槲皮素诱导膀胱癌细胞凋亡的机制研究

为了探讨槲皮素对膀胱癌细胞株生长的影响,运用细胞培养、光镜观察、MTT法测定细胞活性等方法,对比不同浓度槲皮素对膀胱癌细胞的生长抑制情况,为寻找新的膀胱癌细胞的治疗药物提供实验依据。结果表明高浓度的槲皮素对膀胱癌细胞具有明显的抑制作用。

Clinical Significance of Cytokeratin 19 and Cytokeratin 20 in Predicting Recurrence of Bladder Transitional Cell Carcinoma

Objective： To investigate the expressions of cytokeratin 19 （CK19） and cytokeratin 20 （CK20） in bladder transitional cell carcinoma （TCC） and their clinical significance. Methods： The expression of CK19 and CK20 was detected in 54 cases of TCC by immunohistochemical methods and image processing techniques. Results： The expression of CK19 and CK20 was significantly stronger in the recurrent group than in the non-recurrent group （P〈0.01, P〈0.001, respectively）. Conclusion： The expression of CK19 and CK20 was obviously related with biological behaviors of TCC, suggesting that CK19 and CK20 could be used to predict the recurrence of TCC.

Laser Capture Microdissection Combined with RNA in vitro Linear Amplification Detecting the Relevant Genes of Bladder Cancer

Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determinc a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cclls from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods： Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results： That RNA integrity was good after LCM was confirmed by control experiment Ⅰ; By control experiment Ⅱ, the correlation between the number of LCM-shooting and RNA quantity undcr arranged conditions was preliminarily confirmed. About 0.5-2.5kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion： Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.

Preparation of Superparamagnetic Dextran-coated Iron Oxide Nanoparticles used as a Novel Gene Carrier into Human Bladder Cancer Cells'

Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticles (SDION) and the feasibility of SDION used as a novel gene carrier for plasmid DNA in vitro. Methods: SDION were prepared by chemical coprecipitation and separated by gel filtration on Sephacryl S-300HR, characterized by TEM, laser scattering system and Vibrating Sample Magnetometer Signal Processor. The green fluorescent protein (pGFP-C2) plasmid DNA was used as target gene. SDION-pGFP-C2 conjugate compounds were produced by means of oxidoreduction reaction. The connection ratio of SDION and pGFP-C2 DNA was analyzed and evaluated by agarose electrophoresis and the concentration of pGFP-C2 in supernatant was measured. Using liposome as control, the transfection efficiency of SDION and liposome was respectively evaluated under fluorescence microscope in vitro. Results: The diameter of SDION ranges from 3 nm to 8 nm, the effective diameter was 59.2 nm and the saturation magnetization was 0.23 emu/g. After SDION were reasonably oxidized, SDION could connect with pGFP-C2 to a high degree. The transfection efficiency of SDION as gene carrier was higher than that of liposome. Conclusion: The successes in connecting SDION with pGFP-C2 plasmid by means of oxidoreduction reaction and in transferring pGFP-C2 gene into human bladder cancer BIU-87 cells in vitro provided the experimental evidence for the feasibility of SDION used as a novel gene carrier.

Expression and Significance of PTEN in Bladder Transitional Cell Carcinoma

Objective： To explore the expression of anti-oncogene PTEN in bladder transitional cell carcinoma and its clinical significance. Methods： Using immunohistochemical S-P methods, the expression of PTEN gene was detected in 62 specimens of bladder cancer and 18 specimens of normal bladder tissue. Results： In the 62 bladder cancers, the positive rate of PTEN was 53.2% （33/62）. All 18 normal bladder tissues were positive for the PTEN expression. The expression of PTEN was negatively correlated to tumor grades （P〈0.05）. Conclusion： The reduced expression of PETN might play an important role in carcinogenesis and progression of bladder cancer. Detection of PTEN might be useful for judgement of tumor development and prognosis.

The Implicaiton of the Expression of Three Cytokines in Carcinoma of Bladder

Objective To explore the expression of CK19, CK20 and bFGF in transitional cell carcinoma of the bladder (TCC) and their biological implication.Methods Samples from 54 patients with TCC were examined for expression of CK19, CK20 and bFGF.Results There was a significant difference in CK19 expression between relapse and non-relapse groups ( P < 0.01) ; no significant difference was found among groups with different pathological grades and clinical stages ( P > 0.05). There were significant differences in CK20 expression among groups with different pathological grades ( P < 0.05) and clinical stages ( P < 0.01) and between the relapse and non-relapse groups( P < 0.000 1) . The expression of bFGF was significantly different among groups with different pathological grades and clinical stages and between the relapse and non-relapse groups ( P < 0.05) .Conclusion The expression of CK19, CK20 and bFGF is closely associated with their biological behavior and among them bFGF is primarily secreted by TCC tumor tissues.

