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Objective The aims of this study were to investigate the expression of microRNA-30a(miR-30a)in human bladder cancer cell lines and their effects on the proliferation,apoptosis and migration of human bladder cancer cells.Methods The expression levels of miR-30a in bladder cancer cell lines(5637 and T24)and bladder epithelial immortalized cells(SV-HUC-1)were detected by real-time quantitative PCR(qRT-PCR).The expression of miR-30a was up-regulated or down-regulated by T24 cells transfected with miR-30a mimic or 5637 cells transfected with miR-30a inhibitors and controls using NC mimic or NC inhibitor.The effects of miR-30a expression on the proliferation,apoptosis and invasion of bladder cancer cells were investigated by flow cytometry,MTT and Transwell assays.Results The expression level of miR-30a in two bladder cancer T24 and 5637 cell lines was significantly lower than that in normal bladder SV-HUC-1 cell line(P<0.05),and the expression level of miR-30a was lower in the high degree of malignancy in bladder cancer T24 cells than that in malignant degree of relatively low 5637 cells.After 72h transfection,the values of optical density(OD)in the miR-30a mimic group(0.83±0.09)was significantly lower than that in NC mimic group(1.21±0.12)in T24 cells(P<0.01).The OD values of miR-30a inhibitor group(1.28±0.14)was significantly lower than that in the NC inhibitor group(1.09±0.14)in 5637 cells(P<0.01).The apoptotic rate of miR-30a mimic group in T24 cells(21.27±2.42)% was significantly higher than that in the NC mimic group(10.61±1.29)%(P<0.01).The apoptotic rate of the miR-30a inhibitor group in 5637 cells(6.78±2.57)% was significantly lower than that in the NC mimic group(13.42±1.40)%(P<0.01).The number of transmembrane cells in miR-30a mimic group in T24 cells(183.57±16.61)was significantly lower than that in NC mimic group(465.80±9.20)(P<0.01).The number of transmembrane cells in the miR-30a inhibitor group in 5637 cells(581.25±11.02)was significantly lower than that in NC mimic group(397.13±7.57)(P<0.01).Conclusion Up-regulation of miR-30a can inhibit the proliferation of bladder cancer cells,promote cell apoptosis and reduce the ability of migration and invasion in bladder cancer cells.The low expression of miR-30a in bladder cancer cells may be related to the development and metastasis in bladder cancer.
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[Objective] To investigate the characteristics of PD-1/PD-L1 expressed on T cell and bladder cancer cell and clinical significance.[Methods] 64 patients with primary bladder cancer were into experiment group and 10 normal people were into control group.Peripheral bloods were used to test the PD-1 expressed on CD8+ T lymphocytes by flow cytometry.Immunohistochemistry staining was used to detect the PD-L1 expression in tumor and normal specimen.[Results] PD-1 expressed on CD8+T lymphocytes was (2.25 ± 0.60)％ in experiment group and (0.68 ± 0.17)％ in control group,respectively (P ＜ 0.001).And the PD-1 expression on T cell in invasive bladder cancer patient was significant higher than superficial bladder cancer patients [(3.04 ± 0.46)％ vs (0.68± 0.17)％,P ＜ 0.001].The expression of PD-L1 in experiment group was higher than control group,(26/64 vs 0/15,P ＜ 0.001).But there was no different between invasive and superficial bladder cancer patients,(41.3％ vs 38.8％,P ＞ 0.01).[Conclusions] Expression of negative stimulatory molecule PD-1 in CD8+T lymphocytes of peripheral blood is significantly correlated with bladder cancer advanced.Bladder cancer cell was strongly expressed PD-L1,and this expression is not related to cancer advanced.
