Human Bladder Transitional Cell Carcinoma Research Articles

Anticancer properties of cannabidiol and Δ9-tetrahydrocannabinol and synergistic effects with gemcitabine and cisplatin in bladder cancer cell lines

IntroductionWith the legalization of cannabis in multiple jurisdictions throughout the world, a larger proportion of the population consumes cannabis. Several studies have demonstrated anti-tumor effects of components present in cannabis in different models. Unfortunately, little is known about the potential anti-tumoral effects of cannabinoids in bladder cancer and how cannabinoids could potentially synergize with chemotherapeutic agents. Our study aims to identify whether a combination of cannabinoids, like cannabidiol and Δ9-tetrahydrocannabinol, with agents commonly used to treat bladder cancer, such as gemcitabine and cisplatin, can produce desirable synergistic effects. We also evaluated if co-treatment with different cannabinoids resulted in synergistic effects.MethodsWe generated concentration curves with several drugs, including several cannabinoids, to identify the range at which they could exert anti-tumor effects in bladder cancer cell lines. We tested the cytotoxic effects of gemcitabine (up to 100 nM), cisplatin (up to 100 μM), and cannabinoids (up to 10 μM) in T24 and TCCSUP cells. We also evaluated the activation of the apoptotic cascade and whether cannabinoids have the ability to reduce invasion in T24 cells.ResultsCannabidiol, Δ9-tetrahydrocannabinol, cannabichromene, and cannabivarin reduce cell viability of bladder cancer cell lines, and their combination with gemcitabine or cisplatin may induce differential responses, from antagonistic to additive and synergistic effects, depending on the concentrations used. Cannabidiol and Δ9-tetrahydrocannabinol were also shown to induce apoptosis via caspase-3 cleavage and reduce invasion in a Matrigel assay. Cannabidiol and Δ9-tetrahydrocannabinol also display synergistic properties with other cannabinoids like cannabichromene or cannabivarin, although individual cannabinoids may be sufficient to reduce cell viability of bladder cancer cell lines.DiscussionOur results indicate that cannabinoids can reduce human bladder transitional cell carcinoma cell viability, and that they can potentially exert synergistic effects when combined with other agents. Our in vitro results will form the basis for future studies in vivo and in clinical trials for the development of new therapies that could be beneficial for the treatment of bladder cancer in the future.

Hispolon Methyl Ether, a Hispolon Analog, Suppresses the SRC/STAT3/Survivin Signaling Axis to Induce Cytotoxicity in Human Urinary Bladder Transitional Carcinoma Cell Lines.

Bladder cancer is a leading human malignancy worldwide. Signal transducer and activator of transcription (STAT) 3 is an oncogenic transcription factor commonly hyperactivated in most human cancers, including bladder cancer. Notably, preclinical evidence has validated STAT3 blockade as a promising therapeutic strategy for bladder cancer. Hispolon Methyl Ether (HME) is a structural analog of hispolon, an anticancer component of the medicinal mushroom Phellinus linteus. Thus far, HME's anticancer activity and mechanisms remain largely unknown. We herein report HME was cytotoxic, more potent than cisplatin, and proapoptotic to various human bladder transitional carcinoma cell lines. Of note, HME blocked STAT3 activation, evidenced by HME-elicited reduction in tyrosine 705-phosphorylated STAT3 levels constitutively expressed or induced by interleukin-6. Significantly, HME-induced cytotoxicity was abrogated in cells expressing a dominant-active STAT3 mutant (STAT3-C), confirming STAT3 blockage as a pivotal mechanism of HME's cytotoxic action. We further revealed that survivin was downregulated by HME, while its levels were rescued in STAT3-C-expressing cells. Moreover, survivin overexpression abolished HME-induced cytotoxicity, illustrating survivin as a central downstream mediator of STAT3 targeted by HME. Lastly, HME was shown to lower tyrosine 416-phosphorylated SRC levels, suggesting that HME inhibits STAT3 by repressing the activation of SRC, a STAT3 upstream kinase. In conclusion, we present the first evidence of HME's anti-bladder cancer effect, likely proceeding by evoking apoptosis through suppression of the antiapoptotic SRC/STAT3/survivin signaling axis.

LINC00958 Inhibits Autophagy of Bladder Cancer Cells via Sponge Adsorption of miR-625-5p to Promote Tumor Angiogenesis and Oxidative Stress.