PTEN induces anoikis through its phosphatase activity in human bladder transitional carcinoma cells BIU-87

Objective:To investigate the effects and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of human bladder transitional carcinoma cells BIU-87.Methods:BIU-87 cells were transfected with GFP plasmids containing wild-type PTEN or phosphatase inactivating mutant PTEN(C124A-PTEN)in vitro.The PTEN expression and the phosphorylation levels of focal adhesion kinase(FAK)and protein kinase B(PKB/Akt)were detected by Western blotting.Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis in adherent and non-adherent cells.Results: Compared with the control group,PTEN expression in the cells transfected with wild-type PTEN increased to 210%–260%, while the phosphorylation level of FAK and Akt decreased 59%(P<0.01)and 89%(P<0.01),respectively.And the anoikis percentage increased from 8.32±0.57%to 37.62±2.12%.In the cells transfected with C124A-PTEN,neither the phos- phorylation of FAK and Akt nor the anoikis percentage had obviously changed,although the PTEN expression enhanced remarkably in comparison with the control.Conclusion:Through its phosphatase activity,tumor suppressor gene PTEN can suppress the phosphorylation of FAK and Akt,and induce anoikis in human bladder transitional carcinoma cells BIU-87.

Clinical Significance of p53, Proliferating Cell Nuclear Antige and bcl-2 Expression in Bladder Transitional Cell Carcinoma

Objective: To correlate the frequency of p53 mutations, bcl-2 expression and the proliferation status (proliferating cell nuclear antigen, PCNA) in patients with bladder cancer with cell proliferation, apoptosis and their clinico-pathologic ?ndings. Methods: Para?n-embedded sections from 39 super?cial (T1G1-G3) and 23 invasive (T2-T4a G3 N0M0) primary transitional cell carcinomas (TCC) in the bladder were investigated immunohistochemically for p53, bcl-2 and PCNA. The median follow-up was 37 months; 24 had recurrences. The proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL- positive tumor cells. Results: P53 mutation was identi?ed in 50 patients (80.6%). The mutation was most common in tumors of grade 3 (91.3%) as compared to grade 2 (78.5%) and grade 1 (72.7%, P<0.05). Stage pT2 tumors had a higher frequency of p53 mutation (95.7%) as compared to pTa-1 tumors (74.3%, P<0.01). Only 14 tumors (22.5%) expressed bcl-2; grade 3 tumors expressed bcl-2 signi?cantly more frequently (P<0.05); there was no correlation between bcl-2 and tumor stage. There was no interrelation between p53 mutation and bcl-2 expression (P>0.05). The PI ranged from 17.2% to 41.8% (median 22.4%) and the AI from 1.9% to 3.5% (median 2.9%) in bladder cancer. Statistical analyses revealed a close associations between PI, AI and tumor grade and stage of bladder cancer. Conclusion: P53 mutation correlates with invasion. P53 and PCNA overexpression may o?er valuable additional prognostic information in bladder tumors. With the progression of the tumor grade, cell proliferation may be accompanied by frequent apoptosis in bladder cancer, but the PI increased much more than the AI.

Expression of caveolin-1 and MMP-2 in bladder cancer and its clinical significance

Objective： To explore the expression of caveolin-1 （CAV-1） and matrix metalloproteinase-2 （MMP-2）in bladder cancer and its clinical significance. Methods： The expression of CAV-1 and MMP-2 were detected by the SP immunohistochemical method in 77 cases of bladder cancer. Results： The positive rates of CAV-1, MMP-2 in bladder transitional cell carcinoma （BTCC） and bladder adenocarcinoma were significantly high than those of normal bladder mucosa, the positive rates in the deeper cancer invasion was significantly high than those of superficial bladder cancer （P 〈 0.01）, the positive rates of CAV-1 in grades Ⅰ, Ⅱ and Ⅲ of bladder transitional cell carcinaoma （BTCC） were 15%, 40% and 68%, respectively; the positive rates of MMP-2 in grades Ⅰ, Ⅱ and Ⅲ of bladder transitional cell carcinoma （BTCC） were 20%, 40% and 72%, respectively （P 〈 0.01）. CAV-1 was positive associated with MMP-2 in bladder cancer （r = 0.598, P 〈 0.001）, but not in bladder tissue. Conclusion： CAV-1 and MMP-2 were associated with stage and grade of bladder cancer, which suggested that CAV-1 might improve secretion of MMP-2.