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Objective: To investigate the anticancer activity of Luteolin (Lu) and its synergism effect with bacillus calmette-guerin (BCG) on human bladder cancer cell line BIU-87. Methods: Cultured BIU-87 cells were treated with different concentrations of Lu alone or the combination of Lu with BCG. MTT assay was used to measure the cell proliferation inhibition, and IC50 was calculated. Cell cycle and apoptosis were analyzed by lfow cytometry with propidiumiodide (PI) staining and Annexin-V FITC/PI dual parameter markers to clarify the mechanism of inhibiting cell proliferation and inducing apoptosis. Caspase-3 and phosphorylated c-Jun N-terminal kinases (P-JNK) expression were measured to detect the apoptosis signal pathways of Lu in cancer cells. Results: Both Lu and BCG apparently inhibited the cell proliferation and induced the apoptosis dose-dependently, and microscope observation showed morphological changes in the apoptosis. Flow cytometry indicated that Lu arrested the cell cycle at G2 phase (P<0.05). It sensitized BCG-induced cytotoxicity and cell apoptosis, and upregulated expression of caspase-3 and activation of JNK (P<0.05). Conclusion: As an effective anticancer agent, Lu can sensitize the effect of BCG by inducing the cell cycle arrest and apoptosis. hTis synergism effect is achieved by activation of caspase-3 and JNK. Combination of Lu with BCG may be one of the potential treatment for bladder cancer.
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OBJECTIVE: To observe the inhibitor effects of isorhapontigenin (ISO) on the proliferation of bladder cancer cells and expression of X-linked inhibitor of apoptosis (XIAP), and the sensitizing effect of chemotherapy of docetaxel to bladder cancer cells In ISO. METHODS: The ATPase assay and anchorage-independent growth assay were used to detect the cytotoxicity and proliferative capacity of ISO on the T24T bladder cancer cell line, respectively. The reverse transcription polymerase chain reaction (RT-PCR) and Western-blotting methods were used to detect XIAP gene expression after pretreated with ISO. Pretreatment with different concentration of docetaxel and ISO on T24T bladder cancer cell, ATPase methods were performed to measure in vitro cell viability. And the morphological changes and apoptosis rates were detected by phase contrast microscope and flow cytometry assay, respectively. RESULTS: ISO could exhibit significant inhibitory effects on human bladder cancer cell growth through ATPase assay and anchorage-independent growth assay. Further proved by Western-blotting and RT-PCR methods, the protein and mRNA level of XIAP gene in T24T cells were both significantly decreased. After pretreatment with ISO, the IC50 of T24T cells for docetaxel was (1.02 ± 0.38) nmol · L-1, significantly lower than that docetaxel without ISO, which was (8.78 ± 1.32) nmol · L-1 for 24 h( P < 0.01). Compared with the control group, the typical apoptosis morphological changes were observed in the T24T cells pretreatment with ISO combined with docetaxel. And the index of apoptosis was (21.07 ± 2.79)% at 2.5 nmol · L-1 docetaxel, markedly lower than those of T24T cells pretreatment with 2.5 nmol · L-1 docetaxel and ISO, which was (49.59 ± 5.67)% (P < 0.01). CONCLUSION: ISO could exhibit significant inhibitor) effects on human bladder cancer cell proliferation. And the XIAP gene could be down-regulated significantly by ISO, which could increase docetaxel-induced apoptosis and cytotoxic activity significantly.
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Quercetin (3,3',4',5,7-pentahydroxyflavone) is an attractive therapeutic flavonoid for cancer treatment because of its beneficial properties including apoptotic, antioxidant, and antiproliferative effects on cancer cells. However, the exact mechanism of action of quercetin on ion channel modulation is poorly understood in bladder cancer 253J cells. In this study, we demonstrated that large conductance Ca2+-activated K+ (BKCa) or MaxiK channels were functionally expressed in 253J cells, and quercetin increased BKCa current in a concentration dependent and reversible manner using a whole cell patch configuration. The half maximal activation concentration (IC50) of quercetin was 45.5+/-7.2 microM. The quercetin-evoked BKCa current was inhibited by tetraethylammonium (TEA; 5 mM) a non-specific BKCa blocker and iberiotoxin (IBX; 100 nM) a BKCa-specific blocker. Quercetin-induced membrane hyperpolarization was measured by fluorescence-activated cell sorting (FACS) with voltage sensitive dye, bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3); 100 nM). Quercetin-evoked hyperpolarization was prevented by TEA. Quercetin produced an antiproliferative effect (30.3+/-13.5%) which was recovered to 53.3+/-10.5% and 72.9+/-3.7% by TEA and IBX, respectively. Taken together our results indicate that activation of BKCa channels may be considered an important target related to the action of quercetin on human bladder cancer cells.
