Transitional Cell Carcinoma Cell Lines Research Articles

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.

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.

Reduction in mitochondrial oxidative stress mediates hypoxia-induced resistance to cisplatin in human transitional cell carcinoma cells

Tumor hypoxia is known to promote the acquisition of more aggressive phenotypes in human transitional cell carcinoma (TCC), including drug resistance. Accumulating evidence suggests that mitochondria play a central role in the chemoresistance of TCC. However, the role of mitochondria in the hypoxia-induced drug resistance in TCC remains elusive. The present study investigated the function of mitochondria in the drug resistance using a TCC cell line under hypoxic conditions. In vitro hypoxia (0.1% O2, 48 h) was achieved by incubating TCC cells in air chamber. Mitochondrial events involving hypoxia-induced drug resistance were assessed. Hypoxia significantly reduced the cisplatin-induced apoptosis of TCC cells. Additionally, hypoxia substantially decreased the level of mitochondrial reactive oxygen species (ROS) generated by cisplatin treatment. Analogously, elimination of mitochondrial ROS significantly rescued cells from cisplatin-induced apoptosis. Hypoxia enhanced mitochondrial hyperpolarization, which was not related to ATP production or the reversal of ATP synthase activity. The mitochondrial DNA (mtDNA) amplification efficiency data illustrated that hypoxia significantly prevented oxidative damage to the mitogenome. Moreover, transmission electron microscopy revealed that cisplatin-induced disruption of the mitochondrial ultrastructure was abated under hypoxic conditions. Notably, depletion of mtDNA by ethidium bromide abrogated hypoxia-induced resistance to cisplatin. Taken together, the present study demonstrated that TCC cells exposed to hypoxic conditions rendered mitochondria less sensitive to oxidative stress induced by cisplatin treatment, leading to enhanced drug resistance.

Antitumor activity of an oncolytic measles virus against canine urinary bladder transitional cell carcinoma cells

The prognosis of canine transitional cell carcinoma (TCC) of urinary bladder is generally poor because it is difficult to diagnose at early stages and conventional therapies, such as surgical resection and/or chemotherapy, are often not curative treatments. Based on our previous report that recombinant measles virus (rMV-SLAMblind) therapy could be a new treatment for canine mammary tumor, the applicability of rMV-SLAMblind in canine urinary bladder TCC was examined in this study. A canine TCC cell line was established from a TCC patient dog by transplanting a piece of the tumor mass into an immunodeficient mouse and then isolating the primary TCC cells from the grown tumor mass. The primary cultured cells, named TCC-NU1, express nectin-4, a receptor for rMV-SLAMblind infection. The rMV-SLAMblind infected TCC-NU1 cells, and dose-dependently showed cell cytotoxicity. Moreover, intratumoral administration of rMV-SLAMblind in a xenograft model bearing TCC-NU1 cells significantly suppressed the tumor growth reducing the endpoint mass of tumors in treated mice compared to control mice. These results suggest that virotherapy with rMV-SLAMblind be a new candidate therapy for canine TCC.

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.

Identifying the ErbB/MAPK Signaling Cascade as a Therapeutic Target in Canine Bladder Cancer.

Transitional cell carcinoma (TCC) of the bladder comprises 2% of diagnosed canine cancers. TCC tumors are generally inoperable and unresponsive to traditional chemotherapy, indicating a need for more effective therapies. BRAF, a kinase in the mitogen-activated protein kinase (MAPK) pathway, is mutated in 70% of canine TCCs. In this study, we use BRAF mutant and wild-type TCC cell lines to characterize the role of BRAF mutations in TCC pathogenesis and assess the efficacy of inhibition of the MAPK pathway alone and in combination with other gene targets as a treatment for canine TCC. Analysis of MAPK target gene expression and assessment of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation following serum starvation indicated constitutive MAPK activity in all TCC cell lines. BRAF mutant TCC cell lines were insensitive to the BRAF inhibitor vemurafenib, with IC50 values greater than 5 μM, but exhibited greater sensitivity to a paradox-breaking BRAF inhibitor (IC50: 0.2-1 μM). All TCC cell lines had IC50 values less than 7 nM to the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor trametinib independent of their BRAF mutation status. ERK1/2 phosphorylation decreased after 6-hour treatments with MAPK inhibitors, but rebounded by 24 hours, suggesting the presence of resistance mechanisms. Microarray analysis identified elevated expression of the ErbB family of receptors and ligands in TCC cell lines. The pan-ErbB inhibitor sapitinib synergized with BRAF inhibition in BRAF mutant Bliley TCC cells and synergized with MEK1/2 inhibition in Bliley and BRAF wild-type Kinsey cells. These findings suggest the potential for combined MAPK and ErbB receptor inhibition as a therapy for canine TCC. SIGNIFICANCE STATEMENT: The results of this study (1) identify a novel combination strategy for canine bladder cancer treatment: targeting the ErbB/MAPK signaling cascade and (2) establish the utility of canine bladder cancer as a naturally-occurring model for human MAPK-driven cancers.

