LIN28A和LAMP1在膀胱癌细胞系中的表达及意义

目的:探讨LIN28A和LAMP1在膀胱癌细胞系中表达情况,以及两者之间的关系,推测其可能临床意义及对肿瘤进展的影响。方法:采用RT-PCR检测膀胱癌细胞系LIN28A、LIN28B和LAMP1表达,免疫荧光检测LIN28A和LAMP1二者蛋白表达定位;LIN28A敲减后通过qRT-PCR检测LAMP1的mRNA表达变化。结果:5个癌细胞系T24、UM-UC3、J82、5637和SW780和正常移行上皮细胞系SV-HUC-1均表达LIN28A,其中J82也表达LIN28B;5个癌细胞系均表达LAMP1,SV-HUC-1不表达LAMP1;LIN28A和LAMP1蛋白均定位在胞浆;LIN28A敲减后对LAMP1的mRNA表达变化无明显影响,相应蛋白变化需要进一步验证。结论:4个膀胱癌细胞系T24、5637、UM-UC3和SW780可以用于LIN28A与肿瘤相关的机制研究,而J82可用于LIN28B的机制研究。LIN28A对肿瘤细胞和干细胞的调控方面可能具有相似性,敲减后对其靶点mRNA表达量无明显影响,LAMP1蛋白可能对肿瘤细胞侵袭转移具有抑制作用。     

LncRNA DANCR regulates lymphatic metastasis of bladder cancer via the miR-335/VEGF-C axis

BackgroundSubstantial evidence indicate that long non-coding RNA (lncRNA) and microRNA (miRNA) act as key role in bladder cancer. Differentiation antagonistic ncRNA (DANCR) could be used as a biomarker in the occurrence and development of cancer. This study aims to explore the mechanism of DANCR/miR-335/VEGF-C axis affecting lymphatic metastasis of bladder cancer.MethodsqRT-PCR detects the expression of DANCR in bladder cancer cell lines (SW780, 5637, T24, UM-UC-3) and normal bladder cell lines (SV-HUC-1), and selects T24 cell lines for subsequent experiments. The expression levels of DANCR, miR-335 and VEGF were measured by qRT-PCR, and the dual luciferase reporter gene verified the targeted regulation of DANCR on miR-335 and miR-335 on VEGF. CCK-8, Transwell and Wound healing assay detect the proliferation, invasion and migration ability of bladder cancer cells, Endothelial cell adhesion assay and Western blot further prove the lymphatic metastasis of bladder cancer.ResultsIn this study, DANCR was highly expressed in bladder cancer cell lines. Transfection of si-DANCR significantly inhibits the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells. Dual luciferase assay confirmed that DANCR targets miR-335/VEGF-C. Transfection of miR-335 mimic promotes the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells, overexpression of DANCR eliminates the promotion of miR-335 mimic on bladder cancer cells. Further experiments proved that inhibition of miR-335 and overexpression of VEGF-C can reverse the inhibitory effect of silencing DANCR on bladder cancer cells.ConclusionsIn bladder cancer, DARCR plays an important role, which regulates the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells through the miR-335/VEGF-C molecular axis.     

High expression of polo-like kinase 1 is associated with the metastasis and recurrence in urothelial carcinoma of bladder

