Expression and significance of m1A transmethylase,hTrm6p/hTrm61p and its related gene hTrm6/hTrm61 in bladder urothelial carcinoma

Objective: To discuss the expression of hTrm6p/hTrm61p in bladder urothelial carcinoma tissue and its relationship with m1A level in urine, as well as the influences of hTrm6/hTrm61 on the proliferation and apoptosis of cancer cell line on urothelium. Methods: m1A levels in urine of 32 patients of bladder urothelial carcinoma and normal people were detected by HPLC/ESI-Q-TOF-MS, hTrm6p/hTrm61p expression levels in cancer tissue and para-carcinoma tissue of the same patient were detected by western blotting, and hTrm61 expressions of cancer cell line T24, 5637, and EJ of bladder urothelium and kidney cell line HEK-293 of human embryo were detected by RT-qPCR. The hTrm61 high-expression cell line is selected to detect the situation of proliferation and apoptosis by CCK-8 and flow cytometry (FCM) after knocking out hTrm61 with siRNA. Results: m1A level in urine of carcinoma of urinary bladder is significantly higher than that of normal people, and hTrm6p/hTrm61p expression level in cancer tissue is significantly higher than that in para-carcinoma tissue and has linear correlation with m1A level in urine. The hTrm61 expression in cell line 5637 is significantly higher than that of T24, EJ, and HEK293, and apoptosis is significantly affected after hTrm61 is knocked out from 5637 cell line. Conclusion: High-level expression of hTrm6p/hTrm61p is an important reason for high emission of m1A in urine, and hTrm6/hTrm61 promotes the occurrence of carcinoma of urinary bladder.     

ALKBH2, a novel AlkB homologue,contributes to human bladder cancer progression by regulating MUC1 expression

The ALKBH family of proteins are highly expressed in various types of human cancer where they are involved in tumor growth and progression. However, multiple isoforms of ALKBH exist and the effect of individual isoforms on the development of urinary bladder cancer is unknown, particularly the molecular mechanisms involved in the progression from a noninvasive to invasive phenotype. We examined the role and function of ALKBH2 in human bladder cancer development in vitro and provide the first report that suppression of ALKBH2 in a human urothelial carcinoma cell line, KU7, reduces the expression of the transmembrane mucin protein, MUC1, and induces G1 cell cycle arrest. Moreover, reduction of ALKBH2 suppressed epithelial to mesenchymal transition (EMT) via increasing E‐cadherin and decreasing vimentin expression. Transfection of MUC1 siRNA inhibited cell proliferation and EMT to the same extent as ALKBH2 gene silencing in vitro. ALKBH2 knockdown significantly suppressed MUC1 expression and tumor volume of bladder cancers in vivo as assessed in an orthotopic mouse model using ALKBH2 shRNA transfected KU7 cells. Immunohistochemical examination showed high expression levels of ALKBH2 in human urothelial carcinoma samples, especially in high‐grade, superficially and deeply invasive carcinomas (pT(1) and >pT(2)), and in carcinoma in situ but not in normal urothelium. This study demonstrates that ALKBH2 is an upstream molecule of the oncoprotein, MUC1, and regulates cell cycle and EMT, resulting in progression of urothelial carcinomas.     

CXCR4 expression reflects tumor progression and regulates motility of bladder cancer cells

Transitional cell carcinoma of the urinary bladder remains life threatening due to the high occurrence of metastases. Emerging evidence suggests that chemokines and their receptors play a critical role in tumor metastases. In our study, we performed a systematic analysis of the mRNA and protein expression levels of all 18 chemokine receptors in normal urothelium and bladder cancer. CXCR4 was the only chemokine receptor whose mRNA expression level was upregulated in bladder cancer cell lines as well as in invasive and locally advanced bladder cancer tissue samples (pT2-pT4). In contrast, superficial bladder tumors (pTa and pT1) displayed low CXCR4 expression levels and normal urothelial cells were negative for CXCR4. Immunohistochemistry of a bladder cancer tissue microarray (TMA) confirmed that a subgroup of invasive bladder cancers revealed a high CXCR4 protein expression, while superficial bladder tumors showed low immunoreactivity. To investigate the functional significance of CXCR4 expression, we performed migration and invasion assays. Exposure of CXCR4-positive bladder cancer cells to CXCL12 in a Boyden chamber type assay provoked a significant increase in migration as well as invasion across a Matrigel barrier. Enhanced migration and invasion were inhibited by a CXCR4-specific blocking antibody. In contrast, normal urothelial cells did not respond to CXCL12 and lacked chemotactic migration. In conclusion, bladder cancer cells express CXCR4 progressively with advanced tumorigenesis and this receptor interacts with CXCL12 to mediate tumor chemotaxis and invasion through connective tissue. These properties identify CXCR4 as a potential target for the attenuation of bladder cancer metastases.     

