Synergistic Effect Of COX-2 Selective Inhibitor Combined With DDP On Esophageal Cancer Cells

Esophageal cancer is an aggressive tumor that is resistant toconventional modes of treatment with chemotherapy, surgery or radiation.Research into the molecular pathways involved in the development ofesophageal cancer should yield information that will guide therapeuticdecisions. It has been found recently that cyclooxygenase-2 (COX-2) areinvolved in the cancerization of multiple tumors. The inhibition of COX-2,therefore, could be an effective strategy for reducing cell growth in thosetumor lines expressing the molecular marker. The purpose of the presentstudy is to investigate the anti-tumor effect of COX-2 selective inhibitor,NS-398 on esophagus cancer cells. To achieve it, after administrationNS-398 and DDP as single agent or in combination the response of Ec-109cells was measured at different treated conditions respectively.To primary assess the effects of DDPand DDPcombined with NS-398acting on Ec-109 cells, in the present study the growth state of the cells thattreated with DDP and NS-398 was observed firstly under an invertedmicroscope. It suggested that the cultured Ec-109 cells displayedmonolayer growth and presented with polygon and clear outline. As theextension of growth time, adherent cells became increasingly dense. Aftertreated by DDP and NS-398, the cancer cells gradually became round withuneven size. Moreover, the adherent cells were floating and even becomedebris and these changes were positively correlated with the doses of DDPand NS-398.Then, the cell viability of Ec-109 cells was observed in DDP andNS-398 alone or in combination conditions using a CCK-8 kit after being treated for 24 hours. Drug concentrations were 1.25, 2.50, 5.00, 10.00,20.00 mg?L-1 for DDP and DDP combined with NS-398 of 1.00, 10.00μmol?L-1. Afterwards, early apoptotic cells were measured usingAnnexin-V and propidium iodide (PI) double staining by Flow cytometryafter the insult of 1.25, 2.50, 5.00, 10.00 and 20.00 mg?L-1 DDP alone or incombination with 10.00μmol?L-1 NS-398 respectively. The next, agarosegel electrophoresis was carried out to detect the DNA Ladder after Ec-109cells treated by NS-398 and DDP alone or in combination. And then, theultrastructure changes were detected by transmission electron microscopyafter Ec-109 cells treated by NS-398 and DDP as single agent or incombination respectively.In the experimental process, the possible mechanisms of thesynergistic effect in NS-398 and DDP on Ec-109 cells were furtherexplored. Cell cycles analysis was carried out by Flow cytometry usingpropidium iodide (PI) staining in Ec-109 cells treated by NS-398 and DDPalone or in combination. Simultaneously, the protein expression of COX-2,Bcl-2 and Bax were determined in Ec-109 cells subjected to differenttreatments by the method of immunocytochemistry stain and Flowcytometry respectively. Moreover, Rhodamine 123 staining was used todetermine the mitochondrial membrane potential of Ec-109 cells usingFlow cytometry after treated by NS-398 and DDP as a single agent or incombination.Results from cell growth inhibition test indicated that DDP andNS-398 can both inhibit the cell proliferation dose-dependently (P<0.05),treatment in combination has an obvious synergistic effect in the inhibitionof cell proliferation and that the co-suppression effect increased with thedoses of NS-398. The double-staining of Annexin V and PI is a reliable andquantitative marker for cells of early apoptosis. Through the determinationof Annexin V and PI staining, we found that DDP can induce apoptosis anddeath of Ec-109 cells in dose-dependently. The addition of 10.00μmol?L-1 NS-398 presents a significant synergistic effect when compared with theeffect of the same concentration of DDP. After Ec-109 cells treated byNS-398 and DDP alone or in combination for 48 hours, the typical DNAladder of apoptosis came into being and appeared in agarose gel afterelectrophoresis. After addition of different doses of NS-398, the luminanceof DNA ladders was increased significantly and, moreover, the increase ofluminance of DNA ladders was positive correlation with the addition ofNS-398 doses. Administrations of NS-398 and DDP for 24 hours, thechanges of in ultramicroscopic structure were mainly an increase inheterochromatin and chromatin condensation. Apoptotic body graduallyappeared.Results from cell cycles analysis suggested that cell cycles at S phasein DDP treated cells and at G0/G1 phase in NS-398 treated cells wassignificantly increased when compared with control cells. The calculatedapoptosis rate from cell cycle analysis was enhanced significantly by theapplication of NS-398 in DDP treated cells. The protein expressiondetermined by immunocytochemistry and flow cytometry respectivelyshown that COX-2 and Bcl-2 declined and Bax enhanced significantly inEc-109 cells induced by NS-398 and DDP alone or in combination whencompared with control treated cells. These effects increased in NS-398 andDDP in combination compared with NS-398 and DDP alone. Results fromthe assay of mitochondrial transmembrane potential shown thatfluorescence intensity in control group displays the highest strengthsamong three groups. The mitochondrial transmembrane potential in Ec-109cells after 24 hours treated by NS-398 and DDP alone or in combinationare all declined significantly with the increase of the doses of NS-398 and/or DDP when compared with that in control group. Moreover, there havesignificant differences between DDP plus 40.00μmol?L-1 NS-398 groupand DDP alone group or DDP plus 10.00μmol?L-1 NS-398 group.In summary, NS-398 and DDP both have inhibitory effects on the growth of esophageal carcinoma cells dose-dependently. Application ofNS-398 and DDP in combination can enhance the inhibition of cell growthand the induction of apoptosis significantly when compared with the effectof same doses of DDP and has obvious synergistic effects. The possiblemechanism of the synergistic effects may be involved in the inhibition ofcell cycle progression and enhancement of mitochondrial related apoptosis.This study provides an experimental basis for cox-2 selective inhibitorscombined with DDP or other chemotherapy drugs for clinicalchemotherapy and, moreover, gives us a glimmer of hope for reducing drugconsumption lowering the efficacy of chemotherapy.