Objective This study further explored LINC00958's role in promoting tumor angiogenesis (AG) and oxidative stress (OS) development by inhibiting BC cell autophagy through sponge adsorption of miR-625-5p. Methods BC patients and healthy controls who visited our hospital between June 2017 and February 2019 were selected as the research group (RG) and the control group (CG), respectively, with a total of 133 study subjects. Peripheral blood LINC00958 and miR-625-5p in both cohorts of participants were detected. Additionally, human bladder transitional cell carcinoma cells (T24 and J82) and human normal urothelial cells (SV-HUC-1) were purchased. Alterations in cell biological behavior were observed after transfecting miR-625-5p-mimics, miR-625-5p-inhibition, and miR-625-5p-NC sequences into these cells, respectively. Besides, ELISA was performed to quantify inflammatory factors (IFs), AG indicators, and OS indexes in cells. Subsequently, a double luciferase reporter (DLR) assay was performed to verify the targeting relationship between LINC00958 and miR-625-5p. Finally, BALB/c-nu nude mice were purchased, and T24 cells transfected with silenced LINC00958 and miR-625-5p expression sequences were used to establish subcutaneous tumors to observe tumor growth and pathological changes. Results RG exhibited higher LINC00958 and lower miR-625-5p than CG. LINC00958 and miR-625-5p were strongly linked to myometrial invasion (MI), lymph node metastasis (LNM), distant metastasis (DM), and histology in BC patients, and the increase of LINC00958 and the decrease of miR-625-5p predicted an increased risk of prognostic death in such patients. After miR-625-5p inhibition, the capacity of BC cells to proliferate, invade, and migrate enhanced and the AG, inflammatory response, and OS injury increased, while the apoptosis rate and autophagy ability decreased. The DLR assay revealed inhibited LINC00958WT fluorescence activity by miR-625-5p-mimics, while the biological behavior of BC cells cotransfected with sh-LINC00958 and miR-625-5p-inhibition had no difference with the functions of sh-control and miR-625-5p-NC cotransfected cells. Finally, the nude mouse tumorigenesis experiment showed that the tumor mass, volume, and histopathological features of the sh-LINC00958 group were decreased compared with the sh-control group, while those of the miR-625-5p-inhibition group were increased versus miR-625-5p-NC. Conclusions In BC, LINC00958 is highly expressed while miR-625-5p is underexpressed. LINC00958 can inhibit cell autophagy to enhance cell activity; promote OS, inflammation, and AG; and regulate tumor immunity by targeting miR-625-5p, thus participating in the development of BC.

Anti-cancer properties of cannflavin A and potential synergistic effects with gemcitabine, cisplatin, and cannabinoids in bladder cancer

IntroductionSeveral studies have shown anti-tumor effects of components present in cannabis in different models. Unfortunately, little is known about the potential anti-tumoral effects of most compounds present in cannabis in bladder cancer and how these compounds could potentially positively or negatively impact the actions of chemotherapeutic agents. Our study aims to evaluate the effects of a compound found in Cannabis sativa that has not been extensively studied to date, cannflavin A, in bladder cancer cell lines. We aimed to identify whether cannflavin A co-treatment with agents commonly used to treat bladder cancer, such as gemcitabine and cisplatin, is able to produce synergistic effects. We also evaluated whether co-treatment of cannflavin A with various cannabinoids could produce synergistic effects.MethodsTwo transitional cell carcinoma cell lines were used to assess the cytotoxic effects of the flavonoid cannflavin A up to 100 μM. We tested the potential synergistic cytotoxic effects of cannflavin A with gemcitabine (up to 100 nM), cisplatin (up to 100 μM), and cannabinoids (up to 10 μM). We also evaluated the activation of the apoptotic cascade using annexin V and whether cannflavin A has the ability to reduce invasion using a Matrigel assay.ResultsCell viability of bladder cancer cell lines was affected in a concentration-dependent fashion in response to cannflavin A, and its combination with gemcitabine or cisplatin induced differential responses—from antagonistic to additive—and synergism was also observed in some instances, depending on the concentrations and drugs used. Cannflavin A also activated apoptosis via caspase 3 cleavage and was able to reduce invasion by 50%. Interestingly, cannflavin A displayed synergistic properties with other cannabinoids like Δ9-tetrahydrocannabinol, cannabidiol, cannabichromene, and cannabivarin in the bladder cancer cell lines.DiscussionOur results indicate that compounds from Cannabis sativa other than cannabinoids, like the flavonoid cannflavin A, can be cytotoxic to human bladder transitional carcinoma cells and that this compound can exert synergistic effects when combined with other agents. In vivo studies will be needed to confirm the activity of cannflavin A as a potential agent for bladder cancer treatment.

