Reactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression. In the present research, we examined the pathobiological role of NADPH oxidase (NOX)4-mediated generation of reactive oxygen species (ROS) in urothelial carcinoma (UC) of the urinary bladder, and demonstrated the utility of ROS labeling in urine cytology.
NOX4 gene was silenced in vivo and in vitro by NOX4 siRNA transfection with or without atlocollagen. Cell cycle and measurement of ROS were analyzed by flowcytometry. Orthotopic implantation animal model was used in vivo experiment. NOX4 expression in urothelial carcinoma cells was observed by immunohistochemical analysis using surgical specimens of human bladder cancer. Urine cytology was performed after treatment with ROS detection reagents in addition to Papanicolaou staining.
NOX4 was overexpressed in several UC cell lines and the NOX inhibitor, diphenylene iodonium reduced intracellular ROS and induced p16-dependent cell cycle arrest at the G1 phase. Moreover, silencing of NOX4 by siRNA significantly reduced cancer cell growth in vivo as assessed in an orthotopic mouse model. Immunohistochemistry demonstrated high expression of NOX4 in low grade/non-invasive and high grade/invasive UC including precancerous lesions such as dysplasia but not in normal urothelium. Then, we assessed the usefulness of cytological analysis of ROS producing cells in urine (ROS-C). Urine samples obtained from UC cases and normal controls were treated with fluorescent reagents labeling the hydrogen peroxide/superoxide anion and cytological atypia of ROS positive cells were analyzed. As a result, the sensitivity for detection of low grade, non-invasive UC was greatly increased (35% in conventional cytology (C-C) vs. 75% in ROS-C), and the specificity was 95%. Through ROS-C, we observed robust improvement in the accuracy of follow-up urine cytology for cases with previously diagnosed UC, especially in those with low grade/non-invasive cancer recurrence (0% in C-C vs. 64% in ROS-C).
This is the first report demonstrating that ROS generation through NOX4 contributes to an early step of urothelial carcinogenesis and cancer cell survival. In addition, cytology using ROS labeling could be a useful diagnostic tool in human bladder cancer.
mPGES-1 (microsomal prostaglandin E synthase-1) is a stimulus-inducible enzyme that functions downstream of COX (cyclo-oxygenase)-2 in the PGE2 (prostaglandin E2)-biosynthesis pathway. Although COX-2-derived PGE2 is known to play a role in the development of various tumours, the involvement of mPGES-1 in carcinogenesis has not yet been fully understood. In the present study, we used LLC (Lewis lung carcinoma) cells with mPGES-1 knockdown or overexpression, as well as mPGES-1-deficient mice to examine the roles of cancer cell- and host-associated mPGES-1 in the processes of tumorigenesis in vitro and in vivo. We found that siRNA (small interfering RNA) silencing of mPGES-1 in LLC cells decreased PGE2 synthesis markedly, accompanied by reduced cell proliferation, attenuated Matrigel™ invasiveness and increased extracellular matrix adhesion. Conversely, mPGES-1-overexpressing LLC cells showed increased proliferating and invasive capacities. When implanted subcutaneously into wild-type mice, mPGES-1-silenced cells formed smaller xenograft tumours than did control cells. Furthermore, LLC tumours grafted subcutaneously into mPGES-1-knockout mice grew more slowly than did those grafted into littermate wild-type mice, with concomitant decreases in the density of microvascular networks, the expression of pro-angiogenic vascular endothelial growth factor, and the activity of matrix metalloproteinase-2. Lung metastasis of intravenously injected LLC cells was also significantly less obvious in mPGES-1-null mice than in wild-type mice. Thus our present approaches provide unequivocal evidence for critical roles of the mPGES-1-dependent PGE2 biosynthetic pathway in both cancer cells and host microenvironments in tumour growth and metastasis.
