrated the presence of unconventional membrane receptors for testosterone (mAR) besides the classic iAR. Binding sides for testosterone on the surface of LNCaP cells were clearly revealed with the membrane-impermeable testosterone-BSA-FITC by confocal laser-scanning microscopy and flow cytometry. Furthermore, we found that testosterone was able to induce a rapid activation of extracellular signal-related kinase 1 and 2 (ERK1/2), but not altered p38 MAPK and c-jun N-terminal kinase (JNK) activity. The testosterone-induced phosphorylation of ERK1/2 could not be inhibited by the AR antagonist cyproterone, excluding the involvement of iAR. Consistent with this finding, the impeded testosterone-BSA was also capable of rapidly phosphorylating ERK1/2, further revealing membrane receptor being responsible for the activation of ERK1/2. Additionally, testosterone-BSA induced a transient expression of c-Fos, which was in a timescale consistent with the rapid ERK1/2 phosphorylation and could be blocked by ERK1/2 inhibitor PD098059, suggesting an ERK1/2-dependent mechanism. Together, these data demonstrated a novel mode of testosterone signaling on LNCaP cells which was not mediated through the classical androgen receptor response, but through unconventional membrane receptors.

[All-trans retinoic acid enhances bystander effect of suicide-gene therapy against androgen-unresponsive prostate cancer].

OBJECTIVE: To investigate the enhancing effect of all-trans retinoic acid (ATRA) on the bystander effect of the herpes simplex virus thymidine kinase(HSV-TK)/ganciclovir (GCV) against androgen unresponsive prostate cancer. METHODS: The bystander effect of the HSV-TK/GCV system was measured by methyl thiazolyl tetrazolium (MTT) assay on PC-3 cells before and after ATRA treatment. The growth and the histopathology of transplant tumors were observed in 4 groups of nude mice with prostate cancer. RESULTS: ATRA augmented significantly the bystander effect of the HSV-TK/GCV system by reducing TK positive PC-3 cells from 50% to 30% (P < 0.05). HSV-TK showed an inhibiting effect, while ATRA with the HSV-TK/GCV system produced significant effect on prostate cancer 1 week earlier than the former (P < 0.05). CONCLUSION: ATRA can argument the in vivo and in vitro bystander effect of the HSV-TK/GCV system in the treatment of androgen unresponsive prostate cancer.

[Effects of survivin small interfering RNA on biological behaviors of bladder cancer T24 cells].

BACKGROUND & OBJECTIVE: Bladder cancer, a common tumor of urinary system, is related to genetic mutations that deregulate cell cycle and render tumor cells resistant to apoptosis. Survivin, a new member of the inhibitor-of-apoptosis (IAP) family, participates in the genesis of several human cancers, including bladder cancer, through inducing cell proliferation and inhibiting apoptosis. This study was to explore the effects of small interfering RNA (siRNA) targeting survivin on the biological behaviors of bladder cancer T24 cells. METHODS: A pair of survivin siRNA were designed and synthesized, and transfected into T24 cells with increasing concentrations (50-200 nmol/L) and injected in tumor-bearing mice at different doses (5 and 50 microg). The effects of survivin siRNA on the biological behaviors of T24 cells were assessed. RESULTS: Survivin siRNA efficiently down-regulated the expression of survivin mRNA. Its maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%, the inhibition rate of cell proliferation was 55.29% (P<0.01), and the apoptosis rate was significantly higher in siRNA-transfected cells than in control cells (45.70% vs. 2.8%, P<0.01). Intratumoral injection of 50 microg survivin siRNA significantly inhibited the growth of bladder cancer in mice (P<0.01). CONCLUSIONS: Survivin siRNA could markedly inhibit survivin expression in T24 cells, induce cell apoptosis and inhibit the growth of transplanted tumor in mice. It might be a new gene therapy tool for bladder cancer.

ATRA enhances bystander effect of suicide gene therapy in the treatment of prostate cancer.

OBJECTIVES: All-trans retinoic acid (ATRA) has been shown to inhibit the growth of many malignancies by altering gap junctional intercellular communication (GJIC) and the expression of connexin (Cx) 43. Here, we report that the alteration of GJIC by ATRA may directly enhance the bystander effect (BE) of suicide gene therapy against prostate cancer in vitro and in vivo. METHODS: PC-3 cells were exposed to different concentrations of ATRA for varying lengths of time in culture. Flow cytometry was performed to measure Cx43-positive cells and the GJIC function of the cells was examined with the scrape-loading dye transfer assay. Cells were treated with ATRA in combination with an adenovirus/ganciclovir (Ad-TK/GCV) system encoding herpes simplex virus-thymidine kinase, and the BE was assessed in the treatment of androgen-independent prostate cancer both in vitro and in vivo. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to assess the expression of Cx43 mRNA and protein in tumor tissues. RESULTS: ATRA significantly increased the amount of Cx43-positive cells in a time- and dose-dependent manner (P < 0.05). GJIC functions were enhanced 3- to 5-fold in the presence of ATRA, although ATRA did not augment GCV toxicity of PC-3 cells. In the mixing assay, ATRA significantly increased cell killing when the ratio of TK-positive cells in the coculture ranged from 30% to 60% compared with ATRA-untreated cell (P < 0.05), and attained 50% cell killing cells when the ratio of TK-positive cell was 30%, but the same result did not appear until the ratio of TK-positive cell was up to 60% in the ATRA-untreated cell. Mice treated with a combination of ATRA and GCV had significantly smaller Ad-TK infected tumors than those treated with GCV or ATRA alone after 3-weeks of therapy (P < 0.05). However, from the fourth-week of therapy, there was no difference in tumor growth inhibition between GCV treatment and GCV + ATRA treatment (P > 0.05), as two tumors in the latter group started to grow more quickly than tumors in the control group. This phenomenon was not found in other groups. CONCLUSIONS: ATRA could enhance the efficiency of cell killing in suicide gene therapy against prostate cancer by strengthening the BE in vitro and in vivo. Induction of Cxs and GJIC by ATRA might provide an element of selectivity to suicide gene therapy. Future studies should focus on safety and tailoring this cooperative therapy to the patient.

[Experimental study on bladder cancer by the small interfering RNA targeting survivin].

OBJECTIVE: To study the influence of small interfering RNA (siRNA) targeting survivin on the biological behavior of bladder cancer. METHODS: One pair of survivin target sequence-specific siRNA was designed, then siRNA/liposome complex was used to transfect bladder cancer cell line-T24. The efficiency of transfection and the apoptosis were detected by flow cytometry. The transcriptional level of survivin was analyzed using real time PCR. DNA sequence corresponding to siRNA targeting survivin was cloned into pRNAT-U6.1/Neo to produce plasmid targeting surviving. RESULTS: The ratio of T24 cells releasing fluorescence in total cells were 92.3%; siRNA-survivin efficiently down-regulated survivin expression (mRNA) in a dose-and time-dependent manner. Its maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down regulated by 75.91%. Similar results were found in the inhibition ratio of cell growth, which was 55.29%(P< 0.01). Simultaneously the apoptotic rate was markedly increased, which was 45.70%(P< 0.01). After cutting the vector with Bam H I and Hin d III and ligating the vector with the insert by using T4 ligase, the recombinant vector was confirmed by restriction digestion and DNA sequencing. CONCLUSION: The application of siRNA-survivin can markedly inhibit survivin expression in bladder cancer cell line, induce apoptosis and inhibit the growth of the tumor. It may be a new gene therapy tool for bladder cancer. The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique, and lay a foundation for further studies on the function of surviving.