ObjectivesPolo-like kinase 1 (Plk1) has been widely pursued as an oncology target because it is overexpressed in several human tumor types. To investigate whether Plk1 plays a general role in bladder urothelial carcinoma, we examined the expression of Plk1 protein in bladder urothelial carcinoma and cell lines, and analyzed the relationship among Plk1 protein expression, metastasis, and recurrence of urinary bladder urothelial carcinoma.MethodsImmunohistochemistry was used to detect the expression of Plk1 in 120 bladder urothelial carcinoma. Moreover, the expression of Plk1 was analyzed by Western blot in 60 bladder urothelial carcinoma and 21 normal epithelial tissues. MTT assay and flow cytometry and transwell assay were used to examine the proliferative and invasive ability of bladder cancer cells with the treatment of scytonemin (the inhibitor of Plk1). Statistical analysis was used to discuss the association between Plk1 expression and clinicopathologic parameters, tumor metastasis and recurrence, and the proliferative and invasive ability and cell cycle process of the bladder cancer cells.ResultsThere was a significantly higher Plk1expressions in bladder urothelial carcinoma and highly invasive bladder T24 cells than those in bladder normal tissues and the superficial bladder BIU-87 cells. Plk1 expression was positively correlated with histologic grade, pT stage, recurrence, and metastasis. With the increasing concentration of scytonemin, we found that not only the cell proliferation and invasion activity decreased significantly, but also the cell cycle was blocked at G2/M stage.ConclusionPlk1 expression status was closely correlated with important histopathologic characteristics (grades and stages) and the recurrence and metastasis of bladder urothelial carcinomas. Furthermore, Plk1 played an important function on the bladder cancer cells' proliferation by regulating the cancer cell cycle from G1/S to G2/M and probably promoted the invasion and metastasis of bladder cancer.     

一氧化氮对组织特异性溶瘤腺病毒转染膀胱肿瘤细胞的影响

目的:研究一氧化氮(NO)对肿瘤组织特异性溶瘤病毒转染过程的影响及对外源基因表达的调节作用。方法:构建组织特异性溶瘤腺病毒,常规培养膀胱肿瘤BIU-87和5637细胞株,以硝普钠作为外源性NO的供体。应用PTIO和L-NMMA分别作为内源性NO的清除剂和诱导型一氧化氮合酶(NOS)的抑制剂。采用Nitrate/Nitrite Assay Kit检测NO和(或)病毒作用前后膀胱肿瘤细胞培养液中的NO水平。应用四甲基偶氮唑盐(MTT)法检测NO对重组病毒抗肿瘤细胞增殖的影响;透射电镜观察腺病毒颗粒进入细胞情况和亚细胞结构变化。结果:膀胱肿瘤细胞BIU-87和5637本身培养液中NO水平很低,给予外源性NO供体后NO水平随时间延长而升高。重组病毒Ad-UPⅡ-E1A能通过E1A基因发挥溶瘤作用。NO能够促进该病毒转染入BIU-87、5637细胞。50μmol/L和100μmol/L的NO联合Ad-UPⅡ-E1A 30MOI作用后能够促进肿瘤细胞的增殖,而200μmol/L的NO联合重组腺病毒作用后则促进肿瘤细胞的死亡。NO对Ad-UPⅡ-E1A的作用具有时间依赖性。透射电镜观察发现,重组病毒Ad-UPⅡ-E1A能够进入并在膀胱肿瘤细胞内复制,而NO能够提高病毒的转染效率并引起肿瘤细胞自吞噬和凋亡。结论:NO能够促进溶瘤腺病毒Ad-UPIIE1A转染膀胱肿瘤细胞的效率,但NO因其浓度不同对溶瘤腺病毒的溶瘤效果具有双向调节作用,低剂量的NO能够下调重组病毒E1A的表达从而促进肿瘤细胞增殖,而高剂量的NO通过上调E1A的表达因而发挥溶瘤效应。     

Overexpression of lncRNA TINCR is associated with high-grade,invasive, and recurring tumors,and facilitates proliferation in vitro and in vivo of urothelial carcinoma of the bladder

ObjectivesThe aberrant expression of long noncoding RNAs (lncRNAs) plays roles in cancer development. In this work, we measured the expression of lncRNA terminal differentiation-induced non-coding RNA (TINCR) in urothelial carcinoma of the bladder (UCB), determined its impact on the proliferation of UCB in vitro and in vivo and identified its possible targets.MethodsThe expression levels of genes were measured by Real-Time quantitative Polymerase Chain Reaction (qPCR). Cell proliferation, cell motility, and cell apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, wound healing assay, and ELISA, respectively. Tumor growth in vivo was determined by xenograft formation assay in nude mice. The predicted binding site between TINCR and hsa-miR-125b was confirmed by dual luciferase reporter assay.ResultsThe expression levels of TINCR were higher in cancerous tissues than that in paired noncancerous tissues of UCB. Higher expression levels of TINCR were positively associated with high-grade, invasive, and recurring tumors. Depletion of TINCR retarded proliferation, decreased motility, increased apoptosis in UCB cells, and markedly reduced tumor growth in xenograft nude mice. The predicted binding site between TINCR and hsa-miR-125b was functional. TINCR downregulated hsa-miR-125b in UCB cells. Hsa-miR-125b mimic reversed the proliferation caused by TINCR up-expression in UCB cells.ConclusionsUp-regulation of TINCR may act as an unfavorable indicator for the diseasing status of UCB. TINCR facilitates bladder cancer proliferation in vitro and in vivo. Hsa-miR-125b is a target for TINCR in UCB.     