Highly Expressed LINC00958 Modulates the Growth and Epithelial-Mesenchymal Transition of Bladder Cancer Cells Through SAPK/JNK Signaling Pathway.

Objective: To determine the expression of LINC00958 (BLACAT2) in bladder cancer (BC), the most common malignancy in the urinary system, and to determine its exact mechanism of action, so as to provide novel references for future clinical diagnosis and treatment of BC and lay a foundation for the follow-up research on LINC00958. Materials and Methods: Human bladder transitional cell carcinoma cells (T24 and J82) and human normal urothelial cells (SV-HUC-1) were purchased to detect the expression of LINC00958 and SAPK/JNK signaling pathway-related proteins. sh-LINC00958 targeting to silence LINC00958 expression and corresponding negative blank (sh-Control) were transfected into T24 and J82. Additionally, BC cells cultured with SP600125 (SP600125 group), a specific inhibitor of SAPK/JNK signaling pathway, and those cultured with the same amount of normal saline (Blank group) were also constructed. Cell growth capacity, cell invasiveness, and expression of epithelial-mesenchymal transition (EMT)-associated proteins were determined using CCK-8 & clone formation assays, Transwell assay, and Western blot, respectively. Results: The online databases Gene Expression Profiling Interactive Analysis, European Bioinformatics Institute, and StarBase revealed elevated LINC00958 expression in BC, and a potential association between LINC00958 and patient prognosis and survival. PCR results showed that LINC00958 was increased in T24 and J82 compared with the sh-Control group (p < 0.05). The results of biological behavior test revealed that the proliferation and invasiveness capacity of the sh-LINC00958 group decreased, while that of the SP600125 group increased compared with the Blank group (both p < 0.05). In the rescue experiment, the influence of sh-LINC00958 on BC cells was completely reversed by SP600125 (p > 0.05); In addition, the expression of E-cadherin, an EMT marker protein, was lower compared with the SH-LINC0958 group, while the Vimentin expression was higher (p < 0.05). Similarly, the wound-healing assay determined reduced cell healing rate in the sh-LINC00958 group (p < 0.05), and there was no difference between the sh-LINC00958+SP600125 group and the sh-Control group (p > 0.05). Conclusion: LINC00958 shows elevated expression in BC and promotes the growth and EMT of BC cell via inhibiting the SAPK/JNK signaling pathway, which has important potential as a new clinical diagnostic marker and therapeutic target for BC.

CASC5 is a potential cancer-testis gene in human urinary bladder transitional cell carcinoma.

Urinary Bladder cancer (UBC) is a diversified disease with an array of clinicopathological attributes. Several studies have shown that cancer susceptibility candidate 5 (CASC5) plays important roles in various types of malignancies; however its expression and clinical significance in human UBC remain largely unknown. This research study was intended to explore mRNA/protein expression pattern of CASC5 as a member of the cancer-testis (CT) gene family and assess its clinical utility in diagnostic management of patients with UBC. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) was employed to appraise the detailed expression profile of CASC5 in patients with UBC. The mRNA over expression of CASC5 was detected in testis tissue and relatively high frequency 59.2% (45 of 76) of CASC5 mRNA was detected in UBC tissues. CASC5 mRNA relative mean fold expression was also significantly (p < 0.01) higher in the muscle-invasive tumor tissues compared to non-muscle-invasive tumor tissues (12.26 ± 9.53 vs. 4.64 ± 2.50, p = 0.005). Heterogeneous staining pattern of CASC5 protein was exclusively detected using IHC. The frequency of CASC5 protein over expression was detected in 67.7% (44 of 65) UBC patients and negative in benign prostatic hyperplasia (BPH). Further, CASC5 protein expression was significantly (p < 0.001) associated with cigarette smoking habit in UBC patients. Our study findings testified that CASC5 over expression among patients with UBC as compared to controls and concludes that CASC5 is a potential CT gene in UBC.

Frequent expression of a novel cancer testis antigen, protein kinase human monopolar spindle 1 (hMps1/TTK) in human urinary bladder transitional cell carcinoma.