Targeting the ErbB/MAPK Signaling Cascade in Canine Bladder Cancer Cell Lines

Canine transitional cell carcinoma (TCC) of the bladder accounts for 2% of diagnosed canine cancers. Most TCCs are inoperable and unresponsive to traditional chemotherapy. Median survival time is typically less than a year for all treatments indicating a need for more effective therapies. Recent studies have discovered that approximately 70% of canine TCCs harbor mutations in the proto‐oncogene BRAF, a kinase involved in the mitogen‐activated protein kinase (MAPK) pathway that controls cell proliferation, differentiation, and apoptosis. BRAF mutations are present in several human cancers, most of which exhibit adaptive or intrinsic resistance to BRAF‐targeted inhibitors. The goals of this study were to (1) further characterize the role of mutant BRAF in canine TCC and (2) determine whether inhibition of the MAPK pathway alone or in combination with other gene targets may be an effective therapy for TCC treatment.Analysis of ERK1/2 phosphorylation following serum starvation indicates that TCC cell lines exhibit constitutive MAPK pathway activation independent of their BRAF mutation status. MAPK activity was further quantified using gene expression analysis of ten MAPK target genes, revealing that TCC cell lines have significantly higher MAPK pathway activity compared to other canine cancer cell lines. These data suggest a causative role for MAPK signaling in TCC pathogenesis. To determine whether the MAPK pathway could be a therapeutic target for TCC treatment we assessed the effect of BRAF and MEK inhibition on TCC cell proliferation and ERK1/2 phosphorylation. Four BRAF mutant human cell lines with varying degrees of sensitivity to BRAF‐targeted agents were used to determine the relative sensitivity of canine TCC cell lines. BRAF mutant TCC cell lines were sensitive to BRAF inhibition with the “paradox‐breaking” inhibitor PLX7904 (IC50: 0.2–1.2μM), but not vemurafenib (IC50: 7–21μM). Both BRAF wild type and mutant TCC cell lines were sensitive to MEK inhibition with selumetinib (IC50: 15–420nM) and trametinib (IC50: 0.4–8nM). ERK1/2 phosphorylation decreased after 6‐hour treatments with MAPK inhibitors, but rebounded by 24 hours suggesting the presence of resistance mechanisms. Microarray analysis indicated that the ErbB family of receptors and ligands are up‐regulated in TCC cell lines relative to other canine cancer cell lines (fold‐change > 2, q < 0.05). Combined BRAF and ErbB inhibition synergized in the BRAF mutant Bliley TCC cell line, while combined MEK and ErbB inhibition synergized in both Bliley and BRAF wild type Kinsey cells. These findings suggest that targeting ErbB receptors with MAPK inhibition is a potential therapy for canine TCC treatment. Additionally, our data indicate that canine TCC may serve as a naturally‐occurring model for the study of resistance mechanisms to MAPK inhibition in human cancers.Support or Funding InformationMorris Animal Foundation, Shipley University Chair in Comparative OncologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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.

In vitro evaluation of a simulated pneumoperitoneum environment using carbon dioxide on canine transitional cell carcinoma.