结直肠癌表观沉默蛋白Bmi1与Notch信号通路调控的关系

目的 探讨人结直肠癌中表观沉默蛋白Bmi1和Notch1的蛋白表达与结直肠癌病理特征的关系,观察Notch通路对结直肠癌细胞增殖凋亡及Bmi1表达的影响。方法 应用免疫组织化学技术(SP法)检测85例结直肠癌组织及其邻近正常肠黏膜中Notch1及Bmi1的蛋白表达;将Notch1通路中γ-分泌酶抑制剂DAPT,作用于结肠癌SW480细胞株,运用噻唑蓝(MTT)比色法检测细胞增殖状态;流式细胞仪观察其对细胞凋亡的影响,Western blot检测ICN及Bmi1蛋白的表达。结果 结直肠癌组织中Notch1和Bmi1蛋白表达的阳性率明显高于正常肠黏膜(P＜0.05),分别为61.2％ (52/85)对15.3％ (13/85)和56.5％ (48/85)对17.6％ (15/85);Bmi1表达率与Notch1表达率呈正相关(r =0.625,P＜0.01),并与肿瘤分化程度、分期及淋巴结转移有关(P＜0.05);阻断Notch1通路(DAPT)可抑制SW480细胞的增殖,诱导其凋亡,作用12、24、36 h后其增殖抑制率分别为13.1％、17.5及22.6％,而凋亡率为32.7％、45.6％及67.2％;同时Notch1胞内活性段ICN随作用时间延长而下降,而Bmi1表达水平也逐渐降低。结论 结直肠癌中Bmi1与Notch1表达密切相关,阻断Notch通路可抑制Bmi1表达,同时可抑制结肠癌细胞的增殖,促进其凋亡。     

Expression of survivin in bladder cancer cell lines using quantitative real-time polymerase chain reaction

ObjectivePrevious studies have reported that survivin expression is significantly associated with various malignancies including bladder cancer. However, the relationship between the expression of survivin and the tumor stage and grade of bladder cancer still require further study.MethodsTo determine whether survivin plays a role in the differentiation of bladder cancer cells, we conducted a preliminary study to examine the expression of survivin in bladder cancer cell lines.ResultsIn this study, we observed that the gene expression fold changes of survivin ranged from 3.2 to 16.7 in various tumor grades (G1–G4) of bladder cancer cell lines, which were higher than that in normal human urothelial cell line. With the worse differentiation of bladder cancer cell lines, the gene expression fold changes of survivin increased significantly (3.2-fold in RT4, 5.8-fold in 5637, 6.6-fold in T24, and 16.7-fold in HT1197). In addition, we observed different genotypes among various cell lines (C/C in HUC4449, C/G in RT4, C/G in 5637, G/G in T24, and C/C in HT1197). The relationship between survivin ?31 C/G polymorphism and various bladder cancer cell lines was non-significant. However, the overexpression of survivin may be associated with aggressive features of bladder cancer.ConclusionOur findings suggest that survivin could be a potential therapeutic target through the inhibition of cell proliferation in bladder cancer.     

