Suppression Of COX-2 Sensitizes Human Hepatoma Cells To Endoplasmic Reticulum Stress-induced Apoptosis

Background The endoplasmic reticulum (ER) stress response consists of a set of adaptive pathways that can be triggered by disparate perturbations of normal ER function, such as accumulation of unfolded proteins, lipid or glycolipid imbalances, or changes in the ionic conditions of the ER lumen. The primary purpose of the ER stress is to alleviate the stressful disturbance and restore proper ER homeostasis; however, in the case of intense or persistent ER stress, these pathways will trigger programmed cell death or apoptosis. It was reported that ER stress induced insignificant apoptosis in tumor cells, which indicated that tumor cells could adapt to ER stress and were resisted to the ER stress-induced apoptosis.Resistance of cell to apoptosis is one feature of hepatocellular carcinoma (HCC), which play an important role in hepatocarcinogenesis. Many evidences suggest the pivotal role for cyclooxygenase-2 (COX-2) in apoptotic resistance. Upregulation of COX-2 has been demonstrated in various tumor tissues such as colorectal, gastric, esophageal, pancreatic and lung cancers including HCC. Interestingly, Hang et al.founded that, the ER stress induced by TM and brefeldin A results in increased expression of COX-2 in breast cancer cell line MCF-7. The present study was designed to investigate the possible mechanism that hunman hepatoma cell line HepG2 resisted to the ER stress-induced apoptosis, in order to develop an effective therapy for HCC. Objective:To clarify the changes of COX-2 expression after hepatoma carcinoma cell in response to ERS and the effect on apoptosis.Methods:Cell viability was analyzed by the MTT assay. Cell apoptosis was evaluated by TUNEL method and flow cytometry. Glucose regulated protein78 (GRP78) and cycloxygenase-2 (COX-2) expressions were measured by western bloting.Results1. The changes of cell proliferation after hepatoma carcinoma cell and hepatocyte cell under ERS Hepatoma carcinoma cell line HepG2 and normal hepatocyte cell line HL-7702 cells were treated with different concentrations of TM for 48 h and cell survival was analyzed by conventional MTT assays. TM insignificantly reduced viability of HepG2 cells, HL-7702 cells displayed more sensitive to ERS, the inhibitory rate was remarkly increased on HL-7702 cells.2. The changes of apoptosis after hepatoma carcinoma cell and hepatocyte cell under ERS2.1 The morphological change of apoptosis followed by TUNEL Morphological evidence of apoptosis was assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL). The typically apoptotic changes include chromatin condensation and deformed and fragmented nuclei. The results suggested that the proliferation was prosperous in control group, and there was no significently change when HepG2 cells under ERS. However, the cell density was decreased significantly and the number of apoptotic cells increased obviously in HL-7702 cells.2.2 The apoptotic rate assay by FCM A Flow Cytometry (FCM) assay was performed to analyze apoptotic rate. The sub-G1 peak, which appeared before the G0/G1 phase that represents apoptotic cell population,'was slightly increased in HepG2 cells in response to ERS. The apoptotic peak was dramatically increased when HL-7702 cells were in response to ERS.3.The changes of COX-2 expression after hepatocellular carcinoma in response to ERSThe expression of COX-2 and GRP78 were assayed by Western Blot. HepG2 cells were treated with 3unol/L TM to induce ERS for 3,6,9,12,24 h, and the expression of COX-2 was determined by Western Blot. The expression of GRP78, an unfolded protein response chaperone, was enhanced in response to ERS. protein expression of COX-2 gradually increased with the TM treatment time prlonged, while this effect was not detected in HL-7702 cells.4. Effect of celecoxib on cell proliferation of human hepatoma cells under ERSA series of concentration of celecoxib (5,10,20,40umol/L) on HepG2 cells under ERS, and cell proliferation inhibiting rate of MTT were 333.58±2.91%,44.72±2.84%,68.92±2.23% and 84.83±0.17%, it was significant when the celecoxib in concentration of lOumol/L.5. Effect of celecoxib on apoptosis of human hepatoma cells under ERS Selected 10umol/L celecoxib and 3umol/L TM for research. TUNEL results indicated that, the apoptosis index of HepG2 cells was significant increased after celecoxib treatment. Besides, the FCM results showed that, the apoptotic peak was significant increased after celecoxib treatment, and the apoptotic rate was 30.57±3.56%. But celecoxib without obvious effect on HL-7702 cells.6. Effect of celecoxib on expression of COX-2By the combined action of 10umol/L celecoxib and 3umol/L TM for 24h, celecoxib inhibited the expression of COX-2.Conclusion1. COX-2 plays an important role in the resistance of hepatoma carcinoma cells to ERS induced apoptosis.2. Suppression of COX-2 can sensitize hepatoma carcinoma cells to ERS induced apoptosis.