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Humans , Flow Cytometry , Ion Channels , Large-Conductance Calcium-Activated Potassium Channels , Membranes , Peptides , Quercetin , Tea , Tetraethylammonium , Urinary Bladder , Urinary Bladder NeoplasmsABSTRACT
Objective To study the effect of wild type p53 gene on centrosome hyperamplification in bladder cancer cells.Methods A wild type p53 gene recombinant adenovirus vector AdCMVp53 was constructed,and then trangfected into the human bladder cancer cell line T24.The cells were stained with the monoclonal antibody against pericentrin by indirect immunofluorescence method.The change of centrosome hyperamplification was observed under the fluorescence microspcope.Results Introduction of wild type p53 could suppress the centrosome amplification of T24 cell line.Conclusion p53 might play an important role in the regulation of centrosome hyperamplification.The loss of p53 might be one of the mechanisms involved in chromosome instability and contribute to the genesis and development of the bladder carcinoma.
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Summary: The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection in vitro. The expression of CH50 polypeptide was detected by using immunohistochemical S-P method. The expression of the transfected gene was identified by RT-PCR. Cell invasion assay kit was applied to detect the effect of CH50 polypeptide on the invasion ability of BIU-87. The results showed that the BIU-87 cells transfected with pCH510 could express the CH50 polypeptide, while in the control group, no CH50 polypeptide was detectable. In the transfection group, the invasion ability of BIU-87 in vitro was lower than in control group (P<0.05). It was concluded that CH50 polypeptide was successfully expressed in BIU-87 cells by gene transfection, by which the in vitro invasion ability of BIU-87 was inhibited.
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Objective To study on the mechanisms that photodynamic therapy with laser-activated BPD-MA induces human bladder cancer BIU-87 cell apoptosis.Methods Human bladder cancer BIU-87 cells at log phase were randomized into 4 groups.The cells of BDP-MA-PDT groups were added with BPD-MA and then activated with 632.8 nm He-Ne laser;PDT group without BDP-MA addition was only irradiated;BDP-MA group without irradiation;normal control group without BDP-MA addition or irradiation.Photosensitization of BPD-MA was activated by laser with red light(632.8 nm) delivered at 10 mw/cm~(2) to give a total dose of 2.4 J/cm~(2).The expression of Bcl-2 and Bax proteins in human bladder cancer BIU-87 cells were detected by immunohistochemical staining.Results The Bcl-2 protein expression in BPD-MA-PDT group was lower than that of other groups(P0.05),therefore the ratio of Bax to Bcl-2 obviously increased(P
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AIM To investigate the effect of cisplatin on apoptosis in Scaber cell. METHODS The apoptotic cells were detected by TUNEL,HE,eletronic micrpscopy. RESULTS Treatment of Scaber cells with CDDP resulted in characteristics typical of apoptosis. CDDP induced apoptosis of Scaber cells in time and concentration dependent manner. To further investigate the mechanism of apoptosis induced by CDDP, the expressions and activity of apoptosis associated proteins such as bcl 2, bax and caspase 3 were examined using S P method.The results showed: CDDP caused time and concentration dependent decreases in bcl 2 and increased in bax proteins.CDDP bcl 2 and its translocation to perinuclei and nuclei. The expression of caspase 3 in Scaber cell were determined during apoptosis induced by CDDP. CONCLUSION Our investigetion showed that the apoptosis induced by CDDP is related to the increase of bax protein, and the decrease of bcl 2 protein. and its translocation to perinuclei and nuclei.