An experimental model of the epithelial to mesenchymal transition and pro-fibrogenesis in urothelial cells related to bladder pain syndrome/interstitial cystitis

BackgroundSuitable in vitro models are needed to investigate urothelial epithelial to mesenchymal transition (EMT) and pro-fibrogenesis phenotype in bladder pain syndrome/interstitial cystitis (BPS/IC). This study is to establish a novel experimental BPS/IC cell model and explore how different concentrations of tumor necrosis factor (TNF)-α influence the EMT and pro-fibrogenesis phenotype of urothelial cells.MethodsSV-HUC-1 urothelial cells were cultured with 2, 10, or 50 ng/mL TNF-α to mimic chronic inflammatory stimulation. The EMT and pro-fibrogenesis phenotype, including production of collagen I and pro-fibrosis cytokines, were estimated after 72 h of culture.ResultsThe bladder urothelial cells of BPS/IC exhibited upregulated vimentin, TNF-α and TNF receptor, downregulated E-cadherin, and increased collagen I. Higher concentrations of TNF-α (10 and 50 ng/mL) produced an obvious mesenchymal morphology, enhanced invasion and migratory capacity, increased expression of vimentin, and decreased expression of E-cadherin. Collagen I was increased in cells treated with 2 and 10 ng/mL TNF-α after 72 h. Secretion of interleukin (IL)-6 and IL-8 was promoted with 10 and 50 ng/mL TNF-α, while that of IL-1β or transforming growth factor-β was unaffected. Slug and Smad2 were upregulated by TNF-α after 72 h. The Smad pathway was activated most strongly with 10 ng/mL TNF-α and Slug pathway activation was positively correlated with the concentration of TNF-α.ConclusionsSustained 10 ng/mL TNF-α stimulation induced the EMT and pro-fibrogenesis phenotype resembling BPS/IC in SV-HUC-1 cells. Minor inflammatory stimulation induced the pro-fibrogenesis phenotype while severe inflammatory stimulation was more likely to produce significant EMT changes. Different degrees of activation of the Slug and Smad pathways may underlie this phenomenon.     

极低密度脂蛋白受体基因促进膀胱癌细胞迁移的作用及机制

目的:探讨极低密度脂蛋白受体(VLDLR)对人膀胱癌细胞迁移活性的影响及机制。方法:采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测人源膀胱癌细胞(J82、UMUC3与T24)与正常尿路上皮细胞(SV-HUC-1)VLDLR的表达水平;采用小干扰RNA(siRNA)建立VLDLR敲低T24细胞模型,通过划痕实...     

Devazepide suppresses cell proliferation and migration,and induces apoptosis in bladder carcinoma

BackgroundThis study aimed to examine the effects of devazepide on the proliferation, migration, and apoptosis of human bladder cancer (BC) 5637 cells, and its mechanism.MethodsA cell counting kit-8 (CCK-8) for cell viability assays, a colony formation assay, and immunofluorescence were applied to detect the effects of devazepide on the proliferation of 5637 cells. Cell cycle assay, cell apoptosis assay and wound healing assay were performed to detect the effects of devazepide on the cell cycle, apoptosis, and migration of 5637 cells. The protein expression of CyclinD1, Bcl-2-associated X protein (Bax), poly ADP-ribose polymerase 1 (PARP1), and Cleaved Caspase-3 in 5637 cells was detected by a western blot assay.ResultsThe proliferation of 5637 cells was significantly inhibited (P<0.001) after incubation with 12, 25, and 50 µM devazepide for 48 and 72 h. A treatment of 25 µM devazepide for 48 h induced G1–S cell cycle arrest and apoptosis (P<0.01), and inhibited cell migration (P<0.05). By western blot assay, we found that devazepide can down-regulate CyclinD1 expression, and up-regulate Bax, PARP1, and Cleaved Caspase-3 expression.ConclusionsDevazepide inhibits the migration and proliferation of human BC 5637 cells by arresting the G1–S cell cycle, and induces cell apoptosis.     

