| Part IExpressions of HIF-1??VEGF?MMP-2 and Bcl-2 and their correlation in BTCCObjective To investigate the expressions of HIF-la, VEGF, MMP-2 and Bcl-2 in BTCC with regard to the clinical significance.Methods Expressions of HIF-la, VEGF, MMP-2 and Bcl-2 were detected by immunohistochemical staining in 42 cases of BTCC and 9 cases of normal bladder tissues as control. The relevance between the expressions of HIF-la, VEGF, MMP-2 and Bcl-2 and the pathologic grade and clinical stage were also studied.Results Immunohistochemical staining demonstrated that the positive rate of HIF-la, VEGF, MMP-2 and Bcl-2 were 61.9%(26/42), 69.0%(29/42),61.9%(26/42) and 64.3%(27/42) in BTCC respectively, which were significantly higher than those in the normal bladder tissue with 22.2%(2/9) (P<0.05),22.2%(2/9) (P<0.01),22.2%(2/9) (P< 0.05) and 11.1%(1/9) (P<0.01) respectively. The positive rate of HIF-la, VEGF, MMP-2 and Bcl-2 were significantly positive correlated with the pathologic grade and clinical stage except that no correlation was found between the positive rate of Bcl-2 and the clinical stage. Significantly positive correlations were found between the positive rate of HIF-1a and VEGF (r=0.429, P<0.01), MMP-2 (r=0.495, P<0.01), Bcl-2 (r=0.336, P <0.05).Conclusions1. The expressions of HIF-1a, VEGF, MMP-2 and Bcl-2 were significantly higher in BTCC than those in normal bladder tissues.2. The expressions of HIF-la, VEGF, MMP-2 and Bcl-2 were significantly positive correlated with the tumor grade.3. The expressions of HIF-la, VEGF and MMP-2 were significantly positive correlated with the clinical staging except that Bcl-2 was independent of the clinical stage. Part IIEffects of YC-1 on the mRNA and protein expressions of HIF-la in human bladder cell line T24 under hypoxiaObjective To explore the effects of YC-1 on the mRNA and protein expressions of HIF-1a in human bladder cell line T24 under hypoxia.Methods T24 cells were incubated with different concentrations of YC-1 (0?mol/L,1?mol/L,10?mol/L,50?mol/L,100?mol/L) addition for different times (12h,24h,48h) under hypoxia (1 group incubated for 12h with 0?mol/L of YC-1 addition as control). The mRNA and protein expressions of HIF-1a were detected by RT-PCR and Western blotting respectively.Results The mRNA and protein expressions of HIF-la were both decreased in T24 cells with different concentrations YC-1 (0?mol/L,1?mol/L,10?mol/L,50?mol/L,100?mol/L) addition for different times (12h,24h,48h). After T24 cells were incubated with different concentrations of YC-1 (0?mol/L,1?mol/L,10?mol/L,50?mol/L,100?mol/L) addition for 12h, the expression ratio of HIF-la mRNA (HIF-la/GAPDH) were 1.258±0.049,1.019±0.014,0.866±0.034, 0.580±0.018,0.460±0.024; The expression ratio of HIF-1a mRNA were 1.439±0.050,0.983±0.030,0.667±0.026,0.406±0.041,0.387±0.018 for 24 h; The expression ratio of HIF-la mRNA were 1.516±0.036, 0.731±0.032,0.615±0.034,0.351±0.024,0.317±0.019 for 48h. Compared with the control group, the groups incubated with different concentrations of YC-1 addition for different times were all decreased. In addition, The significant time-dependent and dose-dependent effects were observed (P <0.01). After T24 cells were incubated with different concentrations of YC-1 (0?mol/L,1?mol/L,10?mol/L,50?mol/L,100?mol/L) addition for 12h, the expression ratio of HIF-1a protein (HIF-1a/GAPDH) were 0.192±0.003,0.176±0.004,0.115±0.003,0.075±0.002,0.056±0.003; The expression ratio of HIF-1a protein were 0.203±0.004,0.150±0.003, 0.088±0.003,0.055±0.004,0.035±0.002 for 24h. The expression ratio of HIF-1a protein were 0.243±0.005,0.149±0.003,0.069±0.003, 0.040±0.002,0.032±0.002 for 48h. Compared with the control group, the groups incubated with different concentrations of YC-1 addition for different times were all deceased. In addition, The significant time-dependent and dose-dependent effects were also observed (P< 0.01).Conclusions The mRNA and protein expressions of HIF-la were both inhibited by