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To explore the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the human bladder cancer T24 cells in vitro.The methods of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of ING4 in human bladder urothelial carcinoma T24 line. The influence of Ad-ING4 transfection on cell proliferation was evaluated by MTT assay and cell apoptosis by Hochest33258 staining and flow cytometry. RT-PCR and Western blot were performed to detect the transcriptional level of such apoptosis-related genes as Bcl-2, Bax, p53, HIF-1α and caspase-3.Human ING4 was successfully transcribed in T24 cell. Apoptosis rate of T24 cell in Ad-ING4 group was significant higher than that in control groups (17.2% ± 4.1% vs 4.7% ± 1.2% and 4.8% ± 1.2%, P0.05). Ad-ING4 not only up-regulated the expression of p53 and Bax(1.40 ± 0.11 vs 0.27 ± 0.04, 1.50 ± 0.12 vs 0.60 ± 0.05) and down-regulated the expression of Bcl-2 and HIF-1α (0.19 ± 0.02 vs 1.20 ± 0.08, 0.33 ± 0.03 vs 0.98 ± 0.06, all P0.05), but also enhanced the caspase-3 activation and eventually led to apoptosis.Adenovirus-mediated ING4 gene exhibits anti-tumor capacity in T24 human bladder cancer cell and induces in vitro apoptosis. It may be related to the up-regulation of P53 and Bax/Bcl-2, and the down-regulation of HIF-1α. Thus the caspase-3 activation is enhanced so as to lead to apoptosis.

To investigate the in vitro inhibitive effect and apoptotic mechanism of BDI-1 monoclonal antibody targeted SEA on human bladder cancer cell line BIU-87.Human peripheral blood mononuclear cells (PBMC) and human bladder cancer cell line BIU-87 were used as effector and target cells respectively in three experimental groups, control group, SEA group and monoclonal antibody targeted SEA group, treated with different concentrations of SEA and targeted SEA respectively in the latter two. The inhibition rates of target cells were measured by cell counting kit-8 (CCK-8) assay. Apoptosis was determined by fluorescent Hocehst 33258 staining and flow cytometry (FCM). The expression of Bax, Bcl-2 proteins and cytokine concentration of co-culture supernatants were detected by Western blot and ELISA respectively.Compared with those of control group (from 5.8% +/- 3.0% to 16.5% +/- 4.0%) and SEA group (from 20.7% +/- 2.1% to 68.6% +/- 4.0%), the inhibition rates in targeted SEA group increased significantly (from 31.2% +/- 2.6% to 88.7% +/- 3.0%, P0.05). Characteristic changes of apoptosis were observed in both experimental treatment groups, especially in targeted SEA group. FCM also showed more prominent apoptosis induced in targeted SEA group (from 14.6% +/- 0.7% to 66.5% +/- 3.3%), significantly higher than those of control group (from 4.6% +/- 0.7% to 6.5% +/- 0.3%) and SEA group (from 9.1% +/- 0.6% to 29.9% +/- 1.3%, P0.05). In addition, Western blot analysis indicated that the ratio of Bax/Bcl-2 significantly increased to (3.29 +/- 0.34) by targeted SEA treatment at an extremely low concentration, significantly higher than those of SEA group (1.21 +/- 0.05) and control group (0.45 +/- 0.15) (P0.05). While at the same time ELISA assay showed no significant difference of IL-2, TNF-alpha, IFN-gamma concentrations between SEA and targeted SEA groups.Monoclonal antibody targeted SEA significantly increases the inhibitive effect of BIU-87 cell line possibly through the up-regulation of Bax/Bcl-2 ratio to promote the cellular apoptosis.

To investigate the influence of mycophenolate mofetil (MMF) on the maturation and allostimulatory activity of cultured dendritic cell progenitors (DCPs) and to evaluate the efficacy of pretreatment of donor dendritic cells (DCs) with MMF in tolerance induction in allograft recipients and its possible mechanism.DCPs of Balb/c mice were cultured in culture fluid containing recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF), and then divided into 4 groups: control group, MMF group, lipopolysaccharide (LPS) group, and MMF + LPS group. Seven days later, flow cytometry was used to analyze the phenotypes of the DCs. ELISA was used to examine the level of IL-12 in the supernatant. T cells from the spleens of C57/BL6 mice were cultured together with inactivated DCs from Balb/c mice, and added with [(3)H]-TdR. Mixed lymphocyte reaction (MLR) was analyzed. The DCs and MMF-DCs cultured for 5 days were co-cultured with T hybridoma cells of the line MF2.2D9 in culture fluid containing ovalbumin (OVA). Twenty-four hours later, the supernatant was collected and ELISA was used to measure the level of interleukin (IL)-12 so as to reflect the antigen-presenting ability of the DCs. OVA immunized C57/BL6 mice for 4 times. 21 days after T cells were collected from the spleens and co-incubated with DCs and MMF-DCs, [(3)H]-TdR was added and the values of counts per minute (cpm) were calculated so as to analyze the antigen-specific proliferation. Twenty-four Balb/c mice were randomly divided into 3 groups: group A (without treatment), group B (treatment with intravenous injection of untreated DCs of Balb/c mice), and group C (treatment with intravenous injection of DCs of Balb/c mice treated with MMF), and then transplanted with the hearts of C57/BL6 mice. The functions of the transplanted hearts were evaluated by touching the arterial pulse and histological examination. ELISA was used to detect the levels of Th1 cytokines, such as IL-12, IL-4, IL-10, IL-2, and IFN-gamma, in the cultured DCs and in the sera of the recipients 7 and 14 days after culture or transplantation.The immunophenotypic analysis showed that in comparison with those in the control group and the LPS group the expressions of the costimulatory molecules, Ia(d), CD80, and CD86, of the DCPs were significantly weaker in the MMF-group and MMP + LPS group. The IL-12 levels in the supernatant of the MMF and MMF + LPS groups of DCPs were significantly lower than those in the other groups (P0.01). The IL-12 level of the MF2.2D9 cells treated with MMF-treated DCs was significantly lower than control group (P0.01). The ability to stimulate proliferation of T cells of the same genotype in the MMF-DC group was significantly inhibited (P0.01). The survival time of the transplanted heart was 30.50 +/- 3.25 days in the C57/BL6 mice injected with untreated DCPs of Balb/c mice (21.25 +/- 2.12, P0.01) and that in the control C57/BL6 mice (8.63 +/- 1.06 days, P0.01) and with a significant difference between the latter 2 groups too (P0.01). The levels of, such as IL-2 and IFN-gamma, 7 and 14 days after heart transplantation of the group B were all significantly lower than those of the group A (both P0.05). The IL-2 and IFN-gamma levels 7 days after the heart transplantation were similar to those in the group B (both P0.05) and even lower than those of the group C (both P0.05). The IL-10 level in the groups B and C were all higher than those in the group A (all P0.05) with a significant difference between the group B and group C. The level of IL-4 was not significantly different among the 3 groups.MMF has a significant suppressive effect on the maturation and function of DCs, which leads to a donor-specific tolerance in transplant recipients.