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0.05).Conclusion: The method of constructing a highly invasive subline of bladder neoplasm is reliable,and EGFP transfection does not change the biological behavior of the cells.The obtained sublines may provide a valuable experimental platform for further study on the molecular mechanisms of bladder neoplasm metastasis.
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AIM: To study the boosting effect of Bombina maxima protein on bladder cancer cell lines growth and expression of telomerase. METHODS: Cell culture was used to dectect the effects of 3 blad der cancer cell lines growth; TRAP-PCR-ELISA method was used to detect the telomerase express under the ID50 culture concentration. RESULTS: Bombina maxima protein had the strong effect on growth of cell lines; particularly in the first 48 h; no effect was found in telomerase expression. CONCLUSION: Bombina maxima protein has the direct inhibitory effect on bladder cancer cell lines growth; The mechanism of antitumor of Bombina maxima protein is not by telomerase.
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Gonadotropin releasing hormone (GnRH) is believed to be pivotal hormone in hypothalamo-pituitary gonadal axis and the hypothalamus is believed as the exclusive organ producing GnRH and pituitary is for GnRH re ceptor until recently. Some reported the exptra-hypothalamic GnRH or extra-pituitary GnRH receptors from decades ago. The aims of this study are to confirm the existence of the GnRH receptor in bladder epithelial cancer cell, HT-1197 and HT-1376, and evaluated the possible role of the GnRH on cell cycle. The GnRH and GnRH receptor were detected by immunohistochemical staining and the effect of GnRH on cell cycle change in both cell line were studied by fluorescence activated cell sorter (FACS). The control cells were cultured at media supplemented with normal serum, and experimental group were cultured at media supplemented with charcoal stripped serum (CSS) which excluding peptide hormones except exogenous GnRH with different concentration. The GnRHs and GnRH receptors were detected at both cell lines and the cell cycle analysis showed that there were little difference in proportion of cell cycle among examined 10,000 cells in both cell lines, neither control nor experimental groups. This study shows that the GnRHs and GnRH receptors exist in bladder cancer cells and GnRH did not influence on the cell cycle progression. With this study, we suppose that the bladder cancer cells produce the GnRH and GnRH receptors and the role of the GnRF produced from the bladder cancer cells might be the autocrine rather than endo-or paracrine factor.
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Axis, Cervical Vertebra , Cell Cycle , Cell Line , Charcoal , Fluorescence , Gonadotropin-Releasing Hormone , Gonadotropins , Gonads , Hypothalamus , Peptide Hormones , Receptors, LHRH , Urinary Bladder , Urinary Bladder NeoplasmsABSTRACT
1.0 mg?L -1 ),the intracellular calcium became increased,and kept on a higher level.Conclusion PUPS participates in the process of calcium message transduction.
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Objective:To construct eukaryotic expression vectors with the pSilencer2.0 vector for inhibiting human survivin gene by RNA interference,and to detect the effect of the silenced survivin gene on EJ cells.Methods:Two target gene segments were synthesized and cloned into the pSilencer2.0 vector respectively to construct two recombinant eukaryotic expression vectors,pSilencer2.0-SVV1and pSilencer2.0-SVV2,which were identified by enzyme digestion analysis and DNA sequencing.Then the EJ cells were transfected with the recombinant vectors by lipofection and the interference effect was detected by RT-PCR and Western-blot.Results:Enzyme digestion analysis and DNA sequencing showed that two target segments were cloned into pSilencer2.0 vectors correctly.The results of RT-PCR and Western blot indicated that pSilencer2.0-SVV1 and pSilencer2.0-SVV2 vectors could significantly knock down the transcription and expression of survivin gene(P