Enterovesical fistula caused by a bladder squamous cell carcinoma

Enterovesical fistulas are not uncommon in patients with inflammatory or malignant colonic disease, however, fistulas secondary to primary bladder carcinomas are extremely rare. We herein reported a patient presenting with intractable urinary tract infection due to enterovesical fistula formation caused by a squamous cell carcinoma of the urinary bladder. This patient underwent en bloc resection of the bladder dome and involved ileum, and recovered uneventfully without urinary complaint. To the best of our knowledge, this is the first case reported in the literature.

Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity

A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

The Expression and Significance of KAl1 and Ki67 in Bladder Transitional Cell Carcinoma

OBJECTIVE To explore the expression and significance of the tumor metastatic suppressor gene the KAI1 and Ki67 antigen in bladder transitional cell carcinoma （BTCC）. METHODS Two-stepped immuno-histochemical staining was used to detect the expression of the KAI1 protein and Ki67 antigen. The reverse transcription polymerase chain reaction （RT-PCR） was used to detect the KAI1 mRNA in 54 BTCC specimens and 32 normal bladder counterparts. 13-actin was the internal control. RESULTS The KAI1 protein was mainly expressed on cell membranes at cell-to-cell borders, with uniform and continuous staining in normal bladder transitional epithelium. However, the number of positive-staining cells was greatly decreased in BTCC epithelium, and with an increase in the stage and Grade and appearance of lymph node metastasis the staining was non-uniform and discontinuous. The Ki67 antigen was expressed in the nucleus, and with an increase in the stage and Grade and appearance of lymph node metastasis, the Ki67 expression increased. The Ki67 antigen was negatively related to the expression of KAI1 （P〈0.01）.The expression level of KAI1 mRNA was much greater in normal bladder transitional epithelium compared to BTCC, moreover, with an increase in the Grade, infiltration depth and appearance of lymph node metastasis, the expression of KAI1 mRNA was reduced. CONCLUSION The expression of KAI1 protein may be used as a prognostic marker to indicate the degree of infiltration and metastasis. Measurement of KAI1 and Ki67 expression together may be helpful in evaluating the metastatic potential and prognosis of BTCC patients.

Growth Inhibition and Apoptosis Induced by Retinoic Acid Combined with Interferon Alpha-2a on Transitional Cell Carcinoma of Bladder

Objective:To identify new favorable agents and develop novel approaches for the chemoprevention and treatment of superficial bladder cancer and investigate the effects of combination of retinoids and interferon α-2a on growth inhibition and apoptosis induction in bladder cancer cell lines. Methods:Four bladder cancer cell lines,grade 1 to 3,and two retinoids,all-trans-retinoic acid(ATRA),9-cis retinoic acid(9cRA),combined with interferon α-2a(INF),were used in the study.We compared the competence of these agents to inhibit growth,induce apoptosis,affect the expression of nuclear retinoid receptors,and modulate STAT1 protein. Results: Most of the bladder cancer cell lines were resistant to the effect of ATRA and 9cRA on growth inhibition and apoptosis induction,even at higher concentration(10 -5M).The effects of ATRA and 9c RA on cell growth and apoptosis were enhanced by INF α- 2a. Combination of ATRA and IFNα-2a induced RARβ and Stat 1 expression in three bladder cancer cell lines. Conclusion:The results demonstrated that INFα-2a synergize with the inhibitory effect of ATRA and 9c RA on the growth inhibition and apoptosis of bladder cancer cells in vitro,which suggested that it has a potential interest for the treatment of transitional cell carcinoma of bladder.