哺乳动物雷帕霉素靶蛋白抑制剂temsirolimus对膀胱癌作用的实验研究

目的 研究哺乳动物雷帕霉素靶蛋白( mTOR)抑制剂temsirolimus对膀胱癌T24、BIU-87细胞株的作用,探讨以mTOR为靶点治疗膀胱癌的应用前景。 方法 膀胱癌T24、BIU-87细胞株经不同浓度temsirolimus作用后,采用噻唑盐法检测temsirolimus对细胞株增殖的影响;流式细胞技术检测temsirolimus对细胞周期、细胞凋亡的影响;细胞迁移实验及transwell细胞侵袭实验检测temsirolimus对肿瘤迁移和浸润的影响;蛋白质印迹法检测mTOR的表达情况;制作裸鼠T24细胞株移植瘤模型,检测temsirolimus对移植瘤生长的作用,免疫组化检测移植瘤Ki-67表达情况。 结果 temsirolimus能够抑制膀胱癌T24、BIU-87细胞株增殖,呈浓度和时间依赖性;temsirolimus能够抑制膀胱癌细胞迁移能力,temsirolimus作用于T24、BIU-87细胞株24 h后,0 nmol/L组划痕修复率分别为(88.9±14.1)％、(83.6±16.3)％,高于5 nmol/L组的(42.7±11.6)％、(36.9±9.7)％,差异有统计学意义(P＜0.05);temsirolimus能够抑制膀胱癌细胞浸润能力,temsirolimus作用于T24、BIU-87细胞株24h后,0 nmol/L组浸润细胞数(26.5±5.8)、(28.2±4.6),高于5 nmol/L组的(19.0±3.8)、(21.3±5.1),差异有统计学意义(P＜0.05);temsirolimus导致膀胱癌细胞周期阻滞于G0/G1期,temsirolimus作用T24、BIU-87细胞株48 h后,5 nmol/L组G0/G1期细胞分别占(77.46±6.11)％、(73.39±4.94)％,高于0 nmol/L组的(65.99±5.01)％、(60.15±3.98)％,差异有统计学意义(P＜0.05);各组细胞凋亡率差异无统计学意义(P＞0.05);temsirolimus能够阻断mTOR的磷酸化,T24、BIU-87细胞株0 nmol/L组p-mTOR/β-actin值分别为0.92±0.09、1.01±0.08,明显高于5 nmol/L组的0.47±0.05、0.04±0.01,差异有统计学意义(P＜0.05);temsirolimus能够抑制裸鼠T24细胞株移植瘤生长,给药21 d后,给药组移植瘤体积为(147.6±74.4) mm3,对照组为(351.1±139.9) mm3,差异有统计学意义(P＜0.05);给药组移植瘤Ki-67表达阳性细胞百分率为(35.5±6.7)％,低于对照组的(67.3±8.4)％,差异有统计学意义(P＜0.05)。 结论 mTOR抑制剂temsirolimus对膀胱癌细胞具有明显的抑制作用,可考虑将mTOR作为膀胱癌治疗的靶点。     

Multifactorial Involvement of Multidrug Resistance Protein, DNA Topoisomerase II and Glutathione/Glutathione-S-Transferase in NonP- Glycoprotein-Mediated Multidrug Resistance in Human Bladder Cancer Cells

Background :
Multiple mechanisms are important in multidrug resistance in urothelial cancers. We investigated the acquisition of a multidrug resistance phenotype in human bladder cancer cells exposed to doxorubicin.
Methods :
Human bladder cancer cell line 5637 and 2 doxorubicin drug-resistant sublines (5637/DR5.5 and 5637/DR50) were used. Measurements were made of the steady state mRNA levels of the multidrug resistance gene ( mdr 1), multidrug resistance-associated protein (MRP), glutathione-S-transferase-π and DNA topoisomerase II (topo II) genes, P-glycoprotein (PgP) and MRP expression, glutathione (CSH) and GSH enzyme activity, and topo II catalytic activity. The pharmacokinetics were compared between the parent and the drug-resistant sublines.
Results :
5637/DR5.5 and 5637/DR50 cells were 7.6- and 1 6.2-fold more resistant to doxorubicin and 16.7-and 48.3-fold more resistant to etoposide, respectively, compared with 5637 cells. A dose escalation of doxorubicin increased the MRP expression, CSH levels and glutathione-S-transferase (GST) activity, although no PgP expression was observed in any cell line. Resistance was brought about by decreased drug accumulation through drug efflux, although intracellular daunorubicin concentrations were similar between DR5.5 and DR50 cells. Topo II catalytic activity was undetectable in DR50 cells, but maintained in both the parent and DR5.5 cells.
Conclusion :
Reduced drug accumulation in doxorubicin-resistant cells was mediated by MRP instead of PgP indicating that MRP-mediated drug efflux functions in a limited manner for drug resistance. An increase in drug efflux via MRP, reduced topo II activity, and increased GSH levels/GSH-related enzyme activities may play major roles in nonPgP-mediated multidrug resistance in urothelial cancers treated with anthracyclines.     