Adjuvant Chemotherapy for Invasive Urothelial Cancer: Experience with a Methotrexate, Vincristin, Cisplatin, Cyclophosphamide, Adriamycin and Bleomycin (MVP-CAB) Regimen: A Preliminary Report

MVP-CAB chemotherapy (methotrexate, vincristine, cisplatin,cyclophosphamide, adriamycin and bleomycin) was administeredto 28 patients with high grade, locally-invasive or regionallymph node- involved urothelial cancer as an adjuvant therapyafter radical surgery. The median follow-up period of all theevaluated patients was 24 (range, 5–62) months. The mediandisease-free durations in patients with pT1b+2+ 3 bladder andupper urothelial cancer were 26 and 22 months, respectively.In contrast, all patients with pT4 disease or lymph node metastaseshad a recurrence within 24 months of surgery. The median disease-freedurations in patients with pure transitional cell carcinoma(grade 3) of the bladder and upper urothelial cancer were 19and 18 months, respectively. The median disease-free durationin patients with grade 3 pT1b+2+3 pure transitional cell carcinomawas 19 months. In contrast, the median disease-free durationin bladder cancer patients with pT1b+2+3 squamous cell carcinomacomponents was 35 months. The three-year actuarial survivalrates were 79 and 89% for pT1b+2+3 bladder and upper urothelialcancer, respectively, while the three-year actuarial survivalrates of patients with pT4 bladder cancer and pT4 upper urothelialcancer were 0 and 100%, respectively. The two-year actuarialsurvivals in the bladder cancer and upper urothelial cancerpatients with lymph node involvement were 0 and 100%, respectively.The three-year actuarial survivals of patients with pure transitionalcell carcinoma (grade 3) were 53 and 80% in bladder cancer andupper urothelial cancer patients, respectively. The three-yearactuarial survival rate in patients with squamous cell carcinomaor adenocarcinoma components which did not recur was, however,100%. Although randomized studies comparing MVP-CAB and M-VAC(methotrexate, vinblastin, adriamycin and cisplatin) or otherchemotherapeutic regimens will be necessary, we believe ourresults indicate that MVP-CAB chemotherapy may be useful asan adjuvant therapy for patients with urothelial cancer, includingthose with squamous cell carcinoma and adenocarcinoma components.More intensive MVP-CAB chemotherapy, i.e., increasing the doseof cisplatin and giving at least five courses, as well as theuse of granulocyte colony stimulating factor and a new antiemeticdrug (granisetron), will, however, be necessary for patientswith pT4 or lymph node-involved disease.     

High-throughput tissue microarray analysis of CMYC amplificationin urinary bladder cancer

Alterations of chromosome 8, preferentially deletions of 8p and gains of 8q, belong to the most frequent cytogenetic changes in bladder cancer. CMYC on 8q24 is a candidate oncogene in this region. Little is known about the clinical significance of CMYC copy number changes in urinary bladder cancer because its frequency is low and a limited numbers of tumors were analyzed so far. To investigate the impact of CMYC alterations on tumor progression and patient prognosis in bladder cancer, we applied FISH to a tissue microarray containing 2317 bladder cancer samples. Presence of CMYC copy number increase was associated with advanced stage and high grade. CMYC amplifications were seen in 3 of 467 pTa (0.6%), 10 of 247 pT1 (4%) and 11 of 201 pT2-4 urothelial carcinomas (5.5%; p < 0.0001), as well as in 1 of 123 G1 (0.8%), 8 of 470 G2 (1.7%) and 17 of 365 G3 urothelial carcinomas (4.7%; p < 0.0001). CMYC gains were present in 49 of 467 pTa (10.5%), 39 of 247 pT1 (15.8%) and 43 of 201 pT2-4 urothelial carcinoma (21.4%; p < 0.0001), as well as in 7 of 123 G1 (5.7%), 56 of 470 G2 (11.9%) and 72 of 365 G3 urothelial carcinomas (19.7%; p < 0.0001). CMYC copy number changes were unrelated to prognosis of bladder cancer patients. We conclude that alterations of the CMYC gene, including copy number gains and amplifications, are linked to genetically unstable bladder cancers that are characterized by a high histologic grade and/or invasive growth. Patient prognosis was not affected by CMYC gene copy number changes.     