Urothelial bladder cancer (UBC) is a frequently occurring malignancy of the urinary tract. The present study was undertaken to evaluate the mRNA and immunohistochemical (IHC) expression of protein kinase human monopolar spindle 1 (hMps1/TTK) gene in transitional cell carcinoma (TCC) of the bladder and correlate its expression with the clinicopathological characteristics of patients. In the present study, quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate TTK mRNA expression in TCC. IHC analysis of TTK was also evaluated. Independent Student's t, ANOVA and chi-square (χ2) tests were used to analyze the data statistically. The frequency of TTK mRNA over expression was detected in 50% of UBC (38/76) by qRT-PCR. Relative mean fold expression of TTK mRNA was found significantly (p < 0.05) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients (8.96 ± 4.51 vs. 5.64 ± 3.53, p = 0.03). Moreover, IHC reveals heterogenous immunostaining pattern of TTK in TCC tissues. The frequency of TTK protein over expression was detected in 56.9% (37 of 65) UBC patients. No significant IHC expression of TTK was detected among adjacent noncancerous tissues (ANCTs) and benign prostatic hyperplasia (BPH) used as control. Collectively our study observations conclude that TTK is a novel cancer/testis antigen (CTA) as a diagnostic marker for early diagnosis of UBC.

Selective Susceptibility of Human Bladder Transitional Cell Carcinoma T24 Cells towards NBD Peptide

Bladder cancer is a recurrent disease that often develops chemoresistance and is on the rise in developing countries. After diagnosis surgery followed by immunotherapy and/or chemo- radiation therapy are the available treatments. These treatments do not provide protection against disease progression or recurrence thus highlighting the need to identify and develop drugs that are capable of inhibiting bladder cancer. T24 cell line (human bladder transitional cell carcinoma) was treated with NEMO Binding Domain (NBD) peptide and tested for its cytotoxicity, cell cycle inhibition, apoptosis and migration, in vitro. The cytotoxicity of NBD peptide was tested on non-cancerous cells (NIH-3T3-L1, CHO, Vero) and the peptide localization in T24 cells was confirmed using confocal microscopy. The effects of NBD peptide and the standard drugs (cisplatin, gemcitabine) were compared with untreated control. Peptide based chemotherapy for human bladder cells was tested that include combinations of NBD and standard drugs. Statistical significance was assessed between the different treatment groups. The viability of T24 cells after NBD peptide treatment was reduced to 50% at a dose of 18.5 µM after 48 h. The NBD peptide elicited selective cytotoxicity dose-dependently in T24 cells while being non-toxic on normal (non-cancer) cell lines. Sub G0-G1 accumulation of T24 cells was seen where they underwent necrosis and lost the ability to migrate when treated with 100 µM NBD. T24 cell death increased to ~78% when treated with a combination of NBD with cisplatin and gemcitabine (standard drugs) as opposed to ~50% when treated with NBD peptide alone. NBD peptide exhibits specific anti-cancer activity on T24 cells in vitro and found to be non-cytotoxic to select normal cell lines. Combining NBD with standard drugs demonstrated excellent inhibition of malignant growth than the individual anti-neoplastic agents.

Corrigendum] MicroRNA‑497 inhibits the proliferation, migration and invasion of human bladder transitional cell carcinoma cells by targeting E2F3.

Subsequently to the publication of the above article, the authors have realized that Fig.4E in their paper contained errors. The image selected to represent the experiment showing the UM‑UC‑3 cells of the siE2F3 group (0h) was chosen incorrectly during the figure compilation process. A corrected version of Fig.4 is shown opposite. Note that this error affected neither the interpretation of the data nor the reported conclusions of this work, and all the authors agree to this Corrigendum. The authors apologize to the Editor and the readership of the Journal for this unintentional error, and for any inconvenience caused. [the original article was published in Oncology Reports 36: 1293‑1300, 2016; DOI: 10.3892/or.2016.4923].

Effects of Epigallocatechin Gallate (EGCG) on Urinary Bladder Urothelial Carcinoma-Next-Generation Sequencing and Bioinformatics Approaches.