A conventional preclinical schedule of cisplatin is more effective than a metronomic frequent bolus schedule for urothelial carcinoma

ObjectivesGiven the frequent inability to administer a conventional 3-weekly schedule of cisplatin to human subjects with urothelial carcinoma, we evaluated a frequent bolus metronomic schedule in a preclinical system of transitional cell carcinoma (TCC) of the urothelium. We hypothesized that the anti-angiogenic and anti-cell-migratory activity of lower concentrations of cisplatin may confer similar anti-tumor activity and demonstrate less nephrotoxicity than conventional cytotoxic concentrations.Materials and methodsWe evaluated the activity of cisplatin in vitro against human urothelial carcinoma (5637) and endothelial cells (human umbilical vein endothelial cells, HUVECs). The MTT assay was employed to assess viability, and flow cytometry with Annexin-FITC labeling was employed to assess apoptosis. Cell migration in monolayer culture was assessed by scratch assay. Murine xenografts (n = 12 per group) bearing measurable subcutaneous tumors of 5637 cells were administered either no therapy, cisplatin 2 mg/kg/3 days a week (metronomic) or 6 mg/kg/ once a week (standard) intraperitoneal (i.p.) for 4 weeks. Blood was collected from 5 mice per group at baseline and at the end of therapy to measure changes in serum creatinine.ResultsSignificant antiproliferative activity, but poor pro-apoptotic activity, was observed against 5637 urothelial carcinoma cells in vitro by MTT assay with cisplatin at concentrations lower than those attainable in vivo. Anti-proliferative activity against HUVECs required higher concentrations than attainable in vivo. Anti-migratory activity was observed against 5637 and HUVECs at earlier time points and with concentrations lower than anti-proliferative concentrations. In murine 5637 xenografts, standard cisplatin was significantly better at inhibiting tumor growth than the no treatment control (P = 0.005), while metronomic therapy was not better than control (P = 0.22). Standard cisplatin was also significantly nephrotoxic (P = 0.016), while metronomic cisplatin (P = 0.374) or no therapy (P = 0.178) did not demonstrate significant nephrotoxicity compared with baseline.ConclusionsCisplatin exhibited anti-cell-migratory activity against urothelial carcinoma and endothelial cells at lower than cytotoxic concentrations. However, a standard preclinical schedule was better than control in vivo for inhibiting tumor growth, while a metronomic schedule was not better. Despite the lower nephrotoxicity of the metronomic schedule, its lower anti-tumor activity suggests that a standard clinical schedule of cisplatin should not be routinely substituted by a split schedule without definitive clinical data.     

In vivo and in vitro effects of RAD001 on bladder cancer

ObjectiveTo evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines.MethodsICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms.ResultsThe incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G₀/G₁ phase arrest, as well as a statistically significant induction of apoptosis (P = 0.001), was only observed in the 5637 cell line.ConclusionRAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.     