PRR11在膀胱癌组织中的表达及其对膀胱癌T24细胞增殖和凋亡的影响

目的 探讨富含脯氨酸蛋白11(PRR11)在膀胱癌组织中的表达及其基因沉默对膀胱癌T24细胞增殖和凋亡的影响.方法 采用免疫组织化学法检测57例膀胱尿路上皮癌组织及其癌旁组织中PRR11蛋白的表达,并分析PRR11蛋白表达水平与患者临床病理特征的关系.qRT-PCR和Western blot检测人永生化膀胱上皮细胞株S...     

miR-623在膀胱癌中的表达及靶向Fascin1抑制膀胱癌细胞迁移和侵袭

目的:探讨miR-623在膀胱癌中的表达及通过靶向Fascin1对膀胱癌细胞迁移和侵袭能力的影响。方法:采用实时荧光定量PCR检测正常膀胱上皮组织、膀胱癌组织、正常永生化膀胱上皮细胞系(SV-HUC-1)和膀胱癌细胞系（T24、UMUC3）中miR-623的表达量;采用脂质体瞬时转染miR-623 mimics,划痕实验和Transwell侵袭实验检测miR-623过表达后膀胱癌细胞迁移、侵袭能力的改变;生物信息学预测miR-623的作用靶蛋白。miR-623过表达后Western blot及双荧光素酶报告基因检测其靶点的表达及结合情况;使用Fascin1特异性siRNA观察膀胱癌细胞迁移和侵袭能力的变化,并同时转染miR-623 inhibitor进行恢复实验。结果:miR-623在膀胱癌中的表达水平显著低于在正常膀胱组织中的表达（P＜0.05）,在膀胱癌细胞系（T24、UMUC3）中的表达水平显著低于正常膀胱细胞系(SV-HUC-1)（P＜0.05）;miR-623过表达显著抑制T24和UMUC3细胞的迁移和侵袭能力。生物信息学预测Fascin1为miR-623的靶基因,在T24和UMUC3细胞中过表达miR-623,能够显著降低Fascin1的蛋白水平;荧光素酶报告基因分析结果证实miR-623作用于Fascin1的3'-UTR。下调Fascin1表达能够抑制膀胱癌T24和UMUC3细胞的迁移和侵袭能力,同时抑制miR-623的表达能够提高细胞的迁移和侵袭能力。结论:miR-623在膀胱癌中表达水平降低,是一个抑癌因子;并可能通过靶向Fascin1调节膀胱癌的侵袭和转移能力。     

Can Recurrence and Progression be Predicted by HYAL-1 Expression in Primary T1 Bladder Cancer?

Background: Molecular prognostic markers have been under investigation for the last decade and novalidated marker to date has been proven to be used in daily clinical practice for urinary bladder cancers. Theaim of the present study is to evaluate the significance of HYAL-1 expression in prediction of recurrence andprogression in pT1 urothelial carcinomas. Materials and Methods: Eighty-nine urothelial carcinoma casesstaged as T1 according to 2004 WHO classification were studied. Representative sections from every case werestained immunohistochemically for HYAL-1 and scored between 0 and +3, according to staining density, andgraded as low and high for the scores 0-1 and 2-3, respectively. Results: Of the 89 pT1 bladder cancer patients,HYAL-1 expression was high in 92.1% (82 patients; 72 patients +3 and 10 patients +2) and low in 7.9% (only 7patients; 6 patients +1 and 1 patient 0) of the cases. Of the 89 patients, 38 (42.7%) had recurrence and 22 (24.7%)showed progression. HYAL-1 staining did not show significant characteristics for tumor grade, accompanyingCIS, multiplicity, tumor size, age and sex. HYAL-1 expression did not have any prognostic value in estimatingrecurrence or progression. Conclusions: HYAL-1 expression was found to be high, but did not have any prognosticimportance in T1 bladder urothelial carcinomas.     