Background and objectives: Bladder urothelial carcinoma is the most common type of genitourinary cancer. Patients with bladder cancer may have limited treatment efficacy related to drug toxicity, resistance or adverse effects, and novel therapeutic strategies to enhance treatment efficacy or increase sensitivity to drugs are of high clinical importance. Epigallocatechin gallate (EGCG) is a polyphenolic compound found in green tea leaves, and a potential anti-cancer agent in various cancer types through modulating and regulating multiple signaling pathways. The current study aimed to explore the role and novel therapeutic targets of EGCG on bladder urothelial carcinoma. Materials and Methods: The BFTC-905 cells, human urinary bladder transitional cell carcinoma (TCC) cell line, were treated with EGCG or water for 24 hours, and the expression profiles of mRNAs and microRNAs were analyzed using next generation sequencing (NGS). The enriched biological functions were determined using different bioinformatics databases. Results: A total of 108 differentially expressed genes in EGCG-treated bladder TCC cells were identified, which were mainly involved in nicotinamide adenine dinucleotide (NAD) biogenesis, inflammatory response and oxidation-reduction metabolism. Moreover, several microRNA-mRNA interactions that potentially participated in the response of bladder TCC to EGCG treatment, including miR-185-3p- ARRB1 (arrestin beta 1), miR-3116- MGAT5B (alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase B), miR-31-5p-TNS1 (tensin 1), miR-642a-5p-TNS1, miR-1226-3p- DLG2 (discs large homolog 2), miR-484-DLG2, and miR-22-3p- PPM1K (protein phosphatase 1K). Conclusions: The current findings provide insights into novel therapeutic targets and underlying mechanisms of action of EGCG treatment in bladder cancer.

Induction of G2/M Cell Cycle Arrest and Apoptosis by Genistein in Human Bladder Cancer T24 Cells through Inhibition of the ROS-Dependent PI3k/Akt Signal Transduction Pathway.

We examined the anti-cancer effect of genistein, a soy-derived isoflavone, in human bladder transitional cell carcinoma T24 cells. According to our data, genistein induced G2/M phase arrest of the cell cycle and apoptosis. Genistein down-regulated the levels of cyclin A and cyclin B1, but up-regulated the levels of p21WAF1/CIP1, cyclin-dependent kinase (Cdk) inhibitor, that was complexed with Cdc2 and Cdk2. Furthermore, genistein induced the activation of caspases (caspase-3, -8 and -9), and cleavage of poly (ADP-ribose) polymerase cleavage. However, genistein-induced apoptosis was significantly inhibited by a pan-caspase inhibitor, indicating that the induction of apoptosis by genestein was caspase-dependent. In addition, genistein increased the cytosolic release of cytochrome c by increasing the Bax/Bcl-2 ratio and destroying mitochondria integrity. Moreover, genistein inactivated the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, while LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of genistein. Genistein further increased the accumulation of reactive oxygen species (ROS), which was significantly suppressed by N-acetyl cysteine (NAC), a ROS scavenger, and in particular, NAC prevented genistein-mediated inactivation of PI3K/Akt signaling, G2/M arrest and apoptosis. Therefore, the present results indicated that genistein promoted apoptosis induction in human bladder cancer T24 cells, which was associated with G2/M phase cell cycle arrest via regulation of ROS-dependent PI3K/Akt signaling pathway.

Differential expression of S1P receptor subtypes in human bladder transitional cell carcinoma.

Sphingosine 1 phosphate (S1P), S1P receptors (S1PRs) and their signaling pathways play an important role in the fate of cancer cells. The expression pattern of S1PR subtypes (S1PR1-S1PR5) may alter in cancer development stages, depending on the origin and the pathologic features of tumors. The present study aimed to examine the relationship between plasma S1P levels and the expression of S1PR subtypes in bladder tumors. These changes were evaluated in terms of the pathologic grades and stages of human bladder cancer samples. For this, tumor biopsies from 41 new bladder cancer patients as well as 26 normal-looking bladder tissues were collected and processed for immunohistochemistry (IHC) and quantitative real-time RT-PCR of S1PR subtypes. Plasma S1P level was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results show that tissue S1PR1, S1PR2 and S1PR3 are over-expressed in all tumors regardless of their pathological grade (~ 3, ~ 6 and ~ 104 folds, respectively). These results were corroborated by IHC data showing accumulation of S1PR subtypes 1 and 2 in the tissues. Plasma S1P in the plasma samples from patients was in the range of control samples (Controls; 256 ± 47; patients, 270 ± 41). Overexpression of S1PR1, S1PR2 and S1PR3 in bladder tumor biopsies which were corroborated with the pathological grades and stages may suggest that S1PR profile in tumor biopsies is a promising marker in the diagnosis of bladder carcinoma.