Pseudomonas aeruginosa-mannose–sensitive hemagglutinin inhibits epidermal growth factor receptor signaling pathway activation and induces apoptosis in bladder cancer cells in vitro and in vivo

ObjectivesPseudomonas aeruginosa-mannose–sensitive hemagglutinin (PA-MSHA), a peritrichous P. aeruginosa strain with MSHA fimbriae, has been shown to be a valuable anticancer drug in many kinds of cancers. However, the effect of PA-MSHA on bladder cancer has not been elucidated. In this study, we focused on the antitumor activities and related mechanisms of PA-MSHA on bladder cancer in vitro and in vivo.Materials and methodsSV-40-immortalized normal uroepithelial cells (SV-HUC-1) and human bladder cancer cell lines (T24, 5637, and HT-1376) were treated with PA-MSHA or PA (heat-killed P. aeruginosa). At first, the effect of PA-MSHA on cancer cell proliferation was measured using Cell Counting Assay Kit-8 (CCK-8), whereas the changes of cell morphology were observed by transmission electron microscopy. The early apoptosis induced by PA-MSHA was evaluated by flow cytometry, and the expression level of apoptosis-related molecules was detected using Western blot assay. We then investigated the activation of the epidermal growth factor receptor signaling pathway stimulated by PA-MSHA; the expression and phosphorylation of several key regulators involved in the EGFR signaling pathway were detected. At last, xenograft tumor in nude mice was used to further investigate the antitumor effect of PA-MSHA in vivo.ResultsOur results showed that PA-MSHA could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cell lines. Furthermore, cells stimulated with PA-MSHA exhibited an inactivation of EGFR signaling. In vivo, PA-MSHA treatment significantly suppressed tumor growth and induced apoptosis in xenografts tumor in nude mice.ConclusionsPA-MSHA could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cells in vitro and in vivo, which is associated with the inactivation of EGFR signaling pathway, and it might be used as a potential therapeutic agent for bladder cancer.     

MK2206 potentiates cisplatin-induced cytotoxicity and apoptosis through an interaction of inactivated Akt signaling pathway

ObjectivesTo improve conventional chemotherapeutic efficacy, it is important to detect new molecular markers for chemosensitivity and possible accelerating cell-killing mechanisms. In this study, we investigated how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in urothelial cancer cells.Materials and methodsWe examined bladder cancer cell lines for the expression of phospho(p)-Akt and its downstream targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was examined by transwell invasion assay, and the activities of the Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot.ResultsThe expression of p-Akt and its downstream targets was increased in invasive bladder cancer cell lines vs. in noninvasive bladder cancer cell lines. MK2206 (500 nM) inhibited cell invasion in UMUC3 cell line and significantly increased the susceptibility of bladder cancer cell lines to CDDP. When used in combination with CDDP, MK2206 (500 nM) enhanced CDDP-induced cytotoxicity and apoptosis, with suppressed expression of p-Akt and its downstream targets. In vivo MK2206 combined with CDDP effectively suppressed tumor growth in subcutaneous xenograft models.ConclusionsThese results suggest that concomitant use of MK2206 could promote the CDDP-induced cytotoxicity and apoptosis in urothelial cancer cell lines through the inhibited expression of the Akt pathway. This combined treatment may provide a new therapeutic option to enhance chemosensitivity in bladder cancer.     

粉防己碱对膀胱癌BIU-87/ADM细胞株的耐药逆转作用及其机制

目的 探讨粉防己碱(TET)对膀胱癌耐药株BIU-87/ADM多药耐药性(MDR)的逆转作用及其机制。方法 应用CCK法观察不同浓度TET( 1.0、1.5、2.0、2.5 mg/L)对BIU-87和BIU-87/ADM细胞生长的抑制作用,并测定1 mg/L TET逆转多药耐药的活性;Annexin-V与PI双染法流式细胞术分析1 mg/L TET对细胞凋亡的影响;免疫荧光法和逆转录-聚合酶链反应(RT-PCR)分别检测1 mg/L TET对P-gp蛋白和MDR1表达水平的影响;细胞免疫化学法和RT-PCR法检测1 mg/L TET对BIU-87/ADM细胞Caspase-3和Survivin表达水平的影响。结果 1.0 mg/L无毒剂量下的TET显著增加阿霉素(ADM)对耐药株BIU-87/ADM的细胞毒性作用(P＜0.01),逆转指数(RF)为5.33,而对敏感株BIU-87无明显作用(P＞0.05),RF为1.33;1.0 mg/L TET和1.5 mg/LADM共同作用48 h,能使BIU-87/ADM细胞凋亡率增加至(40.86±4.35)％,与单独应用ADM的(12.42 ±3.24)％比较差异有统计学意义(P＜0.01)。而BIU-87细胞凋亡率为(61.23 ±5.46)％,与单独应用ADM的(57.79±4.73)％比较差异无统计学意义(P＞0.05)。TET能明显抑制mdr1mRNA和P-gp蛋白在BIU-87/ADM的表达;与单用ADM比较,TET和ADM联用能使BIU-87/ADM细胞株中Caspase-3的表达水平明显升高(P＜0.01),Survivin的表达水平显著降低(P＜0.01)。结论 粉防己碱能逆转BIU-87/ADM细胞的多药耐药性,这一作用可能与粉防己碱抑制P-gp的表达和增强化疗药诱导细胞凋亡有关。     