Experimental study on the invasiveness inhibition of bladder cancer cells by nuclear factor-κB decoy——circular dumbbell oligodeoxynucleotides

Objective:To determine whether NF-κB is constitutively activated in human bladder cancer cell and,if so,to deter-mine the invasiveness inhibition of bladder cancer cells by nuclear factor-κB decoy—circular dumbbell oligodeoxynucleotides(CD-ODN).Methods:NF-κBp65 activation was determined by immunohistochemical analysis of formalin-fixed,paraffin-embed-ded specimens from 38 cases of bladder transitional cell carcinoma patients.We quantified nuclear staining of RelA as a marker of NF-κBp65 activation.CD-ODN were transfected into human bladder cancer cell line BIU87 by lipofectamine.Luciferase reporter were applied to detecting NF-κB DNA binding activity.The expression levels of uPA were detected by RT-PCR and the cells’ invasion ability by transwell cell culture chamber.Results:P65 excessive activation existed in tumor cell(P<0.01),the activation degree correlated significantly with the expression of uPA(r=0.89,P<0.01),as well as related to tumor invasion-related clinicopathological features such as lymphatic metastasis(P<0.01)and pathological ranking(P<0.05);After transfection with CD-ODN,the activation of NF-κB in BIU87 cell line was suppressed remarkably,the expression level of uPA was decreased and the cells’ invasiveness was weakened as well.Conclusion:Excessively activated NF-κB is related to tumor progression pos-sibly due to its transcriptional regulation of invasion-related factors such as uPA.CD-ODN can efficiently suppress DNA binding activity of NF-κB to reduce the invasive potency of tumor.

Study of wavy laminar growth of human urinary bladder cancer cell line in vitro

Objective: To observe the ordered growth behavior of human urinary bladder cancer cell line (BIU) under culture in vitro. Methods: The suspension of BIU cells was spread locally in a culture container. When the cells grew a-long the wall to form a cellular colony, macroscopic and microscopic observations complemented with measurements of the parameters including expanding diameter, expanding rate, cell shape. average cell density, average cell size. dehydrogenase activity and sensitivity to pH were conducted dynamically. Results: During cell culture, obvious laminar characteristics appeared in localized growing BIU cell colonies and there was difference between the cells of different zones in shape, size, density, dehydrogenase activity and sensitivity to pH. Conclusion: Space closing and bio-dissipation result in self-organization of BIU cells with ordered growth behavior. The present experiment offers a simple, controllable model for the study of wavy growth of human cells.

Expression of connexin43 and its correlation with the expressions of Bcl-2 and Bax proteins in transitional cell carcinoma of the bladder

Objective: To study the expression of connexin43 (Cx43) gene and its correlation with the apoptosis related genes Bcl-2 and Bax in transitional cell carcinoma of the bladder (BTCC), and to investigate the role of Cx43 in the BTCC. Methods: Reverse transcription polymerase chain reaction was used to detect the expression of Cx43 mRNA and immunocytochemistry technique was used to detect the expressions of Bcl-2 and Bax proteins in 60 cases of BTCC tissues, and compared with that of 15 cases of pericancerous tissues and 15 cases of normal bladder tissues. Results: The positive expression rate of Cx43 mRNA in 60 cases of BTCC tissues was 43.33% which was significantly lower than that in pericancerous tissues (73.33%) and normal tissues (100%) (χ2 = 17.58, P < 0.01). The expression of Cx43 had significant negative correlation with the pathological degree and lymph node metastasis of BTCC (χ2 = 9.33 and 9.74, respectively, P < 0.01). However, no correlation was found between the expression and patient sex, age, clinical staging and diameter and growth pattern of BTCC (P > 0.05). Expression of Cx43 negatively correlated with Bcl-2 protein (r = -0.63, P < 0.01) and positively correlated with Bax protein (r = 0.52, P < 0.01). Conclusion: The down-regulated expression of Cx43 gene was closely associated with the development, invasion and metastasis of BTCC. It could be used as a prognostic indicator for BTCC. Cx43 gene may have antagonistic effects with Bcl-2 gene and synergic with Bax gene in the initiation and progression of BTCC.

MDR1/P-Glycoprotein Overexpression in Bladder Transitional Cell Carcinoma and its Correlation with Expression of Survivin and Fas

OBJECTIVE To explore the expression of the MDR1/P-glycoprotein, Fas and survivin and to examine their correlation with the biologic behavior of bladder transitional cell carcinoma (BTCC). METHODS Immunohistochemistry was used to examine the expression of P-gp, survivin and Fas in BTCC (n=64) and normal bladder mucosa (n=12). RESULTS The expression level of P-gp and survivin in BTCC was higher compared to normal bladder mucosa (P<0.01) and their expression was strongly correlated with clinical grading (P<0.01). In BTCC and normal bladder mucosa Fas expression was 50% and 100%, respectively (P< 0.01). Recurrent BTCC showed higher expression than primary BTCC (P< 0.01) and the expression of P-gp in BTCC had a reverse correlation with Fas expression but no correlation with survivin expression. CONCLUSUON The MDR of BTCC was strongly correlated with the ex- pression of P-gp and Fas, but was not correlated with survivin expres- sion. Thus, enhancing cancer sensitivity to chemotherapy by reversing multidrug resistance with reversal agents or up-regulating Fas expres- sion by apoptotic enhancing agents, might be a potential therapy to pre- vent tumor recurrence and invasiveness.

