Transitional Carcinoma Cell Lines Research Articles

Cryptotanshinone Suppresses the STAT3/BCL-2 Pathway to Provoke Human Bladder Urothelial Carcinoma Cell Death.

Bladder cancer is one of the most common human malignancies worldwide. Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is crucial to driving malignant progression and predicting poor prognosis of multiple human cancers, including bladder cancer, making STAT3 a promising target of cancer therapeutics. Cryptotanshinone (CTS) is an anticancer ingredient of Danshen (Salvia miltiorrhiza), a top-graded Chinese medicinal herb. However, whether CTS targets STAT3 to exert its cytotoxic effect on human bladder cancer remains unknown. Herein, we demonstrated that CTS is cytotoxic to multiple human urinary bladder transitional cell carcinoma (TCC) cell lines while sparing normal human urothelial cells. CTS provoked apoptosis-dependent bladder TCC cytotoxicity, as apoptosis blockage by z-VAD-fmk markedly rescued the clonogenicity of CTS-treated cells. Besides, CTS was found to suppress constitutive and interleukin 6-inducible activation of STAT3, evidenced by the downregulation of STAT3 tyrosine 705 phosphorylation and BCL2, a recognized STAT3 transcriptional target. Notably, ectopic expression of a dominant-active STAT3 mutant (STAT3-C) or BCL-2 alleviated CTS-induced apoptosis and clonogenicity inhibition, thus confirming STAT3 blockade as a pivotal mechanism of CTS's cytotoxic action on bladder TCC cells. Lastly, immunoblotting revealed that CTS lowered the levels of active JAK2, an upstream kinase that mediates STAT3 tyrosine 705 phosphorylation. Altogether, we conclude that the blockade of the JAK2/STAT3/BCL-2 antiapoptotic signaling axis is a vital mechanism whereby CTS provokes bladder cancer cytotoxicity. The current evidence implicates CTS's potential to be translated into a bladder cancer therapeutic agent.

Downregulation of the keratins CK13 and CK14 does not significantly affect cell viability of human urinary bladder carcinoma cells.

Bladder cancer is the ninth most common tumour entity worldwide. Aberrant expression of different keratins has been described in bladder cancer, which is used for diagnostic purposes, but it can also have prognostic value. However, not all keratins have been analysed in bladder cancer, and whether keratins are important for cell viability of bladder cancer tumour cells is not yet known. We analyse the expression of CK10, CK13, and CK14 in 4 different urinary bladder transitional cell carcinoma cell lines via western blot. Furthermore, we downregulate the expression of CK13 and CK14 using siRNAs and evaluate the cell viability of the carcinoma cells. In this study, we show that different urinary bladder transitional cell carcinoma cell lines have distinct expression pattern of the keratins CK10, CK13, and CK14. Using several siRNAs targeting either CK13 or CK14, we show that both keratins have long protein half-lives. Although we achieve a reduction in CK13 and CK14 protein levels, these reductions do not influence the cell viability of the cell lines. In conclusion, we provide evidence that CK10, CK13, and CK14 are expressed on the protein level in different urinary bladder transitional cell carcinoma cell lines, but that their targeting does not affect the viability of the carcinoma cells.

The Regulatory Role of Nuclear Respiratory Factor 1 in Bladder Cancer Cells.