Expression of Med19 in bladder cancer tissues and its role on bladder cancer cell growth

ObjectivesThe human Med19 gene encodes a critical subunit that stabilizes the whole mediator complex. To understand the role of Med19 in bladder cancer, we studied the effects of lentivirus-mediated suppression of Med19 expression on bladder cancer cells in vitro and in vivo.Methods and materialsIn this study, immunohistochemical analysis was used to demonstrate the expression of Med19 in human bladder cancer. The lentivirus vectors containing a small hairpin RNA (shRNA) to target Med19 were constructed. After bladder cancer cells (5637 and T24) were infected, RT-PCR and Western blotting were used to measure Med19 expression. The influence of Med19 on the proliferation of bladder cancer cells were assessed using MTT, BrdU, colony formation and tumorigenicity experiments. Cell cycle was analyzed with flow cytometric assay.ResultsMed19 was up-regulated in human bladder cancers compared with adjacent benign tissues by immunohistochemical analysis, but was strongly inhibited in 5637 and T24 bladder cancer cells infected with lentiviruses delivering shRNA against Med19. The down-regulation of Med19 increased the proportion of cells in G0/G1 phases and attenuated the growth of 5637 and T24 cells in vitro. The tumorigenicity of Med19-suppressed T24 cells was decreased after inoculation into nude mice.ConclusionsOur results suggested that lentiviruses delivering shRNA against Med19 may be a promising tool for bladder cancer therapy.     

Novel synthetic lethality drug target in urothelial bladder cancer based on MTAP genomic loss

BackgroundWhen urothelial carcinoma of the bladder (UCB) presents or progresses to chemo-refractory metastatic disease, the search for new therapeutic targets is paramount. Targeting protein arginine methyltransferase 5 accumulation in tumors with methylthioadenosine phosphorylase (MTAP) genomic loss has been proposed as a new anti-tumor strategy. We evaluated the incidence of patients with MTAP loss and correlate to treatment-guiding targets and biomarkers.MethodsTwo thousand six hundred eighty-three cases of advanced UCB underwent hybrid-capture based comprehensive genomic profiling using the FDA-approved F1CDx assay to evaluate all classes of genomic alterations (GA) among 324 genes. Tumor mutational burden was determined on at least 0.8 Mbp of sequenced DNA and microsatellite instability was determined on at least 95 loci.Results650 (24%) of UCB featured MTAP loss mutations (MTAP-). The gene and age distributions were similar in MTAP intact (MTAP+) and MTAP- UCB. MTAP- UCB contained higher GA/tumor frequency than MTAP+ UCB likely reflecting the frequent co-deletions of cyclin-dependent kinase inhibitor 2A/B. Of potential therapeutic targets, fibroblast growth factor receptor 3, and phosphatase and tensin homolog GA were more frequent in MTAP- UCB. In contrast, biomarkers of immunotherapy response, including higher frequencies of high tumor mutational burden and high programmed death-ligand 1 IHC staining, were observed in the MTAP+ UCB.ConclusionsWhen compared with MTAP+ UCB, MTAP- UCB differs in genomic signatures including an increase in potentially targetable alterations but a lower frequency of immunotherapy drug biomarkers. Thus, the genomic landscape in MTAP- UCB may play a role in the design of clinical trials incorporating combination treatment strategies when targeting protein arginine methyltransferase 5 in MTAP- tumors.