Upregulation of TRAG3 gene in urothelial carcinoma of the bladder

Abstract

Conventional chemotherapy is commonly used for advanced stages of bladder cancer with modest success and high morbidity. Identifying markers of resistance will allow clinicians to tailor treatment to a specific patient population. T24-tumorigenic cell line was grown orthotopically in nude mice and monitored using bioluminescence imaging and microcomputed tomography until they developed metastases. Stable sublines were then developed from primary bladder (T24-P), lung (T24-L) and bone (T24-B) tissues. Chromosomal analysis and DNA microarray were used to characterize these sublines. Real-time quantitative polymerase chain reaction and immunohistochemistry were used for validation. Epigenetic modifiers were used to study gene regulation. The cell viability was quantified with MTT assay. Chromosomal analysis revealed multiple alterations in metastatic cell lines compared to T24-P. DNA microarray analysis showed that taxol resistance-associated gene (TRAG) 3 was the most upregulated gene. From real-time quantitative polymerase chain reaction and immunohistochemistry, TRAG3 was significantly higher in T24-L and T24-B than T24-P. TRAG3 gene expression is likely controlled by DNA methylation but not histone acetylation. Interestingly, T24-B and T24-L cells were more resistant than T24-P to treatment with antimicrotubule agents such as docetaxel, paclitaxel and vinblastine. TRAG3 mRNA expression was higher in 20% of patients with pT2 (n 5 10) and 60% of patients with !pT3 (n 5 20) compared to normal adjacent tissue (p 5 0.05). In addition, the median TRAG3 expression was 6.7-fold higher in !pT3 tumors compared to pT2 tumors. Knowing the status of TRAG3 expression could help clinicians tailor treatment to a particular patient population that could benefit from treatment, while allocating patients with resistant tumors to new experimental therapies.In the United States, urothelial carcinoma of the bladder (UCB) is estimated to affect 68,810 new patients, with 14,100 deaths resulting in 2008.1 Conventional cytotoxic chemotherapy is commonly used as a main regimen for advanced stages of bladder cancer.2 However, frequent appearance of drug resistance due to tumor heterogeneity and serious side effects from current treatment regimens have made treatment outcomes unsatisfactory. It is imperative to identify specific patient populations where particular chemotherapeutic regimens can achieve better therapeutic efficacy, while decreasing the morbidity and mortality related to UCB.Toward this goal, we designed experiments to study cytogenetic properties and differential gene expression in primary and metastatic UCB cells. We developed a bladder tumor model using nude mice by instilling with human UCB cells orthotopically; the mice initially developed superficial tumors, which are most frequently diagnosed in patients. Subsequently, these tumors became invasive. Bioluminescence imaging (BLI) and microcomputed tomography (micro-CT)

HIF-1α promotes ZEB1 expression and EMT in a human bladder cancer lung metastasis animal model

“…All components for cell culture were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). T24-parental (P) and T24-lung (L) bladder cancer cells were obtained from primary tumors and lung metastases according to a protocol previously described ( 10 , 11 ). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific Inc.) and 400 mg/l G418 in a humidified incubator with 5% CO 2 , 95% air at 37°C.…”

“…To address these issues, an orthotopic animal model was established by injecting human bladder cancer T24-tumorigenic (T24-t) cells into a mouse bladder. Subsequently, the primary tumor and lung metastases were excised and plated on tissue culture dishes with G418 (400 µg/ml) to derive the sublines T24-parental (T24-P) and T24-t-lung (T24-L), which were outlined in our previous study ( 10 , 11 ). In the present study, the previously described sublines T24-P and T24-L, were used to investigate the role of downstream gene regulation of HIF-1α and EMT by mimicking human bladder cancer metastasis ( 10 ).…”

Inhibition of REDD1 Sensitizes Bladder Urothelial Carcinoma to Paclitaxel by Inhibiting Autophagy

“…Recent phase III clinical trials have found that a PCG (paclitaxel plus cisplatin plus gemcitabine) regimen, consisting of the addition of paclitaxel to a standard chemotherapy regimen of cisplatin plus gemcitabine (GC), can improve the overall response rate and overall survival rate of patients with BUC (7). However, acquired or de novo resistance to paclitaxel limits its clinical application in the treatment of BUC, and the identification of approaches to sensitize BUC to paclitaxel remains a significant clinical challenge (8,9).…”

Inhibiting bladder tumor growth with a cell penetrating R11 peptide derived from the p53 C-terminus

“…The human UBC cell lines (T24 and 253J) and the immortalized bladder epithelial cell line SV-HUC were obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum in a humidified incubator at 37°C with 5% CO 2 . T24-luc sublines were generated after stable transfection with the luciferase gene vector and G418 selection as our previous reports [ 26 ] and maintained in DMEM supplemented with 5% fetal bovine serum.…”

“…For the orthotopic model, intravesical tumor implantation of T24-luc cells was performed as previously described [ 26 , 27 ]. Distant metastatic tumors were developed by intravenously injection of 0.5 × 10 6 T24-luc cells in a final volume of 100 μL saline.…”