Nuclear respiratory factor 1 (NRF1) is a key mediator of genes involved in mitochondrial biogenesis and the respiratory chain; however, its role in bladder cancer remains unknown. Transitional cell carcinoma, also known as urothelial cell carcinoma, is the most common type of bladder cancer resistant to chemotherapy. An established high-grade and invasive transitional cell carcinoma line from patients with urinary bladder cancer, known as T24, has been extensively used in cancer research. In this study, we aimed to investigate the mechanisms through which NRF1 regulates proliferation and cell migration of bladder cancer cells using the T24 cell line. Cells were transfected with plasmid cloning DNA for NRF1 to evaluate the effect of NRF1 overexpression on bladder cancer cells. Western blot was used to examine epithelial and mesenchymal markers (E-cadherin and α-smooth muscle actin), transcriptional regulators for epithelial-mesenchymal transition (snail family transcriptional repressors), components of transforming growth factor-β1/SMADs signaling, high-mobility group box 1 (HMGB1), and receptor for advanced glycation end-products (RAGE). The in situ expression of E-cadherin, α-smooth muscle actin and SMAD7 was determined using immunofluorescence staining. Cell migration capacity was assessed by wound-healing assay. Transfection with NRF1 expression vector repressed the migration capacity of bladder cancer cells, diminishing HMGB1/RAGE expression and reducing transforming growth factor β-associated epithelial-mesenchymal transition in T24 cells. Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments for bladder cancer.

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.

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.

Ablation of Red Stable Transfected Claudin Expressing Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cell Lines by C-CPE Gold-Nanoparticle-Mediated Laser Intervention.

Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.

Metformin and sodium dichloroacetate effects on proliferation, apoptosis, and metabolic activity tested alone and in combination in a canine prostate and a bladder cancer cell line.

An important approach in tumor therapy is combining substances with different action mechanisms aiming to enhance the antineoplastic effect, decrease the therapeutic dosage, and avoid resistance mechanisms. Moreover, evaluating compounds already approved for the treatment of non-neoplastic diseases is promising for new antineoplastic therapies. Sodium dichloroacetate (DCA) reactivates oxidative phosphorylation in the cancer cell mitochondria, reducing apoptosis resistance in cancer cells. Furthermore, metformin inhibits the proliferation of tumor cells and CD133+ cancer -stem-like cells. In the present study, we evaluated the independent and synergistic effect of metformin and DCA on the metabolic activity, cell proliferation, and apoptosis of a canine prostate adenocarcinoma (Adcarc1258) and a transitional cell carcinoma cell line (TCC1506) in comparison to a primary canine fibroblast culture. Determining metformin uptake in tumor cells was performed by quantitative HPLC. Depending on the dosage, metformin as a single agent inhibited the metabolic activity and cell proliferation of the tumor cells, showing only minor effects on the fibroblasts. Furthermore, 1 mM metformin increased apoptosis over 96 h in the tumor cell lines but not in fibroblasts. Additionally, metformin uptake into the tumor cells in vitro was measurable by quantitative HPLC. Synergistic effects for the combination therapy were observed in both neoplastic cell lines as well as in the fibroblasts. Based on these results, metformin might be a promising therapeutic agent for canine urogenital tumors. Further studies on kinetics, toxicology, bioavailability, and application of metformin in dogs are necessary.

Establishment of Canine Transitional Cell Carcinoma Cell Lines Harboring BRAF V595E Mutation as a Therapeutic Target.

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients.

Citrus aurantifulia extract as a capping agent to biosynthesis of gold nanoparticles: Characterization and evaluation of cytotoxicity, antioxidant, antidiabetic, anticholinergics, and anti‐bladder cancer activity