Abstract 885: Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells

Abstract Doxorubicin (Dox) is a chemotherapy drug widely used for treatment of several cancers including bladder cancer. However, cardiovascular side effects caused by doxorubicin-induced cardiotoxicity has necessitated the synthesis and validation of next generation chemotherapy drugs that are as effective but not cardiotoxic. AD 312 (N-Nitrosureidodaunorubicin; Daunomustine®, Paradox Pharmaceuticals, Inc.) and AD 198 (N- benzyladriamycin-14-valerate; Benzarubicin®, Paradox Pharmaceuticals, Inc.) are novel anthracyclines with confirmed non-cardiotoxicity in mice. Compared to Dox, AD 312 and AD 198 induce apoptosis in cells through distinct mechanisms. The tumor suppressor gene p53 is a key regulator of cell-cycle arrest and apoptosis. Dysregulation of p53 expression and function was identified in over 70% of the bladder cancers and reported as a potential target for therapy. As a result, small molecule compounds that can restore mutated p53 function to wild-type, including PRIMA-1, are under intensive investigation. In this study, we have compared the efficacy of AD 312 and AD 198 to Dox to induce apoptosis in human bladder transitional cell carcinoma (TCC) cells by MTT assay and assessed the possible mechanisms of anthracyclines-induced apoptosis in TCC cells expressing wild-type (wt) p53 (RT4, SW780) and mutated (mt) p53 (UM-UC-3, 5637, J82, and TCCSUP) protein. We have also investigated the effects of co-treatment of anthracyclines with PRIMA-1 in J82 cells, as a representative mt-p53 TCC cell line, and compared the co-treatment effects in RT4, as a representative wt-p53 cell line. Our results demonstrated that anthracyclines inhibited cell viability of all tested TCC cells in vitro, with AD 198 demonstrating highest efficacy. Furthermore, anthracyclines upregulated p53 protein expression in wt-p53 RT4 and SW780 cells, but not in tested mt-p53 UM-UC-3, 5637, J82, and TCCSUP cells. A strong correlation was observed between p53 mutation status and activation of caspase 3/7 and cleavage of PARP. The upregulated caspase 3/7 activity and PARP cleavage was observed in wt-p53, but not in mt-p53 TCC cells. AD198 blocked c-myc expression in mt-p53 TCC cells and no expression of c-myc was detected in wt-p53 TCC cells. Furthermore, sensitivity to AD 198 treatment in mt-p53 J82 cells was significantly improved by a co-treatment with PRIMA-1. However, PRIMA-1 had no effect on cell viability in RT4, the wt-p53 TCC cells. Overall, our results demonstrated that AD 312 and AD 198 are potent inhibitors of bladder cancer cells in vitro and are promising candidates for new therapeutic strategies to replace Dox for bladder cancer treatment. PRIMA-1 even further sensitized mt-p53 cells to AD 198 treatment and thereby this co-treatment improved the inhibition of bladder cancer in vitro. Citation Format: Sony Pandey, Leonard Lothstein, Maria Cekanova. Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 885.

Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro.

PurposeReceptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with highly expressed receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR) and c-Kit receptor. Resistance to targeted therapies is partially due to the activation of alternative pro-survival signaling pathways, including cyclooxygenase (COX)-2. In this study, we validated the effects of two RTKIs, axitinib and AB1010, in combination with COX inhibitors on the V-akt murine thymoma oncogene homolog 1 (Akt) and COX-2 signaling pathways in bladder cancer cells.MethodsThe expression of several RTKs and their downstream signaling targets was analyzed by Western blot (WB) analysis in human and canine bladder transitional cell carcinoma (TCC) cell lines. The effects of RTKIs and COX inhibitors in bladder TCC cells were assessed by MTS for cell viability, by Caspase-3/7 and Annexin V assay for apoptosis, by WB analysis for detection of COX-2 and Akt signaling pathways, and by enzyme-linked immunosorbent assay for detection of prostaglandin E2 (PGE2) levels.ResultsAll tested TCC cells expressed the c-Kit and PDGFRα receptors, except human 5637 cells that had low RTKs expression. In addition, all tested cells expressed COX-1, COX-2, Akt, extracellular signal regulated kinases 1/2, and nuclear factor kappa-light-chain-enhance of activated B cells proteins, except human UM-UC-3 cells, where no COX-2 expression was detected by WB analysis. Both RTKIs inhibited cell viability and increased apoptosis in a dose-dependent manner in tested bladder TCC cells, which positively correlated with their expression levels of the PDGFRα and c-Kit receptors. RTKIs increased the expression of COX-2 in h-5637 and K9TCC#1Lillie cells. Co-treatment of indomethacin inhibited AB1010-induced COX-2 expression leading to an additive effect in inhibition of cell viability and PGE2 production in tested TCC cells.ConclusionCo-treatment of RTKIs with indomethacin inhibited cell viability and AB1010-induced COX-2 expression resulting in decreased PGE2 production in tested TCC cells. Thus, COX inhibition may further potentiate RTKIs therapies in bladder cancer.