To assess the impact of a simulated CO2 pneumoperitoneum environment on the viability and proliferation of canine transitional cell carcinoma (TCC) cells in vitro. In vitro study. A control Madin-Darby canine kidney (MDCK) cell line and 3 canine TCC cell lines were exposed to 100% CO2 at pressure of 0, 5, 10, or 15 mmHg for 2 hours by using an airtight chamber and a mechanical insufflator at 37°C. Culture media pH was measured. Viability and proliferation were assessed by using a resazurin assay and trypan blue dye, respectively. The pH in the media significantly decreased immediately after CO2 exposure but returned to normal within 1 hour. The viability of the cell lines was variably affected at the evaluated pressures. Insufflation pressure of 10 mmHg resulted in significantly decreased cell viability compared with control. The impact of 15 mmHg CO2 was comparable to 0 mmHg and control. CO2 insufflation pressure had no significant effects on proliferation up to 7 days postexposure. Conclusion/Clinical significance: A positive pressure CO2 environment significantly decreased the viability of TCC and MDCK cells under specific conditions without influencing their proliferation up to 7 days postexposure. Investigating these effects in clinical patients undergoing CO2 laparoscopy is essential to assess for port site metastasis or peritoneal carcinomatosis in order to translate these in vitro results to clinical recommendations.

Bladder cancer and Th1 chemokines

Bladder cancer arises from the epithelial lining of the urinary bladder, and it is known as transitional cell carcinoma (TCC). Tobacco smoking is the main known contributor to urinary bladder cancer. However thirty percent of bladder tumors probably result from occupational exposure in the workplace to carcinogens. Immunotherapy by intravesicular delivery of Bacillus Calmette–Guérin (BCG) is used to treat and prevent the recurrence of superficial bladder cancer. Successful BCG immunotherapy for bladder cancer is associated with proper induction of T helper (Th)1 immunity. In bladder cancer patients after intravesicular BCG, urine was found to contain high levels of IP-10, and Interferon (IFN)-γ. TCC and endothelial cell lines were able to secrete IP-10 in response to BCG or IFN stimulation in vitro. Furthermore intravesicular BCG induces a cytokine-rich urinary microenvironment that is inhibitory to human endothelial cells and it is anti-angiogenetic by the induction of Th1 chemokines. Other studies suggest that therapeutic strategies involving Th1 induction and Th2 dampening may improve responses to immunotherapy. Further studies are needed to evaluate the IP-10 in circulation, and urine, as prognostic marker of bladder cancer patients, also in relation to BCG immunotherapy

The effect of dichloroacetate in canine prostate adenocarcinomas and transitional cell carcinomas in vitro.

The Warburg effect describes the ability of cancer cells to produce energy via aerobic glycolysis instead of oxidative phosphorylation of pyruvate. This deviation in mitochondrial metabolism inhibits apoptosis, allowing increased proliferation under conditions of reduced oxygen levels. Dichloroacetate (DCA) was successfully used in several human cancer cell lines to reactivate oxidative phosphorylation in mitochondria. The aim of this study was the characterization and response of canine cancer cell lines after DCA exposure. The effect of 10mM DCA was characterized in vitro on a set of six canine prostate adenocarcinoma and transitional cell carcinoma (TCC) derived cell lines. Cell counts, lactate levels, apoptosis, expression of apoptotic proteins, survival factors and different miRNAs were analyzed. Additionally, metabolic activity, mitochondrial activity and proliferation were investigated. DCA significantly decreased cell number of all but one utilized cell lines and leads to a significant reduction of lactate release. Decreased survivin levels were found in all cell lines, two of which presented a significant reduction in metabolic activity. Increased miR-375 levels were measured in all TCC cell lines. Reactivation of pyruvate dehydrogenase and an elevated mitochondrial activity appear to induce the transition from aerobic glycolysis back to oxidative phosphorylation. Further, these results display that DCA treatment has a suppressant effect on proliferation of canine cancer cells.