Unusual side effect and dramatic response from gefitinib as first-line use in a male-patient with bronchioloalveolar carcinoma

A 45-year-old man with bronchioloalveolar carcinoma was treated with gefitinib. The most severe side effect was hemorrhagic cystitis but he had a dramatic response.

Apoptosis Induced by Ginsenoside Rg3 in a Human Bladder Carcinoma Cell Line

OBJECTIVE This study was conducted to explore the effect of Rg3 on inhibition of proliferation and induction of apoptosis in bladder cancer cells. METHODS The EJ bladder cancer cell line was treated with Rg3 at various concentrations. Cell proliferation was measured by the MTT assay. Morphological changes in the cells were observed by fluorescent staining using Hoechst 33258. The cell cycle and apoptotic rate were analyzed by flow cytometry （FCM） and the expression of caspase-3 in cells was detected by immunocytochemistry. DNA ladder analysis was conducted by agarose gel electrophoresis. RESULTS Rg3 inhibited proliferation of EJ cells in a concentration-dependent manner, resulting in an IC50 for Rg3 at 48 h of 125.5 μg/ml. When treated with 150 μg/ml of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including condensed chromatin, nuclear fragmentation, apoptotic bodies and bright fluorescent granules as well as a higher caspase-3 expression. The FCM assay indicated that Rg3 altered the cell cycle and induced apoptosis of the EJ cells, when treated for 24 h and 48 h with 75 μg/ml of Rg3 as well as for 48 h with 150 μg/ml. The percentages of cells in the S phase and the GJM transition were increased, whereas the percentages of cells in the G0-G1 transition were decreased. The apoptotic rates were increased from （1.05±0.17）% in the control group cells to （8.41 ±0.98）%, （18.57±2.20）% and （33.98±1,64）% respectively. Significant changes in the DNA ladders, showed that the effects of Rg3 were displayed in a dose and time dependent manner. CONCLUSION The results suggest that Ginsenoside Rg3 exerts an inhibitory effect on proliferation of EJ cells by inducing apoptosis.

Effects of dendritic cell vaccines on hematogenous micrometastasis of bladder cancer carrying for PBL-SCID mice

Objective： To study the effect of dendritic cells loaded with whole tumor antigen on hematogenous micrometastasis of bladder cancer model in hu-PBL-SCID mice. Methods： T24-3 ceil subset was selected from human bladder transitional cell carcinoma T24 cell line by Boyden chamber system. The SCID mice intraperitoneally injected with 4 × 10^7 hu-PBL and subcutaneously injected with 3 × 10^6 T24-3 cells were named hu-PBL-T24-3-SCID model. Human IgG level in the blood plasma of mice was detected by ELISA, and human CD3^＋, CD4^＋, CD8^＋ T cells in blood and spleen cells of mice were detected by FCM analysis for human immune reconstruction study. Human CK20 mRNA expression in mice peripheral blood was detected by RT-PCR to investigate metastasis of tumor cells. The PBMCs were isolated from human peripheral blood, and were induced into DCs by co-culture with rhGM-CSF and rhlL-4 in vitro. The DC vaccines were produced by co-culturing with whole tumor antigen which was purified through freezing and melting T24-3 cell subset. After T24-3 cells injected into SCID mice for 5 weeks, the mice were treated with DC vaccines. Results： All mice were initially treated at 5th week. The expression of CK20 mRNA in peripheral blood of DC vaccines treated mice was the lowest. There was 2 mice showing CK20 mRNA expression and 3 mice with metastasis tumor in PBS group. MMP-7 mRNA expression in tumor tissues of DC vaccines treated mice was statistically lower than that of PBS group （P 〈 0.01）. Conclusion： DC vaccines have a good effect on hu-PBL-SCID mice bladder cancer model by reducing hematogenous micrometastasis.