Fluid Shear Stress Induces Cell Cycle Arrest in Human Urinary Bladder Transitional Cell Carcinoma Through Bone Morphogenetic Protein Receptor-Smad1/5 Pathway.

Mechanical force generated from the interstitial fluid flow inside and surrounding tissue has been known to play a significant role in cancer pathophysiology. In this study, we aimed to investigate the role of laminar shear stress (LSS) in modulating the cell cycle of human bladder transitional carcinoma (BFTC-905) cells which are frequently stimulated by not only the interstitial fluid flow, but also the urine flow transported from kidney to bladder in the urinary tract. The BFTC-905 cells were subjected to 0-12 dynes cm-2 LSS for 1, 4, 8, or 12h, respectively, followed by cellular and molecular assays for investigations of cell cycle regulation protein expressions, cell growth rates, and the potential mechanism. The results showed that the LSS with ≥ 8 dynes cm-2 for ≥ 8h significantly increased protein expressions of cyclin B1, Wee1, p21, and p-CDK1(Tyr15) (p < 0.05 for each), but conversely decreased protein expressions of cyclin A2, D1, E1, and CDK-1, -2, -4, and -6 (p < 0.05 for each) in the BFTC-905 cells, indicating that a G2/M cell cycle arrest was obtained after shearing stimulation. Furthermore, our data demonstrated that the LSS-induced G2/M arrest and the corresponding changes in cell cycle regulatory protein expressions were modulated by bone morphogenetic protein (BMP) receptor-Smad1/5 signaling pathway. Our findings provided evidences for the effect of mechanical microenvironment on urothelial cancer pathobiology and generated insights into mechanism of LSS-regulated bladder tumor cell cycle.

Effect of Pretreatment of Morin on the Cisplatin-Induced Toxicity on LLC-PK1 and T24 Cells

Cisplatin is a chemotherapeutic drug broadly used in the treatment of solid tumors. However, patients under this chemotherapy develop severe adverse effects, being nephrotoxicity the most frequent. Cisplatin-induced nephrotoxicity has been associated with oxidative stress, inflammation and apoptosis. Morin, a rather abundant flavonoid, presents antioxidant properties and, at the same time, decreases viability in some tumor cells. Taking into account these facts, it has been hypothesized that morin could attenuate cisplatin-induced kidney injury without interfering with antineoplastic activity of cisplatin. The aim of this study was to evaluate the potential cytoprotective effect of morin against cisplatin-induced damage in the Lilly Laboratories cell-porcine kidney 1 (LLC-PK1) cell line. In addition, the effect of pretreatment with morin on the toxicity of cisplatin on human urinary bladder transitional cell carcinoma T24 cell line was investigated. It was found that 200 μM morin attenuates cisplatin-induced damage on LLC-PK1 cells in a heme oxygenase-1 (HO-1) dependent-way. Moreover, it was found that 200 μM morin alone, decreases T24 cell viability in the same proportion as cisplatin. However, when T24 cells were pretreated with morin, the cell viability did not change compared to the cells treated only with cisplatin.

Wild-type p53-induced phosphatase 1 is a prognostic marker and therapeutic target in bladder transitional cell carcinoma

Wild-type p53-induced phosphatase (Wip1) is an established oncogene and is associated with development of multiple forms of human cancer. However, the expression and role of Wip1 in human bladder transitional cell carcinoma (TCC) remains to be elucidated. In the present study, immunohistochemistry demonstrated that Wip1 was overexpressed in bladder TCC tissues compared with corresponding normal bladder tissues in 106 bladder TCC cases (P<0.0001). Furthermore, high expression levels of Wip1 were significantly associated with increasing tumor size (P=0.002), pathological grade (P=0.025), clinical T stage (P=0.001) and lymph nodal metastasis (P=0.003). Kaplan-Meier survival analysis identified that patients with high Wip1 expression levels exhibited a lower overall survival time (P<0.0001), and Cox proportional hazards regression model analysis demonstrated that Wip1 expression was an independent prognostic factor in patients with bladder TCC (P=0.025). In addition, downregulation of Wip1 expression by transfection with small interfering RNA in bladder cancer cells inhibited cell proliferation, invasion and migration (P<0.05), along with the upregulation of p53 protein levels (P<0.05). These findings suggest that Wip1 may function as a potential prognostic marker and therapeutic target in bladder cancer.