姜黄素诱导人膀胱癌UMUC2细胞株凋亡的作用

目的为寻找有效无毒的膀胱灌注药物用于膀胱肿瘤术后预防肿瘤种植与复发,我们观察了姜黄素对人膀胱癌细胞UMUC2的细胞毒效应及诱导凋亡的体外作用。方法以0~160 μmol/L姜黄素作用于UMUC2细胞1~48 h,应用MTT法、平板克隆实验观察姜黄素的细胞毒效应,荧光染色观察凋亡形态学改变,流式细胞仪进行细胞周期分析及凋亡定量测定,免疫细胞化学染色观察凋亡相关蛋白p53和Survivin的表达。结果所有浓度组姜黄素均对膀胱癌UMUC2细胞有明显的生长抑制作用,160 μmol/L姜黄素作用1 h对全部癌细胞是致命的。荧光染色观察到典型的核凝集、碎裂凋亡形态学改变。流式细胞术定量分析了亚G1峰,后者同时表明姜黄素导致的细胞周期阻滞以G2/M期为主,使S期细胞比例显著减少。免疫细胞化学染色显示姜黄素下调p53、Survivin蛋白的表达。结论姜黄素明显抑制膀胱癌细胞系UMUC2的生长并诱导凋亡,导致细胞G2/M期阻滞,其作用机制与其下调p53、Survivin的表达相关,克隆形成实验证实大剂量姜黄素、短期作用对膀胱癌细胞系UMUC2有致命的细胞毒效应,显示了姜黄素用于膀胱癌患者的化学治疗,尤其是腔内灌注化疗的可能性。     

Two cases of plasmacytoid variant of urothelial carcinoma of urinary bladder: systemic chemotherapy might be of benefit

We report two cases of the plasmacytoid variant of urothelial carcinoma of urinary bladder in which systemic chemotherapy was effective. In the first case, a 76-year-old man presented with dysphasia. Magnetic resonance imaging (MRI) and computed tomography revealed a brain tumor and a bladder tumor. Resection of the brain tumor and transurethral resection of the bladder tumor were performed. The pathological diagnosis was plasmacytoid variant of urothelial carcinoma of urinary bladder with brain metastasis (pT1N0M1). Three cycles of adjuvant MVAC (methotrexate, vinblastine, adriamycin, and cisplatin) chemotherapy were performed. He has no evidence of recurrence 96 months after resection of brain metastasis. In the second case, a 76-year-old man presented with hematuria. MRI revealed a bladder tumor with abdominal wall invasion, and a transurethral biopsy was performed. The pathological diagnosis was plasmacytoid variant of urothelial carcinoma of urinary bladder (cT4bN0M0). After three cycles of neoadjuvant GC (gemcitabine and cisplatin) chemotherapy, MRI demonstrated a complete response. Radical cystectomy was performed, and the pathological diagnosis was pT0pN0. Although there was no evidence of recurrence 9 months after radical cystectomy, he died from other causes. Our two cases suggest that systemic chemotherapy might be effective for the plasmacytoid variant of urothelial carcinoma.     