In this study, new chemotherapeutic Au nanoparticles (AuNPs) were prepared to treat bladder cancer. AuNPs were synthesized using Citrus aurantifulia seed extract as the capping agent. Characterization of AuNPs was performed by Fourier‐transform infrared (FT‐IR), field emission scanning electron microscopy (FE‐SEM), UV–Vis, energy‐dispersive X‐ray spectroscopy (EDS), and transmission electron microscopy (TEM). In UV–Vis, the exact peak in the wavelength of 522 nm revealed the formation of AuNPs. Besides, in the FE‐SEM and TEM images, the AuNPs were spherical with range size of 17 to 24 nm. To investigate the antioxidant properties of AuNPs, the DPPH test was used. AuNPs inhibited half of the DPPH molecules in the concentration of 248 μg/ml. MTT assay was used on bladder cancer (HT‐1376, Grade 3, carcinoma), TCCSUP (Grade IV, transitional cell carcinoma), SCaBER (squamous cell carcinoma), and UM‐UC‐3 (transitional cell carcinoma) cell lines. AuNPs had very low cell viability and anti‐bladder cancer properties dose dependently against HT‐1376, TCCSUP, SCaBER, and UM‐UC‐3 cell lines. The best result of the anti‐bladder cancer properties of AuNPs contrary to the above cell lines was observed in the case of SCaBER cell line. Additionally, these AuNPs were suitable inhibitors of the cholinesterase and α‐glycosidase enzymes with Ki values of 55.32 + 5.32 and 148.78 ± 23.83 μM for α‐glycosidase, 6.68 ± 0.67 and 23.98 ± 3.42 μM for BChE, and 26.29 ± 6.76 and 76.85 ± 6.46 μM for AChE, respectively. After confirming the recent results in the clinical trial studies, AuNPs may be used as antioxidant, antidiabetic, anticholinergics, and anti‐bladder cancer supplements in human.

Investigation of the Inhibitory Effect of Platycodin D in Human Transitional Cell Carcinoma Cell Line 5637

Platycodin D is an active component isolated from Chinese herb Platycodonis radix with various pharmacological activities, such as antitussive, expectorant, anti-inflammatory, and analgesic effects. Interestingly, platycodin D also exerts anticancer effects against several types of cancer. However, few studies on the anti-tumour effects of platycodin against urinary bladder cancer have been reported. In this study, we explored the anti-tumour effect of platycodin D against human bladder cancer and its mechanisms in vitro and in vivo. We found that platycodin D had significant anti-proliferative effects on four types of cancer cells, especially the 5637 bladder cancer cell line, and exerted these effects by preventing cell cycle progression from G0/G1 to S phase, down-regulating Ki-67 and cyclin D1 protein expression and up-regulating P21 protein expression. Furthermore, platycodin D inhibited 5637 cell migration by decreasing twist-related protein 1 (Twist1) and matrix metallopeptidase 2 (MMP2) expression and exerted significant tumour-suppressive effects in tumour-bearing nude mice. Platycodin D also increased caspase-9, caspase-8, caspase-3, and p53 expression and decreased Bcl-2 expression in tumour tissues. Taken together, our results provide a theoretical basis for application of platycodin D in treating urinary bladder cancer.

Re: The CUL4B-miR-372/373-PIK3CA-AKT Axis Regulates Metastasis in Bladder Cancer.

No AccessJournal of UrologyUrological Survey1 Oct 2020Re: The CUL4B-miR-372/373-PIK3CA-AKT Axis Regulates Metastasis in Bladder Cancer Anthony AtalaMD Anthony AtalaAnthony Atala More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001222.01AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "Re: The CUL4B-miR-372/373-PIK3CA-AKT Axis Regulates Metastasis in Bladder Cancer." The Journal of Urology, 204(4), p. 882 Suggested Reading : Transurethral bladder tumor resection alters fibronectin expression in transitional carcinoma cell lines. J Urol 1997; 157: 1136. Link, Google Scholar © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 204Issue 4October 2020Page: 882-882 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Anthony Atala More articles by this author Expand All Advertisement Loading ...

Characterization of six canine prostate adenocarcinoma and three transitional cell carcinoma cell lines derived from primary tumor tissues as well as metastasis.

Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) of prostate and urinary bladder are highly invasive and metastatic tumors of closely neighbored organs. Cell lines are valuable tools to investigate tumor mechanisms and therapeutic approaches in vitro. PAC in dogs is infrequent, difficult to differentiate from TCC and usually characterized by poor prognosis, enhancing the value of the few available cell lines. However, as cell lines adapt to culturing conditions, a thorough characterization, ideally compared to original tissue, is indispensable. Herein, six canine PAC cell lines and three TCC cell lines were profiled by immunophenotype in comparison to respective original tumor tissues. Three of the six PAC cell lines were derived from primary tumor and metastases of the same patient. Further, two of the three TCC cell lines were derived from TCCs invading into or originating from the prostate. Cell biologic parameters as doubling times and chemoresistances to commonly used drugs in cancer treatment (doxorubicin, carboplatin and meloxicam) were assessed. All cell lines were immunohistochemically close to the respective original tissue. Compared to primary tumor cell lines, metastasis-derived cell lines were more chemoresistant to doxorubicin, but equally susceptive to carboplatin treatment. Two cell lines were multiresistant. COX-2 enzyme activity was demonstrated in all cell lines. However, meloxicam inhibited prostaglandin E2 production in only seven of nine cell lines and did neither influence metabolic activity, nor proliferation. The characterized nine cell lines represent excellent tools to investigate PAC as well as TCC in prostate and urinary bladder of the dog. Furthermore, the profiled paired cell lines from PAC primary tumor and metastasis provide the unique opportunity to investigate metastasis-associated changes PAC cells undergo in tumor progression. The combination of nine differently chemoresistant PAC and TCC cell lines resembles the heterogeneity of canine lower urinary tract cancer.

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.

Biosensor device for the photo-specific detection of immuno-captured bladder cancer cells using hexaminolevulinate: An ex-vivo study.

This study investigate the relationship between different regulatory factors (time, concentration, temperature and addition of a fluorescent dye) and Hexaminolevulinate (HAL)-mediated photodynamic diagnosis of bladder cancer (PDD) in vitro. We examine the natural photosensitizer Protoporphyrin IX (PpIX) fluorescence induced by HAL in several human bladder cancer cell lines and one non-cancer foreskin fibroblast cell line and identify the processing conditions that maximise the difference in fluorescence intensity between malign and benign cell types. The detection of HAL induced fluorescence at a single cell level by a selective cancer cell capture platform is also tested. Experiments were performed on cultured monolayer cells and cells in suspension. The cell lines examined included the transitional epithelium carcinoma cell lines HT1197, HT1376, EJ138 and RT4, and the non-cancer foreskin fibroblasts HFF. Cells were incubated with HAL in various doses, time and temperature settings. We also used the nuclear red as a tool to study the PpIX subcellular localization. PpIX fluorescence intensities were measured and analysed using fluorescence microscope software. Finally, we evaluated the possibility of using HAL to discriminate between cancer and non-cancer cells from a mixed cell population using a newly developed immunofunctionalized microfluidic platform. The accumulation of PpIX in bladder cancer cells was significantly higher than in non-cancer cells, both cultured monolayer cells and cells in suspension. Effectively, the fluorescence intensity was 4.5 times brighter in bladder cancer cells than non-cancer foreskin fibroblast cells when incubated in the optimum condition, in which the nuclear stain adjuvant acted as a fluorescence enhancer. Cancer cells displayed PpIX accumulated mainly in mitochondria but none or very little PpIX was observed in non-cancer cells. HAL-induced fluorescence could be used to correctly identify bladder cancer cells within the EpCAM conjugated POx based microfluidic sensor with an 88% capture selectivity rate. These findings prove that the application of HAL-induced PpIX fluorescence can successfully distinguish between cancer and non-cancer cells in vitro. This test can provide advanced second level of confidence on the cancerous nature of cells captured by the immunofunctionalized bladder cancer diagnostic platform.

Thymoquinone suppresses the proliferation of renal cell carcinoma cells via reactive oxygen species-induced apoptosis and reduces cell stemness.