knockout mouse; metastasis; microsomal prostaglandin E synthase-1; prostaglandin E2; tumorigenesis; COX, cyclo-oxygenase; cPGES, cytosolic prostaglandin E synthase; DMEM, Dulbecco's modified Eagle's medium; dmPGE2, 16,16-dimethyl prostaglandin E2; ECM, extracellular matrix; EP, prostaglandin E receptor; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; KD, knockdown; KO, knockout; LLC, Lewis lung carcinoma; MMP, matrix metalloproteinase; mPGES, microsomal prostaglandin E synthase; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; PGES, PGE synthase; RT, reverse transcriptase; siRNA, small interfering RNA; TBS, Tris-buffered saline; TBS-Tween, TBS containing 0.05% Tween 20; VEGF, vascular endothelial growth factor; WT, wild-type
Low nuclear expression of the RNA-binding motif protein 3 (RBM3) has previously been found to be associated with poor prognosis in several cancer forms e.g. breast, ovarian, colorectal, prostate cancer and malignant melanoma. The aim of this study was to examine the prognostic impact of RBM3 expression in urinary bladder cancer.
Immunohistochemical RBM3 expression was examined in tumours from 343 patients with urothelial bladder cancer. Chi-square and Spearman’s correlation tests were applied to explore associations between RBM3 expression and clinicopathological characteristics. The impact of RBM3 expression on disease-specific survival (DSS), 5-year overall survival (OS) and progression-free survival (PFS) was assessed by Kaplan-Meier analysis and Cox proportional hazards modelling.
Reduced nuclear RBM3 expression was significantly associated with more advanced tumour (T) stage (p <0.001) and high grade tumours (p=0.004). Negative RBM3 expression was associated with a significantly shorter DSS (HR=2.55; 95% CI 1.68-3.86)) and 5-year OS (HR=2.10; 95% CI 1.56-2.82), also in multivariable analysis (HR=1.65; 95% CI 1.07-2.53 for DSS and HR=1.54; 95% CI 1.13-2.10 for 5-year OS). In patients with Ta and T1 tumours expressing reduced RBM3 levels, Kaplan-Meier analysis revealed a significantly shorter PFS (p=0.048) and 5-year OS (p=0.006).
Loss of RBM3 expression is associated with clinically more aggressive tumours and an independent factor of poor prognosis in patients with urothelial bladder cancer and a potentially useful biomarker for treatment stratification and surveillance of disease progression.
RBM3; Urothelial bladder cancer; Prognosis
Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.
The orphan GPR87 has recently been matched with its ligand LPA, which is a lipid mediator with multiple physiological functions, including cancer cell proliferation. This study aimed to clarify the role of GPR87 in urothelial carcinoma of the bladder. GPR87 expression was assessed in seven human bladder cancer cell lines. A replication-deficient recombinant adenoviral vector expressing shRNA targeting GPR87 (Ad-shGPR87), was constructed. Gene silencing was carried out using Ad-shGPR87. Immunohistochemical analysis was performed for transurethral resection of bladder tumor samples from 71 patients with non-muscle-invasive bladder cancer. We observed GPR87 expression in five of the seven cell lines, and silencing GPR87 gene expression significantly reduced cell viability. GPR87 expression was positive in 38 (54%) of 71 tumors. Ki-67 index was associated with positive GPR87 staining status (p < 0.0001). Patients with GPR87-positive tumors had shorter intravesical recurrence-free survival than those with GPR87-negative tumors (p = 0.010). Multivariate analysis revealed that GPR87 staining status was an independent prognostic parameter for intravesical recurrence (p = 0.041). Progression from non-muscle-invasive to muscle-invasive tumor was more frequently observed in patients with GPR87-positive tumors, although this trend did not reach statistical significance (p = 0.056). These results warrant further prospective studies to clarify the role of GPR87 expression in intravesical recurrence and progression in bladder cancer.
GPR87; non-muscle-invasive bladder cancer; intravesical recurrence; progression
Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (−)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.
Celecoxib and other non-steroidal anti-inflammatory drugs (NSAIDs) are being evaluated in the prevention of bladder and other cancers. Here we investigate molecular effects of celecoxib independent of cyclooxygenase (COX)-2 expression levels in urothelial carcinoma of the bladder.
Materials and Methods
Low-grade RT-4 and high-grade UM-UC-3 bladder cancer cells were treated with 0–50 μM celecoxib. Growth, cell cycle and apoptosis were measured by crystal violet elution and flow cytometry. Western analysis was performed for COX-2, Rb, cyclin B1/D1, and phosphocyclin B1/D1. COX-2 induction was achieved with phorbol ester.