Abstract 617: Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib

Abstract Transitional cell carcinoma (TCC) is the most common bladder cancer in humans and their canine companions. Canine TCCs are usually papillary infiltrative TCCs of intermediate to high grade. Genetic defects identified in human TCCs aid in the diagnosis and therapy of human bladder cancer. We utilized whole exome sequencing to screen canine TCCs for gene mutations that contribute to pathogenesis. Genomic DNA was isolated from 11 canine TCCs, 3 matched normal tissue samples, and 2 canine TCC cell lines. Whole exome capture was conducted using the Canine Agilent Sure-select in-solution capture system, and captured fragments were sequenced using an Illumina HiSeq2000 platform. The sequences were mapped to the CanFam3.1 canine reference genome and somatic mutations were identified using Freebayes. Somatic mutations were characterized and compared to the Cancer Gene Census (COSMIC). Nonsense, missense, and insertion/deletion mutations were identified in 126 genes shown to be drivers or repressors in human cancer. Missense mutations were further screened using SIFT to identify alterations deleterious to protein function. The genes exhibiting in/dels, nonsense, or missense mutations with SIFT scores < 0.45 in at least 2 sites or 2 samples were: BRAF, RPL5, RANBP2, EWSR1, NONO, PTPRB, LYL1, JAK1, MSH2, PER1, PIM1, and WRN. In fact, an activating BRAF V to E mutation was identified in 5 of the tumors and both cell lines. The RPL5 and RANBP2 mutations were also hotspot mutations identified in 5 and 3 of the samples, respectively. Mutations were confirmed using Sanger sequencing of amplified genomic DNA. Interestingly, deleterious RANBP2 mutations were only observed in non-BRAF mutant samples. Drug sensitivity assays using the BRAF V600E targeting drug, Vemurafenib, were conducted in the BRAF mutant canine cell lines as well as the BRAF mutant human A375 melanoma cell line. As previously described, the IC50 for the sensitive A375 line was approximately 100 nM, while each of the BRAF mutant canine lines had an IC50 value ≥ 10 μM. Reverse transcriptase PCR was used to amplify the coding sequence for BRAF from the Bliley canine TCC cell line. The amplified transcript (2125 bp) was sequenced, confirming the heterozygous expression of the V548 to E mutant form of BRAF in this cell line. The sequence also indicated that the predicted expressed protein (AA 10 - 715 of XP_013975364.1, corresponding to predicted exons 1 through 20 of XM_014119889.1) exhibited 99% homology to human BRAF AA53 - 763 (NP_004324.2). BRAF protein expression in these cell lines was confirmed by Western blot analysis. Thus, insensitivity to Vemurafenib is not due to differences in canine BRAF, reduced expression, or alternative splicing of the expressed transcript as previously observed in some Vemurafenib resistant human melanomas. These data indicate that although constitutively active BRAF is expressed in canine TCC, other factors may contribute to pathogenesis. Citation Format: Belen Hernandez, Kathryn Cronise, James C. Costello, Rodney Page, Susan Lana, Kenneth L. Jones, Dawn L. Duval. Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 617.

Therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer.

Controversial effects of thalidomide for solid malignancies have been reported. In the present study, we evaluate the effects of thalidomide for transitional cell carcinoma (TCC), the most common type of bladder cancer. Thalidomide precipitates were observed when its DMSO solution was added to the culture medium. No precipitation was found when thalidomide was dissolved in 45% γ-cyclodextrin, and this concentration of γ-cyclodextrin elicited slight cytotoxicity on TCC BFTC905 and primary human urothelial cells. Thalidomide-γ-cyclodextrin complex exerted a concentration-dependent cytotoxicity in TCC cells, but was relatively less cytotoxic (with IC50 of 200 µM) in BFTC905 cells than the other 3 TCC cell lines, possibly due to upregulation of Bcl-xL and HIF-1α mediated carbonic anhydrase IX, and promotion of quiescence. Gemcitabine-resistant BFTC905 cells were chosen for additional experiments. Thalidomide induced apoptosis through downregulation of survivin and securin. The secretion of VEGF and TNF-α was ameliorated by thalidomide, but they did not affect cell proliferation. Immune-modulating lenalidomide and pomalidomide did not elicit cytotoxicity. In addition, cereblon did not play a role in the thalidomide effect. Oxidative DNA damage was triggered by thalidomide, and anti-oxidants reversed the effect. Thalidomide also inhibited TNF-α induced invasion through inhibition of NF-κB, and downregulation of effectors, ICAM-1 and MMP-9. Thalidomide inhibited the growth of BFTC905 xenograft tumors in SCID mice via induction of DNA damage and suppression of angiogenesis. Higher average body weight, indicating less chachexia, was observed in thalidomide treated group. Sedative effect was observed within one-week of treatment. These pre-clinical results suggest therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer.

