NS-398 Reverses Multi-drug Resistance Effects Of Colorectal Carcinoma Cells

Colorectal carcinoma has become a serious threat to human life, due to the insidious onset, early diagnosis could not be made in early stage. In recent years, colorectal carcinoma has leapt to the fourth leading cause of cancer death, and the morbidity of it has been increasing. At present,surgery, radiotherapy and chemotherapy remain the mainstay of therapy for colorectal carcinoma, in which chemotherapy plays a major role in the treatment of colorectal carcinoma. However, because of the frequently occurring of multi-drug resistance(MDR) during chemotherapy, the effectiveness of chemotherapy was seriously affected and even disappeared,thus tumor recurrence and/or metastasis was leaded to, which has became a very difficult problem in cancer chemotherapy.The aim of this study was to observe the MDR reversal and the regulating effects of P-glycoprotein(P-gp), multidrug resistance related protein(MRP), cyclooxygenase-2(COX-2) of COX-2 selective inhibitors NS-398 on colorectal carcinoma HCT-8/FU cells and explore its possible mechanism preliminarily. To detect the toxic effect and the MDR reversal fold of NS-398 to colorectal carcinoma HCT-8/FU cells by CCK-8 assay.Drug-induced apoptosis was observed by Caspase 3 activity assay kit and stained Hoechst 33342. ELISA assay was used to detect the PGE2 content variation before and after the effect of NS-398. In order to detect the expression of P-gp, MRP and COX-2 before and after the role of NS-398,immunocytochemical method was used. The following questions were discussed: To discuss the toxic effects of COX-2 selective inhibitors NS-398 and 5-FU on colorectal carcinoma HCT-8/FU cells and identify that which was the effective doses of NS-398 could reverse the MDR inHCT-8/FU cells. COX-2 selective inhibitors NS-398 as common gastrointestinal malignancies(such as colorectal carcinoma) chemotherapy resistance reversal agents, we attempted to investigate whether NS-398 could inhibit HCT-8/FU cells expression of P-gp, MRP and COX-2 and reduce the PGE2 levels in the cell culture, in order to preliminarily discuss the molecular mechanisms of reversion.It was found that the growth inhibition rates of HCT-8/FU cells were5%, 8% when 10, 20 μ mol·L-1 NS-398 were used to treat cultured HCT-8/FU cells for 24 h, the difference was significant(P<0.01), thus it could be observed that the two doses had no significant toxicity to HCT-8/FU cells, moreover, after combined with different concentrations of5-FU, we observed the reversal effects were 2.831 times and 7.629 times respectively; The Caspase 3 activity assay revealed that the percentage of Caspase 3 activity was 386.11% in 20μmol·L-1 NS-398+320μg·m L-1 5-FU group, which was significantly higher than that in 320μg·m L-1 5-FU group(179.94%) and 20μmol·L-1 NS-398 group(125.23%)(P<0.05);Hoechst- 33342 staining results also showed that the nuclear of combination group had visible stain dense, this results demonstrated that the number of cell apoptosis was increased compared with alone groups;Meanwhile, HCT-8/FU cells were treated with 10μmol·L-1, 20μmol·L-1NS-398 for 24 hours, after that we detected the content of PGE2 were189.437ng·L-1, 151.282ng·L-1 respectively through the method of ELISA,which were significantly decreased compared with the 0μmol·L-1 NS-398group(321.659ng·L-1)(P<0.05); the immunocytochemical method found that the strongly positive expression rates of P-gp and MRP significantly decreased to weakly positive expression rates, meanwhile the moderately positive expression rates of COX-2 decreased to weakly positive expression rates after treated by 20μmol·L-1 NS-398 for 24 hours.From the present results we concluded that cyclooxygenase-2selective inhibitor NS-398 could significantly reverse multi-drug resistanceof colorectal carcinoma HCT-8/FU cells, and the sensitivity of HCT-8/FU cells to 5-FU could be enhanced. Furthermore, its reversal mechanism was formed that the cell surface expression of P-gp and MRP were inhibited while suppressing COX-2, which effectively reduced the chemotherapy drugs pump-out, meanwhile NS-398 was able to suppress the generation of PGE2 and restore the effect of the body’s anti-tumor immune response.Therefore NS-398 could fully enhance the clinical efficacy of chemotherapy drugs and provide a new therapeutic approach in patients with colorectal carcinoma in China.