Syndecan-1, a new target molecule involved in progression of androgen-independent prostate cancer

Heparan sulfate proteoglycan syndecan-1 (CD138) is well known to be associated with cell proliferation, adhesion and migration in various types of malignancies. In the present study, we focused on the role of syndecan-1 in human prostate cancer. Immunohistochemical analysis revealed either no or rare expression of syndecan-1 in normal secretory glands and prostate cancer cells at hormone naïve status, whereas the expression was significantly increased in viable cancer cells following neo-adjuvant hormonal therapy. Syndecan-1 expression was much higher in the androgen independent prostate cancer cell lines DU145 and PC3, rather than the androgen-dependent LNCaP, but the level in LNCaP was up-regulated in response to long-term culture under androgen deprivation. Silencing of syndecan-1 by siRNA transfection reduced endogenous production of reactive oxygen species through down-regulating NADPH oxidase 2 and induced apoptosis in DU145 and PC3 cells. Consistently, NADPH oxidase 2 knockdown induced apoptosis to a similar extent. Subcutaneous inoculation of PC3 cells in nude mice demonstrated the reduction of tumor size by localized injection of syndecan-1 siRNA in the presence of atelocollagen. Moreover, the mouse model and chorioallantoic membrane assay demonstrated significant inhibition of vascular endothelial growth factor and tumor angiogenesis by silencing of syndecan-1. In conclusion, syndecan-1 might participate in the process of androgen-dependent to -independent conversion, and be a new target molecule for hormone resistant prostate cancer therapy. ( Cancer Sci 2009; 100: 1248–1254)     

膀胱癌组织中TIPE通过调节VEGFR2表达促进膀胱癌细胞增殖、迁移和血管生成

目的:探究膀胱癌组织中TIPE通过调节血管内皮生长因子受体2（VEGFR2）表达促进膀胱癌细胞增殖、迁移和血管生成的机制。方法:收集膀胱癌组织及其对应的癌旁组织各34例,以人膀胱上皮永生化细胞SV-HUC-1、人膀胱癌细胞株UMUC3和人脐静脉内皮细胞（HUVECs）为体外研究对象,免疫组织化学（IHC）检测组织中TIPE表达;RT-PCR检测细胞中TIPE mRNA表达;Western blot检测组织和细胞中TIPE、PDK1、p-PDK1和VEGFR2蛋白表达;CCK-8检测UMUC3细胞增殖能力;Transwell实验检测UMUC3细胞迁移能力;血管形成实验检测HUVECs血管形成能力。结果:与癌旁组织相比,膀胱癌组织中TIPE IHC评分和蛋白表达明显上调（P＜0.05）。与SV-HUC-1组相比,UMUC3组中TIPE mRNA和蛋白表达水平明显上调（P＜0.05）。与siRNA-NC组相比,siRNA-TIPE组UMUC3细胞增殖、迁移能力、HUVECs的管道交叉数明显降低（P＜0.05）,TIPE、p-PDK1和VEGFR2蛋白表达水平明显降低（P＜0.05）。PDK1抑制剂GSK2334470（简称GSK）的使用浓度为4 μmol/L。与Vector组相比,TIPE组UMUC3细胞TIPE、p-PDK1和VEGFR2蛋白表达水平及细胞增殖、迁移能力和HUVECs的管道交叉数明显升高（P＜0.05）,Vector+GSK组UMUC3细胞p-PDK1和VEGFR2蛋白表达水平及细胞增殖、迁移能力和HUVECs的管道交叉数明显降低（P＜0.05）,GSK可逆转TIPE对UMUC3和HUVECs细胞的作用。结论:TIPE通过磷酸化PDK1上调VEGFR2表达促进膀胱癌细胞增殖、迁移和血管生成。     

siRNA沉默c-FLIP基因表达与表柔比星联合应用对MCF-7细胞凋亡的影响

目的 探讨RNA干扰技术沉默c-FLIP基因表达联合应用表柔比星对人乳腺癌细胞MCF-7凋亡的影响。方法 体外化学合成c-FLIP序列特异性双链小干扰RNA(siRNA),转染MCF-7细胞,应用Western blot方法检测c-FLIP蛋白的抑制水平、检测Caspase-8的表达差异;应用流式细胞术检测细胞凋亡的变化及表柔比星与c-FLIP-siRNA联合应用对细胞凋亡的影响。结果 c-FLIP-siRNA能有效地抑制c-FLIP蛋白的表达(P＜0.05),促进细胞的凋亡(P＜0.05)。c-FLIP-siRNA和表柔比星联合应用较单用表柔比星能明显促进MCF-7细胞的凋亡(P＜0.05)。结论 c-FLIP-siRNA能够促进MCF-7细胞的凋亡,并且能够增强表柔比星的抗肿瘤作用。