The Effect Of NS-398 On Apoptosis And Expression Of Associated Molecules Of Human Colorectal Carcinoma HCT-8 Cell

Colorectal carcinoma, as a global familiar disease, is greatly endangering human health. In recent years, although there has been mading great progress in the studies of colorectal carcinoma, the treatment effect is still not very satisfactory and its morbidity and mortality are still on the rise. As the speed-limiting enzyme for the synthesis of prostaglandins, Cyclooxygenase-2 is found to be over-expressed in many tumors, and it plays a very important role in the occurrence, development, incursions and metastasis. As the specific cyclooxygenase-2 inhibitor, non-steroid anti-inflammatory drug NS-398 can inhibit the expression of cyclooxygenase-2 strongly. Recent studies have found that non-steroid anti-inflammatory drugs have anti-tumor effects mainly through inhibiting cyclooxygenase-2 expression. Survivin is the strongest inhibitor of apoptosis protein, and can inhibit cell apoptosis by adjusting the activity of caspase-3 and caspase-7. From the perspective of cell proliferation and disturbance of apoptosis, the study researched the targets and its mechanism of apoptosis in colon cancer cells induced by cyclooxygenase-2 selective inhibitor, which would provide the theoretical basis of preventing tumorigenesis and neoadjuvant therapy.The aim of this study was to observe the effects of cyclooxygenase-2 selective inhibitor NS-398 on inducing apoptosis of colorectal carcinoma HCT-8 cells and study the regulatory effects of cyclooxygenase-2, survivin and caspase3/7, in order to explore its possible mechanism preliminarily. To detect the inhibition rates of NS-398 on colorectal carcinoma HCT-8 cells by CCK-8 assay; Cell apoptosis was determined by flow cytometry analysis using PI staining; The cells were stained by Hoechst33342 and observed under fluorescent inverted microscope; In order to detect the expression of cyclooxygenase-2 and survivin before and after the effect of NS-398, immunocytochemical method was used; ELISA assay was used to detect the PGE₂ content variation; Caspase3/7 activity was analysed by automatic fluorescence enzyme-linked immunoassay detector. The following questions were discussed: To discuss the effective doses and the inhibitory effect of cyclooxygenase-2 selective inhibitor NS-398 on the growth of colorectal carcinoma HCT-8 cells, and further identify cell apoptosis induced by NS-398. We attempted to investigate whether NS-398 could inhibit the expression of cyclooxygenase-2 and survivin, down-regulate the activity of caspase3/7 in HCT-8 cells and reduce the PGE₂ levels in the cell culture, in order to preliminarily discuss the molecular mechanisms of cell apoptosis.It was found that NS-398 significantly inhibited the proliferation of HCT-8 cells in a time- and concentration-dependent manner, and there were significantly differences among the 40, 80, 160 μmol·L^-1 NS-398 concentration groups（P<0.05）; Flow cytometry assay revealed, after treated by 40, 80, 160 μmol·L^-1 NS-398 for 24 hours, apoptosis rates were 12 %, 17 % and 26 %, in a dose-dependent manner, indicating that NS-398 could induce apoptosis of HCT-8 cells significantly; The morphological changes by Hoechst33342 staining were consistent with it; After treated by 40, 80, 160 μmol·L^-1 NS-398 for 24 hours, the immunocytochemical method found that the strongly positive expression rates of cyclooxygenase-2 and survivin significantly decreased to weakly positive expression rates; The levels of PGE₂ production were 384.915 ng·L^-1, 292.111 ng·L^-1 and 132.408 ng·L^-1 respectively, which significantly decreased compared with 446.818 ng·L^-1 treated by control group; Meanwhile, the protein expression of caspase3/7 was induced by NS-398 in a dose-dependent manner, caspase3/7 activity was strongly activated especially by high-concentration NS-398.From the present results we concluded that cyclooxygenase-2 selective inhibitor NS-398 could significantly inhibit the proliferation of HCT-8 cells mainly by inducing cell apoptosis. Furthermore, its mechanism was formed that the cell expression of survivin was inhibited while suppressing cyclooxygenase-2, and further up-regulated the activity of caspase3/7, meanwhile NS-398 was able to suppress the generation of PGE₂, which could provide the theoretical basis of preventing tumorigenesis and neoadjuvant therapy.