Celecoxib inhibited growth of RT-4 and UM-UC-3, with G1 cell cycle arrest and altered cyclin B1/D1 expression in RT-4, whereas Rb up-regulation occurred in UM-UC-3. Apoptosis occurred in both cell lines.
Celecoxib induces G1 cell cycle arrest in low- and high-grade bladder cancer by different pathways. This heterogeneous molecular response supports combination approaches to prevention and treatment.
Cyclooxygenase (COX)-2; celecoxib; bladder cancer; chemoprevention
Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFRs) and activating downstream signalling pathways, including RAS/MAPK, PLCγ1, PI3K, and STATs. In the last ten years, it has become clear that FGF signalling is altered in a high proportion of bladder tumours. Activating mutations and/or overexpression of FGFR3 are common in urothelial tumours with low malignant potential and low-stage and -grade urothelial carcinomas (UCs) and are associated with a lower risk of progression and better survival in some subgroups. FGFR1 is not mutated in UC, but overexpression is frequent in all grades and stages and recent data indicate a role in urothelial epithelial-mesenchymal transition. In vitro and in vivo studies have shown that FGFR inhibition has cytotoxic and/or cytostatic effects in FGFR-dependent bladder cancer cells and FGFR-targeted agents are currently being investigated in clinical studies for the treatment of UC. Urine-based tests detecting common FGFR3 mutations are also under development for surveillance of low-grade and -stage tumours and for general population screening. Overall, FGFRs hold promise as therapeutic targets, diagnostic and prognostic markers, and screening tools for early detection and clinical management of UC.
The ErbB4 receptor tyrosine kinase is expressed at high levels in human and mouse colitis, and inhibits colon epithelial cell apoptosis in the presence of pro-inflammatory cytokines. In this study, we investigated the molecular mechanisms responsible for ErbB4-induced cell survival. In cultured mouse colon epithelial cells, ErbB4 overexpression resulted in increased levels of cyclooxygenase-2 (COX-2) mRNA and protein; in contrast, ErbB4 knockdown with siRNA blocked COX-2 accumulation in response to tumor necrosis factor. While ErbB4 is expressed as up to four different isoforms in epithelial tissues, its ability to promote COX-2 expression was isoform-independent. ErbB4-stimulated COX-2 induction was associated with an increase in mRNA half-life and was blocked by inhibition of Src, phosphatidylinositol 3-kinase, or epidermal growth factor (EGF) receptor (R). Furthermore, ErbB4 expression promoted EGFR phosphorylation in the presence of heregulin, implicating ErbB4-EGFR heterodimerization in these responses. With regard to the cellular responses to ErbB4 activation, increased survival of ErbB4-expressing cells in the presence of pro-inflammatory cytokines was sensitive to the COX-2 inhibitor celecoxib. Furthermore, ErbB4-overexpressing cells acquired the ability to form colonies in soft agar, indicative of cellular transformation, also in a celecoxib-sensitive manner. Together our data indicate that ErbB4 is a key regulator of COX-2 expression and cellular survival in colon epithelial cells, acting in concert with EGFR through a Src and phosphatidylinositol 3-kinase dependent mechanism. These results suggest that chronic overexpression of ErbB4 in the context of inflammation could contribute to colitis-associated tumorigenesis by inhibiting colonocyte apoptosis.
Cell survival; colon epithelial cells; cyclooxygenase-2; ErbB4; inflammatory bowel diseases; receptor tyrosine kinases
Markers for outcome prediction in bladder cancer are urgently needed. We have previously identified a molecular signature for predicting progression in non-muscle-invasive bladder cancer. ANXA10 was one of the markers included in the signature and we now validated the prognostic relevance of ANXA10 at the protein level.
We investigated ANXA10 expression by immunohistochemistry using a tissue microarray with 249 Ta and T1 urothelial carcinomas. The expression of ANXA10 was also investigated in an additional set of 97 more advanced tumours. The functional role of ANXA10 in cell lines was investigated by siRNA-mediated ANXA10 knockdown using wound-healing assays, proliferation assays, and ingenuity pathway analysis.