YC-1 in T24 cells incubated under hypoxia in a dose-dependent and time-dependent manner. Part IIIEffects of YC-1 on the expressions of HIF-la-mediated genes and the cytobiological behaviors in human bladder cell line T24 under hypoxiaObjective To explore the effects of YC-1 on the expressions of HIF-1a-mediated genes and the cytobiological behaviors in T24 cells under hypoxia, including cell proliferative vitality, apoptosis and migration activity.Methods T24 cells were incubated with different concentrations of YC-1 (0?mol/L,1?mol/L,10?mol/L,50?mol/L,100?mol/L) addition for 48h under hypoxia. The protein expressions of VEGF, MMP-2 and Bcl-2 were detected by Western blotting. The cell proliferative vitality, apoptosis and migration activity were determined by MTT assay, flow cytometry and transwell migration assay respectively.Results After T24 cells were incubated with different concentrations of YC-1 (0?mol/L,1?mol/L,10?mol/L,50?mol/L,100?mol/L) addition for 48h, the protein expression ratio of VEGF (VEGF/GAPDH) were 0.496±0.004,0.391±0.003,0.319±0.002,0.290±0.003,0.171±0.003; The protein expression ratio of MMP-2 (MMP-2/GAPDH) were 0.611±0.006,0.435±0.003,0.323±0.002,0.298±0.002,0.231±0.003; The protein expression ratio of Bcl-2 (Bcl-2/GAPDH) were 0.396±0.006, 0.266±0.003,0.198±0.003,0.156±0.003,0.089±0.003. The significant dose-dependent effect was observed (P<0.01). After T24 cells were incubated with different concentrations of YC-1 (0?mol/L,1?mol/L,10?mol/L,50?mol/L,100?mol/L) addition for 48h, the cell proliferative vitality rates were 100%,94.17%±1.18%,85.55%±1.20%, 82.81%±1.27%,77.33%±0.99%; the apoptosis rates were 5.55%±1.32%, 6.55%±1.35%,9.05%±1.54%,11.83%±2.78%,16.05%±2.26%; the mean migrated cells were 54.0±4.0,48.2±4.7,41.4±4.4,31.4±3.8,25.8±5.3. The significant dose-dependent effect was observed (P<0.01).Conclusions1. The expressions of HIF-la-mediated genes VEGF, MMP-2, Bcl-2 were all inhibited by YC-1 in T24 cells in a significant dose-dependent manner.2. The cell proliferative vitality was inhibited by YC-1 in T24 cells in a significant dose-dependent manner.3. The apoptosis was induced by YC-1 in T24 cells in a significant dose-dependent manner.4. The migration activity was inhibited by YC-1 in T24 cells in a significant dose-dependent manner. Part?Effects of YC-1 on ERK/P38MAPK signaling pathway in human bladder cell line T24Objective To explore whether effects of YC-1 work on ERK/P38MAPK signaling pathway in human bladder cell line T24 to inhibit the expression of HIF-la.Methods The ERK specific inhibitor PD98059 (25 uM) and P38MAPK specific inhibitor SB203580 (15 uM) were used on sustained inactivation of ERK/P38MAPK signaling cascades. The T24 cells were pretreated with and without the aforementioned inhibitors for 1h and then were incubated with and without 50?mol/L of YC-1 for 48h under both normoxia and hypoxia. After that, the protein expression of HIF-1?was examined by Western blotting.Results The protein expression ratio of HIF-1?(HIF-1?/GAPDH) of control group, YC-1 group, YC-1 plus PD98059 group and YC-1 plus SB203580 were 0.166±0.008,0.044±0.008,0.068±0.010,0.155±0.010 under normoxia. The YC-1 group was significant different from the YC-1 plus PD98059 group (P<0.01) and the YC-1 plus SB203580 group (P< 0.01). The protein expression ratio of HIF-1?(HIF-1?/GAPDH) of control group, YC-1 group, YC-1 plus PD98059 group and YC-1 plus SB203580 were 0.346±0.009,0.128±0.009,0.201±0.008,0.221±0.007 under hypoxia. The YC-1 group was significant different from the YC-1 plus PD98059 group (P<0.01) and the YC-1 plus SB203580 group (P< 0.01).Conclusions1. Inhibitory effects of YC-1 on HIF-la in T24 cells can be reversed by the ERK specific inhibitor PD98059 and P38MAPK specific inhibitor SB203580 under both normoxia and hypoxia.2. ERK/P38MAPK signaling pathway may be involved in YC-1 mediated HIF-la expression suppression in T24 cells. |
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