Decreased expression of protein tyrosine phosphatase non-receptor type 12 is involved in the proliferation and recurrence of bladder transitional cell carcinoma.

Protein tyrosine phosphatase non-receptor type 12 (PTPN12) has been shown to be involved in the development of a number of types of carcinoma. However, the effect of PTPN12 on the proliferation and recurrence of human bladder transitional cell carcinoma (TCC) is unclear. The present study aimed to investigate the expression and function of PTPN12 in human TCC. Samples from 164 patients with TCC, in addition to 146 patients undergoing bladder surgery for indications other than TCC, were examined. PTPN12 protein expression was examined using immunohistochemistry and western blotting, and PTPN12 mRNA expression was examined using reverse transcription-quantitative polymerase chain reaction. PTPN12 expression was increased following transfection with the PTPN12-expressing, pcDEF3 vector, and PTPN12 expression was decreased by RNA interference, in four TCC cell lines. The proliferation of TCC cells was analyzed by a WST-1 assay and in xenografts on BALB/C nude mice. The effect of PTPN12 on tumor recurrence was analyzed by adhesion, migration and invasion assays in TCC cell lines. PTPN12 expression was significantly decreased in TCC tissues compared with that in normal urothelium, and the level of PTPN12 expression was negatively correlated with tumor size, pathological grade, clinical stage and tumor recurrence. Furthermore, decreased expression of PTPN12 significantly enhanced the proliferation of TCC cells in vitro and in vivo. TCC cells with lower levels of PTPN12 exhibited greater adhesion, migration and invasion. In conclusion, PTPN12 expression is downregulated in human TCC. Restoring PTPN12 activity may represent a novel therapeutic strategy for this disease.

Hedgehog signaling is activated in canine transitional cell carcinoma and contributes to cell proliferation and survival.

Transitional cell carcinoma (TCC) is the most commonly diagnosed tumor of the canine urinary system. Hedgehog (HH) signaling represents one possible novel therapeutic target, based on its recently identified central role in human urothelial carcinoma. The purpose of this study was to determine if HH mediators are expressed in canine TCC and the effect of inhibition of this pathway on cell growth and survival. HH pathway mediators were found to be expressed in five canine TCC cell lines. Indian HH was expressed in tumor cells in five canine bladder tumor tissues, but not in normal canine bladder tissue. Inhibition of HH signaling with cyclopamine and GANT61 led to significantly decreased cell proliferation but had a smaller effect on apoptosis. These results support future investigation of inhibitors of HH signaling in the treatment of canine TCC.

Effects of sunitinib malate on growth of human bladder transitional cell line T24 in vitro.

To investigate the growth-inhibitory effect of sunitinib malate on human bladder transitional cell carcinoma (TCC) in vitro. Human bladder TCC cell line T24 was cultured and exposed to graded concentrations of sunitinib malate for 72 hours in vitro to determine the sensitivities to drug. Cell viability was measured by MTT assay. Cell apoptotic morphology was observed by fluorescence microscope following DAPI staining. Band expressions of Fas, Fas ligand, poly (ADP-ribose) polymerase (PARP) and β-actin were analyzed by Western blot. Wound healing process of T24 cells exposed to sunitinib malate was assayed. Sunitinib malate exerted a concentration-dependent and time-dependent inhibitory effect on the T24 cell lines. Fluorescence microscopy showed that small vacuoles appeared in the nuclei of T24 cells and the vacuoles were bigger with higher drug concentrations. The expressions of Fas ligand and PARP in T24 cells treated with sunitinib malate exhibited a concentration-dependent increase. Moreover sunitinib malate suppressed the wound healing process in a concentration-dependent manner. Sunitinib malate exerted marked inhibitory activity against bladder cancer cell line T24.

Human urothelial carcinoma cell response to Sunitinib malate therapy in vitro.