Low expression of ANXA10 correlated with shorter progression-free survival in patients with stage Ta and T1 tumours (P<0.00001). Furthermore, patients with more advanced tumours and low ANXA10 expression had an unfavourable prognosis (P<0.00001). We found that ANXA10 siRNA transfected cells grew significantly faster compared with control siRNA transfected cells. Furthermore, a wound-healing assay showed that ANXA10 siRNA transfected cells spread along wound edges faster than control transfected cells.
We conclude that ANXA10 may be a clinical relevant marker for predicting outcome in both early and advanced stages of bladder cancer.
bladder cancer; biomarker; progression; metastasis; ANXA10; S100A4
Cyclooxygenase (COX)-2 has been implicated in tumour progression, angiogenesis and metastasis in non-small cell lung cancer (NSCLC). We speculated that inhibition of COX-2 activity might reduce expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in lung cancer cells.
The levels of IL-8, VEGF and prostaglandin E2 (PGE2) were measured by ELISA. Expression of COX-1 and COX-2 was determined by Western blotting. Inhibition or knockdown of COX-2 was achieved by treating NSCLC cells with specific COX-2 inhibitor NS-398 or COX-2 siRNA, respectively.
We found that NSCLC cell lines produced more IL-8 than VEGF (p < 0.001). In contrast, small cell lung cancer (SCLC) cell lines produced more VEGF than IL-8 (p < 0.001). COX-1 was expressed in all cell lines, but COX-2 was expressed only in NSCLC cell lines. Consistent with this, PGE2 was significantly higher in NSCLC cell lines than SCLC cell lines (p < 0.001). We tested these cell lines with a potent specific COX-2 inhibitor NS-398 at concentrations of 0.02, 0.2, 2, 20 μM for 24 or 48 h. The COX-2 activity was reduced in a dose-dependent fashion as shown by reduced PGE2 production. VEGF was significantly reduced following the treatment of NS-398 in A549 (by 31%) and MOR/P (by 47%) cells lines which expressing strong COX-2, but not in H460 cell line which expressing very low COX-2. However, IL-8 was not reduced in these cell lines. To confirm these results, we knocked down COX-2 expression with COX-2 siRNA in these cell lines. VEGF was significantly decreased in A549 (by 24%) and in MOR/P (by 53%), but not in H460 whereas IL-8 was not affected in any cell line.
We conclude that NSCLC cells produce much higher levels of IL-8 than SCLC cells whereas both NSCLC and SCLC cells produce similar levels of VEGF. COX-2 is only expressed in NSCLC cells, but not in SCLC cells. VEGF is produced in both NSCLC and SCLC cells regardless of COX-2 expression. However, VEGF production is, at least partly, COX-2 dependent in NSCLC cells expressing COX-2. In contrast, IL-8 production is COX-2 independent in both NSCLC and SCLC cells. We speculate that combined targeting of COX-2 and IL-8 may be useful in the treatment of patients with NSCLC and targeting VEGF may be useful in the treatment of patients with SCLC.
Cyclooxygenase2 (COX-2), one isoform of cyclooxygenase proinflammatory enzymes, is responsible for tumor development, invasion and metastasis. Due to its role and frequent overexpression in a variety of human malignancies, including osteosarcoma, COX-2 has received considerable attention. However, the function of COX-2 in the pathogenesis of cancer is not well understood. We examined the role of COX-2 in osteosarcoma.
We employed lentivirus mediated-RNA interference technology to knockdown endogenous gene COX-2 expression in human osteosarcoma cells (SaOS2) and analyzed the phenotypical changes. The effect of COX-2 treatment on the proliferation, cell cycle, invasion and migration of the SaOS2 cells were assessed using the MTT, flow cytometry, invasion and migration assays, respectively. COX-2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) mRNA and protein expression were detected by RT-PCR and western blotting.
Our results indicate that a decrease of COX-2 expression in human osteosarcoma cells significantly inhibited the growth, decreased the invasion and migration ability of SaOS2 cells. In addition, it also reduced VEGF, EGF and bFGF mRNA and protein expression.