MicroRNA-497 inhibits the proliferation, migration and invasion of human bladder transitional cell carcinoma cells by targeting E2F3.

Accumulating evidence indicates that microRNAs (miRNAs) play critical roles in regulating cellular processes, such as cell growth and apoptosis, as well as cancer progression and metastasis. Low expression of miR-497 has been observed in breast, colorectal and cervical cancers. Human bladder transitional cell carcinoma (BTCC) progression typically follows a complex cascade from primary malignancy to distant metastasis, but whether the aberrant expression of miR-497 in BTCC is associated with malignancy, metastasis or prognosis remains unknown. In the present study, we found that miR-497 was markedly downregulated in BTCC tissue samples when compared with that noted in adjacent normal tissues, and low expression of miR-497 was correlated with poor prognosis in BTCC patients. We also found that overexpression of miR-497 inhibited the proliferation, migration and invasion of bladder cancer cells by downregulating E2F3 (an miR-497 target gene) mRNA and protein and that siRNA against E2F3 inhibited cell proliferation, migration and invasion, which was similar to the effect of miR-497 overexpression in the BTCC cells. Our experimental data indicated that miR-497 mediates the invitro proliferation, migration and invasion of BTCC cells. Together, these results suggest that miR-497 may represent a novel prognostic indicator, a biomarker for the early detection of metastasis and a target for gene therapy of BTCC.

AB112. Expression of brain-specific angiogenesis inhibitor 1 and association with p53, microvessel density and vascular endothelial growth factor in the tissue of human bladder transitional cell carcinoma

ObjectiveBrain-specific angiogenesis inhibitor 1 (BAI1) was initially described in 1997, and there have since been a number of studies on its expression in different types of cancer. The aim of the present study was to investigate the expression levels of BAI1 in bladder transitional cell carcinoma (BTCC) at different stages and the mechanism by which it inhibits tumor endothelial cell proliferation.MethodsNormal bladder mucosa biopsy specimens were obtained as the control group, and human BTCC biopsy specimens were used as the study group. Immunohistochemical assays were used to detect the expression levels of BAI1, vascular endothelial growth factor (VEGF) and mutant p53, in addition to microvessel density (MVD) in the tissues. Western blotting was used to analyze the differential expression of BAI1 in the two samples.ResultsStatistical analysis was performed, which indicated that BAI1 expression levels in the normal bladder mucosa group were significantly higher than those in the BTCC group and were associated with clinical staging. BAI1 levels in the T1 stage BTCC tissues were higher than those in the T2–4 stage BTCC tissues (P<0.05). BAI1 expression levels were negatively correlated with those of VEGF (r=−0.661, P<0.001), mutant p53 (r=−0.406, P=0.002) and with the MVD (r=−0.675, P<0.001).ConclusionsBAI1 may be involved in the negative regulation of BTCC microvascular proliferation, and its expression may be associated with a reduction in p53 mutations.

AB147. Wild-type p53-induced phosphatase 1 is a prognostic marker and therapeutic target in bladder transitional cell carcinoma

ObjectiveWild-type p53-induced phosphatase (Wip1) is known as an oncogene and is associated with development of various types of human cancers. However, the expression and role of Wip1 in human bladder transitional cell carcinoma (TCC) remains unclear.MethodsThe expression of Wip1 in bladder cancer patients was determined using immunohistochemistry. Bladder cancer T24 was transfected with Wip1-siRNA or negative control siRNA. Cell proliferation, invasion and migration and were determined using MTT assay, transwell assay, respectively.ResultsIn this study, immunohistochemistry evaluation found that Wip1 was overexpressed in bladder TCC tissues compared with corresponding normal bladder tissues in 106 bladder TCC patients (P<0.0001). Moreover, high expression of Wip1 was significantly associated with increasing tumor size (P=0.002), pathological grade (P=0.025), clinical T stage (P=0.001) and lymph nodal metastasis (P=0.003). The Kaplan-Meier survival analysis revealed that patients with high Wip1 expression showed a lower overall survival time (P<0.0001), and the Cox proportional hazards regression model demonstrated that Wip1 expression was an independent prognostic factor in bladder TCC patients (P=0.025). In addition, downregulation of Wip1 by transfected with small interfering RNA (siRNA) in bladder cancer cells inhibited cell proliferation, invasion and migration (P<0.05), along with the upregulation of p53 protein levels (P<0.05).ConclusionsOur findings suggest that Wip1 may act as a potential prognostic marker and therapeutic target of bladder cancer.