The Effect Of AMPK Activation On COX-2 Expression In Colon Cancer

Background and ObjectiveColon cancer is one of the most common malignant tumors in alimentary system. Recurrence and metastasis due to drug resistance remain major obstacles. It has been proposed that COX-2 expression is increased in chemo-resistant colon cancer cells, and COX-2 inhibition can result in increasing drug chemosensitivity. COX-2 (Cyclooxygenase-2)is a key enzyme of prostaglanding synthesis, and recent studies show that some plants'extraction which activate AMPK can decrease the intracellular synthesis of prostaglanding, so it is supposed to have an anticancer effect through COX-2 signaling pathways. AMPK(AMP-activated protein kinase) is a kind of serine/threonine protein kinase and recently has been shown to inhibit cell proliferation in low energy state. But the role of AMPK in tumor is still unknown,and the relationship between AMPK activation and COX-2 expression is also rarely studied. So we detect the effect of AMPK activation on COX-2 expression in order to look for its contribution in clinic.Methods1. Immunohistochemical method was applied to detect the activation of AMPK and the expression of COX-2 in colon cancer tissues. Meanwhile, the relationship between them and clinical pathology characteristics was analyzed statistically.2. Colon cancer cell line HT-29 were treated with AICAR, and western blot was used to investigate the influence of AMPK activation on the expression of COX-2.3. Colon cancer cell line HT-29 were treated by AICAR and celecoxib separately in different dose and time. Then morphological assessment was performed with microscope and cell survival rate was measured by MTT assay.4. AO staining was used to observe HT-29 cells apoptosis in trentment of AICAR and celecoxib, and cell apoptosis rate was detected by FCM assay.5. AICAR and celecoxib were combined to treat HT-29 cells. Then cell survival rate of different groups was measured by MTT assay. Results1. P-ACC and COX-2 protein were both stained positively in cytoplasm by immunohistochemistry assay. P-ACC was much lower in serosa infiltration group than that in non-infiltration group(p<0.01). COX-2 was much higher in serosa infiltration group than that in non-infiltration group(p<0.01), and also be higher both in lymphnode metastasis group and Dukes C~D group(p<0.05). The data suggested that P-ACC and COX-2 were negative correlated(r=0.655,p=0.001).2. Based on western blot result, the data showed that COX-2 expression of HT-29 cells decreased when AMPK was activated.3. With the increasing concentration of AICAR and celecoxib, cell population decreased gradually and there were some changes in cellular morphous. AICAR and celecoxib both inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner.4. Apoptotic cells were observed with fluorescence microscope after acridine orange staining both in trentment of AICAR and celecoxib. And the cell apoptosis rate both in AICAR group and celecoxib group was much higher than that in control group(p<0.05).5. Cell survival rate was 23.0±0.82% when celecoxib was combined with AICAR. And it was lower both than that in AICAR group and celecoxib group(p<0.05).ConclusionAfter studying the effect of AMPK activation on COX-2 expression and its role in colon cancer, we hypothesized that1. AMPK activation and COX-2 expression were both closely correlated with serosa infiltration. Human colon cancer with inactivation of AMPK and activation of COX-2 has more invasion potential. AMPK activation and COX-2 expression were negative correlated.2. In colon cancer cell line HT-29, AMPK activation could result in COX-2 inhibition.3. AMPK activation could inhibit HT-29 cells'growth and proliferation.4. AMPK activation could induce HT-29 cells'apoptosis.5. AMPK activator and selective COX-2 inhibitor were both proliferation-inhibited and apoptosis-induced to HT-29 cells. Combination with AMPK activator and selective COX-2 inhibitor resulted in synergism.