The COX-2 signaling pathway may provide a novel therapeutic target for the treatment of human osteosarcoma.
Rationale: Th17 cells comprise a distinct lineage of proinflammatory T helper cells that are major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived eicosanoids regulate Th17 cells during allergic lung inflammation.
Objectives: To determine the role of COX metabolites in regulating Th17 cell differentiation and function during allergic lung inflammation.
Methods: COX-1−/−, COX-2−/−, and wild-type mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th17 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time polymerase chain reaction, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown.
Measurements and Main Results: Th17 cell differentiation in lung, lymph nodes, and bronchoalveolar lavage fluid was significantly lower in COX-2−/− mice after ovalbumin sensitization and exposure in vivo. In vitro studies revealed significantly impaired Th17 cell differentiation of COX-2−/− naive CD4+ T cells with decreased Stat3 phosphorylation and RORγt expression. Synthetic PGF2α and PGI2 enhanced Th17 cell differentiation of COX-2−/− CD4+ T cells in vitro. The selective COX-2 inhibitor, NS-398, and PGF2α receptor and PGI2 receptor siRNA knockdown significantly decreased Th17 cell differentiation in vitro. Administration of synthetic PGs restored accumulation of Th17 cells in lungs of allergic COX-2−/− mice in vivo.
Conclusions: COX-2 is a critical regulator of Th17 cell differentiation during allergic lung inflammation via autocrine signaling of PGI2 and PGF2α through their respective cell surface receptors.
Th17 cell; COX-2; asthma; prostaglandins; IL-17
The prognosis of pancreatic cancer is still very poor. No specific effective gene therapy for pancreatic cancer has been found. As a key enzyme of the metabolic process of arachidonic acid, cyclooxygenase-2 (COX-2) has been found to be closely related to the tumorigenesis of epithelial cancers. However, the antitumor effect of small interfering RNA (siRNA) targeting COX-2 in pancreatic cancer has not yet been verified. Therefore, the aim of this study was to investigate the effects of COX-2 gene silencing by siRNA on cell proliferation, cell apoptosis, cell cycle and tumorigenicity of pancreatic cancer cells. COX-2 mRNA was detected by RT-PCR and real-time PCR. COX-2 protein was detected by Western blotting. The cell proliferation was measured by cell counting using microscopy. The cell apoptosis and cell cycle were measured by flow cytometry. The tumorigenicity of Capan-2 pancreatic cancer cells transfected with COX-2 siRNA was evaluated using a nude mouse xenograft model. The expression of COX-2 mRNA as well as COX-2 protein were downregulated after COX-2 siRNA transfection. COX-2 siRNA could inhibit the growth of Capan-2 cells significantly by decreasing the cell proliferation, increasing cell apoptosis and regulating cell cycle as well. In vivo experiments demonstrated that the mean volume and weight of subcutaneous xenografts in nude mice derived from Capan-2 cells transfected with COX-2 siRNA were significantly decreased. COX-2 siRNA could inhibit the growth of Capan-2 pancreatic cancer cells and also decrease the tumorigenicity of Capan-2 cells, implicating a new potential therapeutic target in pancreatic cancer.
pancreatic neoplasm; cyclooxygenase-2; RNA interference; cell proliferation; apoptosis; cell cycle
The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with <1% of cells displaying detectable nuclear localisation of plakoglobin. siRNA knockdown experiments targeting plakoglobin, revealed enhanced migration in all cell lines in the presence and absence of E-cadherin expression. In bladder cell lines expressing low levels of plakoglobin and desmoglein-2, elevated levels of desmoglein-2 were detected following restoration of plakoglobin expression in transfected cell lines. Analysis of wnt signalling revealed no activation event associated with plakoglobin expression in the bladder model. These results show that plakoglobin acts as a tumour suppressor gene in bladder carcinoma cells and the silencing of plakoglobin gene expression in late-stage bladder cancer is a primary event in tumour progression.
plakoglobin; bladder cancer; desmosome; signalling
AIM: To investigate the mechanisms of how cyclooxygenase-2 (COX-2) regulates E-cadherin in gastric cancer cells.