Thymoquinone is a phytochemical compound isolated from Nigella sativa and has various biological effects, including anti-inflammation, antioxidation, and anticancer. Here, we further investigated the anticancer effects and associated molecular mechanism of 2-methyl-5-isopropyl-1,4-benzoquinone (thymoquinone) on human renal carcinoma cell lines 786-O and 786-O-SI3 and transitional carcinoma cell line BFTC-909. Results showed that thymoquinone significantly reduced cell viability, inhibited the colony formation of renal cancer cells, and induced cell apoptosis and mitochondrial membrane potential change in both cancer cells. In addition, thymoquinone also triggered the production of reactive oxygen species (ROS) and superoxide and the activation of apoptotic and autophagic cascade. ROS inhibition suppressed the caspase-3 activation and restored the decreased cell viability of 786-O-SI3 in response to thymoquinone. Autophagy inhibition did not restore the cell viability of 786-O-SI3 suppressed by thymoquinone. Moreover, thymoquinone suppressed the cell sphere formation and the expression of aldehyde dehydrogenase, Nanog, Nestin, CD44, and Oct-4 in 786-O-SI3 cells. The tumor-bearing model showed that thymoquinone in vivo inhibited the growth of implanted 786-O-SI3 cell. All these findings indicate that thymoquinone inhibits the proliferation of 786-O-SI3 and BFTC-909 cell possibly due to the induction of ROS/superoxide and the consequent apoptosis, suggesting that thymoquinone may be a potential anticancer supplement for genitourinary cancer.

MicroRNA Biomarkers for Patients With Muscle-Invasive Bladder Cancer Undergoing Selective Bladder-Sparing Trimodality Treatment

Trimodality therapy with maximal transurethral resection of bladder tumor and definitive chemoradiation reserving cystectomy for salvage of local recurrence is an accepted treatment alternative to upfront cystectomy for selected patients with muscle-invasive bladder cancer. There is a need for molecular biomarkers to predict which patients will respond to bladder preservation therapy. We sought to identify biomarkers with the ability to predict response to chemoradiation and survival after selective bladder preservation therapy in a cohort of 40 patients using a microRNA profiling approach. Invitro experiments were performed using transitional cell carcinoma lines CRL1749, HTB5, and HTB4. We identified a panel of microRNAs associated with overall survival in our bladder preservation cohort and in the TCGA cohort. We also identified several microRNAs, including miR-23a and miR-27a, microRNAs of the miR-23a cluster, to be suggestively associated with complete response to chemoradiation therapy. The microRNAs were significantly associated with overall survival in The Cancer Genome Atlas cohort. Invitro studies suggest that the functional roles of miR-23a and miR-27a involve targeting the SFRP1 protein, a negative regulator of the Wnt signaling pathway. The upregulation of β-catenin in the Wnt signaling pathway mediated proliferation, migration, invasion, and sensitivity to radiation and cisplatin treatment in bladder cancer cells. Our results indicate that miR-23a and miR-27a act as oncomirs, and once independently validated, they may help appropriately triage selected bladder cancer patients to individualize treatment.

Anti-tumour effect of lapatinib in canine transitional cell carcinoma cell lines.

Transitional cell carcinoma (TCC) accounts for >90% of canine malignant tumours occurring in urinary bladder, and the prognosis is poor. Our previous study, using RNA sequencing, showed that human epidermal growth factor 2 (HER2) was the most activated upstream regulator related to carcinogenesis in canine TCC. The aim of this study was to examine the anti-tumour effect of lapatinib, a tyrosine kinase inhibitor of HER2, on canine TCC cell lines in vitro and in vivo. Five canine TCC cell lines (TCCUB, Love, Sora, LCTCC, and MCTCC) were used. Western blotting showed that HER2 protein expression was observed in all of the canine TCC cell lines. Lapatinib inhibited phosphorylation of HER2 and cell growth in a dose-dependent manner. Cell cycle analyses using flow cytometry showed that lapatinib significantly increased the sub-G1 and G0 /G1 phase fractions and significantly decreased the S and G2 /M phase fractions in the cell lines (Sora and TCCUB). For the in vivo experiments, the canine TCC cells (Sora) were subcutaneously injected into nude mice. Six days after inoculation, lapatinib (100 mg/kg) or vehicle was administered daily via intraperitoneal administration for 14 days. Tumour volume was significantly smaller in the lapatinib group compared with the vehicle control group. Histologically, lapatinib significantly increased necrotic areas in the tumour tissues. These findings suggest that lapatinib exerts anti-tumour effects on canine TCC cells by inhibiting HER2 signalling and inducing cell cycle arrest.