ObjectivesBladder transitional cell carcinoma (TCC) is one of the most common solid malignancies in China. This study examined the antitumor effect and underlying mechanism of action of sunitinib malate in human bladder TCC in vitro.MethodsBladder TCC cell lines 5637 and BIU87 were maintained in 1640 medium and T24 cell lines in DMEM/F12 medium. All 3 cell lines were then exposed to graded concentrations (0.625-20 μmol/L) of sunitinib malate, sorafenib and cisplatin for 24–96 hours to determine the sensitivities to each drug. Cell viability was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay, and apoptosis was analyzed by flow cytometry. Cell apoptotic morphology was observed by a fluorescence microscope after DAPI (4′,6-diamidino-2-phenylindole) staining. Protein concentrations were measured by western blot.ResultsSunitinib malate showed a concentration-dependent inhibitory effect on the 5637, T24 and BIU87 cell lines with IC50’s of 1.74 μmol/L, 4.22 μmol/L, and 3.65 μmol/L, respectively. Cisplatin also exhibited good antitumor activity, but whereas sorafenib suppressed proliferation of the cells at concentrations of 10 μmol/L or higher, there was practically no response at lower concentrations. Sunitinib malate treatment resulted in an accumulation of cells in the sub-G1 phase, especially with the T24 and BIU87 cell lines, which induced apoptosis of the cells.ConclusionsSunitinib malate exerted marked inhibitory activity against bladder cancer cells. The cell growth inhibitory effect of the drug was related to induction of apoptosis. These results suggest that clinical application of sunitinib-based therapy for advanced bladder cancer is possible.

SiRNA knockdown of the DEK nuclear protein mRNA enhances apoptosis and chemosensitivity of canine transitional cell carcinoma cells

Transitional cell carcinoma (TCC) in dogs is an aggressive malignant neoplasm, originating in the epithelium of the urinary bladder. The DEK nuclear protein is overexpressed in several types of human bladder cancer, where it is involved in chromatin reconstruction, gene transcription and apoptosis. Since DEK represents a potential therapeutic target for canine TCC, this study was designed to investigate DEK expression in canine TCC and to determine the effects of DEK mRNA silencing on TCC cells in vitro. The gene expression profiles of seven selected cancer-associated genes was assessed in four canine TCC cell lines and expression of DEK protein was evaluated in bladder tissue biopsies from healthy dogs and those affected with cystitis or TCC. After transfection of four canine TCC cell lines with DEK-specific or scrambled siRNA, annexin V staining was performed to evaluate apoptosis, and methylthiazole tetrazolium assays were performed to assess both cell viability and sensitivity to carboplatin.DEK mRNA expression was relatively high in canine TCC cells and expression of the DEK protein was significantly greater in TCC tumours compared with the other tissue samples. After transfection with DEK-specific siRNA, apoptosis, cell growth inhibition, and enhanced sensitivity to carboplatin were observed in all TCC cells assessed. These research findings suggest that DEK could be a potential therapeutic target for canine TCC.

Overexpression of flotillin-1 is involved in proliferation and recurrence of bladder transitional cell carcinoma.

Flotillin-1 (FLOT1) is known to have a role in tumorigenesis; however, the effect of FLOT1 on proliferation and recurrence of human transitional cell carcinoma (TCC) is unclear. Samples from 156 TCC patients and 142 patients undergoing open bladder surgery for indications other than TCC were used in the present study. FLOT1 protein expression was determined by immunohistochemistry and western blot analysis, and mRNA expression was detected by RT-PCR and real-time PCR. A FLOT1-expressing pcDEF3 vector was stably transfected into 4 TCC cell lines and FLOT1 expression was decreased by RNAi. Proliferative analysis of TCC cells was detected by the WST-1 assay and a xenograft model using BALB/C nude mice. The association between FLOT1 expression and TCC recurrence was also analyzed by adhesion, migration and invasion assays. FLOT1 expression in TCC was significantly overexpressed compared to normal urothelial tissue, and the level of FLOT1 expression was significantly correlated with tumor size, pathologic grade, clinical stage and recurrence. In addition, FLOT1 significantly increased the proliferative ability of TCC cells in vitro and in vivo. TCC cells with a high level of FLOT1 expression exhibited a higher level of adhesion, migration and invasion. FLOT1 expression was shown to be upregulated in human TCC. These findings suggest that FLOT1 plays an important role in the proliferation and recurrence of TCC and that silencing FLOT1 expression might be a novel therapeutic strategy.