METHODS: COX-2 expression in human gastric cancer cell lines SGC-7901, BGC-823, MGC-803 and AGS were measured at the mRNA and protein level. COX-2 rich cell line SGC-7901 was chosen for subsequent experiments. siRNA mediated gene knockdown was used to investigate the impact of COX-2 on nuclear factor-κB (NF-κB), Snail, and E-cadherin in gastric cancer cells. Gene expression was determined by Western blot and real-time polymerase chain reaction. To analyze whether NF-κB inhibition could interrupt the modulatory effect of COX-2 or prostaglandin E2 (PGE2) on E-cadherin, gastric cancer cells were treated with celecoxib or PGE2, in the presence of NF-κB specific siRNA.
RESULTS: Highest expression level of COX-2 was found in SGC-7901 cells, both at mRNA and protein levels. siRNA mediated down-regulation of COX-2 led to a reduced expression of NF-κB and Snail, but an increased expression of E-cadherin in SGC-7901 cells. siRNA mediated down-regulation of NF-κB also led to a reduced expression of E-cadherin and Snail in SGC-7901 cells. However, COX-2 expression did not alter after cells were treated with NF-κB specific siRNA in SGC-7901 cells. Treatment of SGC-7901 cells with celecoxib led to a reduced expression of Snail but an increased expression of E-cadherin. In contrast, treatment of SGC-7901 cells with PGE2 led to an increased Snail and a decreased E-cadherin. However, siRNA-mediated knockdown of NF-κB partially abolished the effect of celecoxib and PGE2 on the regulation of E-cadherin and Snail in SGC-7901 cells.
CONCLUSION: COX-2 likely functions upstream of NF-κB and regulates the expression of E-cadherin via NF-κB/Snail signaling pathway in gastric cancer cells.
Cyclooxygenase-2; E-cadherin; celecoxib; Prostaglandin E2; Gastric cancer
Cyclooxygenase-2 (COX-2) enzyme has been involved in the tumorigenesis and in the progression of colorectal cancer (CRC). The use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) or selective COX-2 inhibitors has been proposed for the prevention and the treatment of this relevant neoplastic disease. In the light of an innovative alternative to these pharmacological approaches, we review here the possible strategies to achieve a strong and selective inhibition of COX-2 enzyme by using the mechanism of RNA Interference (RNAi) targeted against its mRNA. Anti-COX-2 siRNA molecules (siCOX-2) can be generated in CRC cells from short hairpin RNA (shRNA) precursors, delivered in vitro by a retroviral expression system, and induce a significant and stable silencing of overexpressed COX-2 in human colon cancer cells. As a safer alternative to viral approach, nonpathogenic bacteria (E. coli) can be engineered to invade eukaryotic cells and to generate siCOX-2 molecules in cancer cells. Moreover, the involvement of miRNAs in COX-2 posttranscriptional regulation opens up the possibility to exploit an endogenous silencing mechanism to knockdown overexpressed COX-2. Thus, these recent strategies disclose new challenging perspectives for the development of clinically compatible siRNA or miRNA capable of selectively inhibiting COX-2 enzyme.
Inflammation may activate stem cells via prostaglandin E2 (PGE2) production mediated by cyclooxygenase-2 (COX-2) expression. We performed an immunohistochemical analysis of the expression of stemness markers (Oct3/4 and CD44v6) and COX-2 in urinary bladder tissues obtained from cystitis and cancer patients with and without Schistosoma haematobium infections. Immunoreactivity to Oct3/4 was significantly higher in S. haematobium-associated cystitis and cancer tissues than in normal tissues. CD44v6 expression was significantly higher in bladder cancer without S. haematobium than in normal tissues. COX-2 was located in the cytoplasmic membrane, cytoplasm, and nucleus of the cancer cells. Interestingly, the nuclear localization of COX-2, which was reported to function as a transcription factor, was significantly associated with the upregulation of Oct3/4 and CD44v6 in bladder cancer tissues with and without S. haematobium infection, respectively. COX-2 activation may be involved in inflammation-mediated stem cell proliferation/differentiation in urinary bladder carcinogenesis.