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.

Expression of phosphatases of regenerating liver-1 and phosphatases of regenerating liver-3 in the bladder transitional cell carcinoma cells

Objective To study the expression of phosphatases of regenerating liver (PRL)-1 and PRL-3 in the different bladder transitional cell carcinoma lines, so as to detect their roles in invasion and metastasis of cancer. Methods Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were used to detect the expression levels of PRL-1 and PRL-3 in the BIU-87 and T24 cell lines. Boyden chamber experiment was used to detect the invasiveness of the two cell lines. Results The relative transcript levels of PRL-1 and PRL-3 mRNA were 0.772±0.037 and 0.304±0.033 in the BIU-87 cells, and 0.828±0.039 and 0.298±0.038 in the T24 cells (t=20.930 and 21.494, P=0.000). The relative transcript levels of PRL-1 and PRL-3 mRNA had the positive correlations in the BIU-87 cells and the T24 cells (r=0.941, P=0.017; r=0.997, P=0.000). The relative transcript levels of PRL-1 and PRL-3 protein were 7.000±1.870 and 4.400±1.140 in the BIU-87 cells, and 8.200±0.836 and 5.200±1.303 in the T24 cells (t=2.654, P=0.029; t=4.330, P=0.003). The relative transcript levels of PRL-1 and PRL-3 protein had the positive correlations in the BIU-87 cells and the T24 cells (r=0.958, P=0.010; r=0.942, P=0.017). The transmembrane cell counts of the BIU-87 and T24 cells were respectively (148.800±6.418) and (102.200±6.340) cells with the difference being significant (t=11.549, P=0.000). Conclusion High expression levels of PRL-1 and PRL-3 are observed in the bladder transitional cell carcinoma cell lines, and they maybe take a very important part in the invasion and metastasis of bladder transitional cell carcinoma. Key words: Phosphatases of regenerating liver-1; Phosphatases of regenerating liver-3; Bladder transitional cell carcinoma; Invasion; Metastasis

Near infrared photoimmunotherapy targeting bladder cancer with a canine anti-epidermal growth factor receptor (EGFR) antibody

Anti-epidermal growth factor receptor (EGFR) antibody therapy is used in EGFR expressing cancers including lung, colon, head and neck, and bladder cancers, however results have been modest. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate which is activated by NIR light. NIR-PIT is in clinical trials in patients with recurrent head and neck cancers using cetuximab-IR700 as the conjugate. However, its use has otherwise been restricted to mouse models. This is an effort to explore larger animal models with NIR-PIT. We describe the use of a recombinant canine anti-EGFR monoclonal antibody (mAb), can225IgG, conjugated to the photo-absorber, IR700DX, in three EGFR expressing canine transitional cell carcinoma (TCC) cell lines as a prelude to possible canine clinical studies. Can225-IR700 conjugate showed specific binding and cell-specific killing after NIR-PIT on EGFR expressing cells in vitro. In the in vivo study, can225-IR700 conjugate demonstrated accumulation of the fluorescent conjugate with high tumor-to-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 µg of can225-IR700 i.v. only; (3) NIR light exposure only; (4) 100 µg of can225-IR700 i.v., NIR light exposure. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and significantly prolonged survival was achieved (p < 0.001 vs. other groups) in the treatment groups. In conclusion, NIR-PIT with can225-IR700 is a promising treatment for canine EGFR-expressing cancers, including invasive transitional cell carcinoma in pet dogs, that could provide a pathway to translation to humans.