The Expression Of Glutamate Cysteine Ligase In Bladder Cancer

BackgroundBladder Urothelial carcinoma (BUC) is one of the most common malignan t tumors of the urinary system, and its incidence is increasing in recent years. Despite extensive clinical trials, the pathogenesis and determination of prognosi s for patients with BUC has remained poor. There are numerous components t hat make up the tumor environment each of which allows for an advantageous environment for survival. Oxidative stress has also been linked to the progress ion of mutations and cancer. In the case of a cancer cell, a significant imbalan ce between ROS/RNS production and antioxidant defense can explain all findin gs associating with tumor growth and a state of high oxidative stress i.e. loss of redox homeostasis. Hypoxia is usually a hallmark of solid tumors. Increased GSH contents have been observed in a number of different human cancer tiss ues including, breast, brain, colon, pancreas, lungs, head and neck cancer and1eukemia. High intracellular GSH levels are important contributors to pathologie s such as, cellular transformation and resistance to radiation and antineoplastic treatments in cancer cells. It is believed that Bladder Urothelial carcinoma (BU C) of the clear cell type also belongs to tumors in which signifiant changes oc cur in cellular redox balance. Thus, in BUC marked oxidative alterations of lip ids, proteins and DNA have been found. Higher GCLC levels were associated with lower presence of intracellular ROS and interestingly also lower rates of cell proliferation. GSH is synthesized de novo in a two-step process catalyzed by glutamate cysteine ligase (GCL, EC6.3.2.2), formerly known as gamma-glu tamylcysteine synthase (GCS), and GSH synthase (GS, EC6.3.2.3). GCL is a heterodimeric enzyme, consisting of a catalytic subunit, GCLc, and a modulator y subunit, GCLm, which are encoded by two distinct genes. GSH can be depl eted by specifically downregulating GCL levels using hammerhead ribozyme. R ibozymes has been shown to deplete GSH levels by inhibiting both GCLC and GCLM and enhance anticancer drug sensitivity.ObjectiveThe objective of the current study was to evaluate the change of glutamate cysteine ligase expression and activity in BUC patients. We detect the gene and protein expression of GCLc and GCLm in bladder cancer tissues through quantitative real-time PCR and western blotting, and analyze the GCL activity between bladder cancer and control groups.MethodsWe examined the protein expression levels of GCL subunits (catalytic subunit, GCLc, and modulatory subunit, GCLm) and GCL activity in bladder cancer tissue.46patients fulfilling the criteria of WHO revised in2004were enrolled. Tumor tissue and adjacent tissue were sampled from all the subjects through surgical operation. Semi-quantative real time-PCR (qRT-PCR) was used to assay the mRNA expression of GCLc and GCLm subunit in bladder cancer and control groups. Protein contents of GCLc and GCLm subunit in bladder cancer and control groups were determined by Western blotting. GCL activity was analyzed by a fluorescence assay. Statistical analysis was performed in the software SPSS13.0(SPSS, Inc., Chicago, IL, USA). Data were expressed as the mean±standard deviation. The difference between subject groups was analyzed using student’s t-test independently.ResultsThe results indicated that the mRNA expression of GCLc and GCLm subunit in bladder cancer has a significant decrease compared with control para-carcinoma tissue groups through Semi-quantative real time-PCR (GCLc:BUC group1±0.028, Control groups3.74±0.545; p<0.01)(GCLm:BUC group1±0.05, Control groups3.07±0.571; p<0.01). The relative contents of GCL in tumor tissue of BUC was significantly declined than the values seen in the adjacent control tissue (GCLc:BUC group0.983±0.022, Control groups1.248±0.076; p<0.05)(GCLm:BUC group1.103±0.014, Control groups1.306±0.033; p<0.05). The average GCL activity in tumor tissue from the BUC patients was331±123mmol/min/mg protein, which significantly increased compared with adjacent normal tissue (96±25mmol/min/mg protein)(p<0.01).ConclusionsWe demonstrated that protein expression of GCL catalytic subunit and modulatory subunit decreased significantly in tumor tissue of BUC patients, and GCL activity also decreased significantly in tumor tissue from BUC patients. These results indicate that the changed expression and enzymatic activity of GCL is closely associated with BUC and suggest an important role for GSH in the pathogenesis of BUC. More mechanistic studies in vitro and in vivo are required for addressing how the interplay between glutathione and pathogenesis may lead to a break intolerance and aggressiveness of BUC disease.