Overexpression of cyclooxygenase (COX-2) is commonly observed in human cancers. In a murine model of metastatic breast cancer, we observed that COX-2 expression and enzyme activity were associated with enhanced tumorigenic and metastatic potential. In contrast to the high COX-2 expression in metastatic tumors, transplantation of poorly tumorigenic tumor cell lines to syngeneic mice results in less COX-2 expression and less COX-2 activity in vivo. Aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in human cancer cell lines and in some human tumors of the gastrointestinal tract.
Using bisulfite modification and a methylation-specific PCR, we examined the methylation status of the COX-2 promoter in a series of four closely-related murine mammary tumors differing in COX-2 expression and metastatic potential.
We showed that line 410, which does not express COX-2 in vivo, exhibited evidence of promoter methylation. Interestingly, the metastatic counterpart of this cell (line 410.4) displayed only the unmethylated COX-2 promoter, as did two additional cell lines (lines 66.1 and 67). The methylation patterns observed in vitro were maintained when these murine mammary tumor lines were transplanted to syngeneic mice. Treatment with the DNA demethylating agent 5-aza-deoxycytidine increased COX-2 mRNA, increased protein and increased enzyme activity (prostaglandin synthesis).
These results indicate that COX-2 promoter methylation may be one mechanism by which tumor cells regulate COX-2 expression. Upregulation of COX-2 expression in closely related metastatic lesions versus nonmetastatic lesions may represent a shift towards the unmethylated phenotype.
cyclooxygenase; promoter methylation; breast cancer
Regulator of Cullins-1 (ROC1) is a key subunit in the Cullin-RING ligase (CRL) protein complex. Overexpression of ROC1 protein is associated with tumor progression and poor prognosis of non-muscle invasive bladder transitional cell carcinoma (NMIBC). This study was designed to assess the effects of ROC1 knockdown in bladder cancer cells and to determine the potential mechanisms involved. A total of 112 bladder cancer tissue specimens were recruited for immunohistochemical analyses of ROC1 overexpression. Bladder cancer cell lines were used to knockdown ROC1 expression using ROC1 siRNA. Our data showed that ROC1 knockdown remarkably inhibited bladder cancer cell growth, arrested cells at the G2 phase of the cell cycle, and induced the p53-dependent cell senescence. Molecularly, G2 arrest was associated with upregulation of p21, p27, cyclin B1, and Cdc2 proteins. ROC1 knockdown induced-senescence functioned through p53/p21 pathway. Knockdown of p21 expression partially rescued ROC1 knockdown-induced growth inhibition in cancer cells. Furthermore, nude mouse xenograft analyses confirmed these in vitro data. In conclusion, data from the current study indicate that ROC1 plays an essential role in bladder cancer progression and could serve as a novel anticancer target for bladder transitional cell carcinoma (BTCC).
The objective of this study was to analyze the expression profile of the angiogenic components, vascular endothelial growth factor-A (VEGFA), basic fibroblast growth factor-2 (FGF2), osteopontin (OPN) and ras homolog gene family, member C (RHOC), in urothelial cell carcinoma (UCC) of the urinary bladder and to examine their role as candidate diagnostic biomarkers. Using qPCR, 77 samples of UCC of the urinary bladder and 77 matched tumor-associated normal samples were investigated to determine the expression of the four angiogenic components. The correlation between gene expression, patient survival and pathological features of the tumors was also examined. The VEGFA and OPN transcript levels were greater in the bladder cancer tissue than in the normal urothelium (P<0.001). Patients with higher VEGFA mRNA levels showed a tendency towards shorter cancer-specific survival. OPN levels showed a gradual increase, the lowest levels being found in non-invasive carcinoma and the highest in muscle invasive tumors. Elevated OPN levels indicated poor prognosis in connection with advanced disease stage (P<0.001). Both superficially invasive and muscle invasive tumors had significantly higher FGF2 levels compared to the control tissues (P=0.018 and P=0.050, respectively). Moreover, FGF2 was significantly higher in the metastatic vs. the non-metastatic tumors (P=0.0097). FGF2 levels exhibited a trend towards a correlation with worse patient survival. RHOC mRNA levels were higher in muscle invasive compared to superficially invasive tumors, as well as in grade III vs. grade I/II tumors. Furthermore, we detected worse overall survival for patients with high RHOC expression levels. VEGFA and FGF2 exhibited the best linear combination in the ROC curves for specificity and sensitivity. Thus, VEGFA and FGF2 may serve as candidate biomarkers for diagnostic purposes. Higher OPN expression may be used as a potential biomarker to predict patient survival relative to advanced tumor stage. However, further studies are required to investigate its role in urinary bladder carcinogenesis.
angiogenesis; VEGFA; FGF2; OPN; RHOC; urinary bladder
The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic infiltration, and COX-2 expression and survival was examined in patients with transitional cell carcinoma of the urinary bladder (n=103). Sixty-one patients had superficial disease and 42 patients had invasive disease. Cancer-specific survival was shorter in those patients with invasive compared with superficial bladder cancer (P<0.001). On univariate analysis, stratified by stage, increased Ki-67 labelling index (P<0.05), increased COX-2 expression (P<0.05), C-reactive protein (P<0.05) and adjuvant therapy (P<0.01) were associated with poorer cancer-specific survival. On multivariate analysis of these significant factors, stratified by stage, only C-reactive protein (HR 2.89, 95% CI 1.42–5.91, P=0.004) and adjuvant therapy (HR 0.29, 95% CI 0.14–0.62, P=0.001) were independently associated with poorer cancer-specific survival. These results would suggest that tumour-based factors such as grade, COX-2 expression or T-lymphocytic infiltration are subordinate to systemic factors such as C-reactive protein in determining survival in patients with transitional cell carcinoma of the urinary bladder.
bladder cancer; Ki-67; C-reactive protein; T-lymphocytes; COX-2; survival
Although the anti-tumour effect of cyclooxygenase-2 (Cox-2) inhibitors in invasive bladder cancer has been confirmed, its mechanisms of action are unclear. Recently, the concept of an epithelial-to-mesenchymal transition (EMT) promoting carcinoma progression has been suggested, and a key feature of the EMT is the downregulation of E-cadherin. In this study, we investigated the effect of Cox-2 inhibitors on reversal EMT and tumour growth inhibition in bladder cancer cells.
We used three Cox-2 inhibitors, etodolac, celecoxib and NS-398 and three human bladder cancer cell lines, T24, 5637 and KK47, in this study. T24 xenograft tumour mouse model was used in the in vivo study.
Within the clinical drug concentrations, only etodolac showed the in vitro growth inhibition in T24 not in the other cell lines. Etodolac reduced SNAIL mRNA and vimentin cell surface expression, and induced E-cadherin mRNA and E-cadherin cell surface expression, in T24. Etodolac also most strongly inhibited the cell migration of T24 in vitro and showed the highest tumour growth inhibition in T24 tumour in vivo.
Etodolac at clinical doses exhibited induced in vitro and in vivo anti-tumour effects and reversal effect of EMT in T24. These results suggest that etodolac is a good candidate for an anti-tumour or chemopreventive reagent for high-grade bladder cancer.
Cox-2 inhibitor; E-cadherin; SNAIL; EMT; bladder cancer
Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required.
We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi).
A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects.
Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.
COX-2; CRC; RNAi; E. coli
Light microscopic evaluation of cell morphology in preparations from urine or bladder washing containing exfoliated cells is a standard and primary method for the detection of bladder cancer and also malignancy from other parts of the urinary tract. The cytopathologic examination is a valuable method to detect an early recurrence of malignancy or new primary carcinoma during the follow-up of patients after the treatment of bladder cancer.
Characteristic cellular and nuclear signs of malignancy indicate invasive or in situ urothelial carcinoma or high-grade papillary urothelial carcinoma. However, low sensitivity of the method reflects the unreliable cytopathologic diagnosis of low-grade urothelial neoplasms as cellular and nuclear signs of malignancy in these neoplasms are poorly manifested. Many different markers were developed to improve the diagnosis of bladder carcinoma on urinary samples. UroVysion™ test is among the newest and most promising tests. By the method of in situ hybridization one can detect specific cytogenetic changes of urothelial carcinoma.
cytology; urine; bladder washing; urothelial carcinoma