The Study Of Effects And Mechanism Of Curcumin Combined With Gefitinib On NCI-H1975

Objective:To examine whether a combinatorial treatment with Curcumin enhances the effects of the epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI) Gefitinib on cell proliferation, clonogenic capacity, expression of P-glycoproein, and apoptosis in the drug-resistant lung cancer cell line NCI-H1975, and further investigate the molecular mechanisms involved.Methods:First of all, MTT assays were performed to evaluate the effects of Curcumin or Gefitinib on NCI-H1975 to determine each IC50 (half maximal inhibitory concentration) index. Preliminary experiments revealed that the Gefitinib IC50 was 8 μmol/L, while the Curcumin IC15 was 10 ng/mL.We use different concentrations (2,4,6,8,10) μmol/L of Gefitinib alone or with 10 ng/ml Curcumin for 24 hours to examine whether a combinatorial treatment with Curcumin can enhances Gefitinib suppressive effect on proliferation capacity of NCI-H1975.And then we use IC15 as working concentration of Curcumin, which was lOng/ml, and select IC50 as concentration of Gefitinib, which was 8 μmol/L. Then their co-treatment and single medicine Gefitinib were compared in following test, we also set up group that treated with single Curcumin at lOng/ml and control group without any additive, which made four groups in total. We defined as follows:Group 1.control group: cells treated without Curcumin or Gefitinib; Group 2.Curcumin-treated group:cells treated with Curcumin at 10 ng/mL; Group 3. Gefitinib-treated group:cells treated with Gefitinib at 8μmol/L, Group 4. Curcumin+Gefitinib-treated group:cells treated with Gefitinb at 8μmol/L and Curcumin at 10 ng/mL. NCI-H1975 cells were treated with Curcumin and Gefitinib alone or in combination, and clonogenic capacity, expression of P-glycoproein and apoptosis were examined using the MTT assay, clone forming experiments, and flow cytometry, respectively, while p38, ERK1/2, NF-κB and AKT phosphorylation were examined using western blotting.Results:1. The treatment with different concentrations (2,4,6,8,10) μmol/L of Gefitinib alone or with 10 ng/ml Curcumin for 24 hours, at the same time set up without medicine as blank group. The results show:compared with Gefitinib alone, the combination of Curcumin and Gefitinib had a stronger suppressive effect on proliferation capacity of NCI-H1975 at each concentration (p<0.05), and combination with 10 ng/ml Curcumin can reduce the Gefitinib IC50 (1.98±0.67μmol/L VS 8.15±2.31μmol/L) (p<0.05).2. Flow cytometry analysis of apoptosis in NCI-H1975 cultures following treatment with Curcumin (10 ng/mL), Gefitinib (8μmol/L), or a combination of the two showed that the apoptosis frequency was higher in the cultures that were treated with both agents (p<0.05), indicating that Curcumin can enhance the effects of Gefitinib in inducing tumor cell apoptosis.3. Using Western Blotting to examine the phosphorylation status of multiple kinases (P13K/AKT, ERK1/2, NF-κB, and p38) in pathways downstream of the EGFR receptor, treatment with concentrations of lOng/ml Curumin,8μmol/L Gefitinib alone and combination on NCI-H1975 cells after 24 hours, set up without medicine for blank group. The results found a significant inhibition of p38, ERK1/2, NF-Kb, and AKT phosphorylation in the cells treated with the combination of Curcumin and Gefitinib, compared to that in cells treated with either agent alone (p<0.05).4. NCI-H1975 cells treated with Curcumin, Gefitinib, or combination of the two on the above mentioned concentrations were cultured at 37℃,100% saturated humidity, 5% CO2 for 10 d, and then the colonies that contained more than 30 cells were counted using an inverted microscope. The results show that:the combination of Curcumin and Gefitinib had a stronger suppressive effect on clonogenic capacity compared to Gefitinib alone (p<0.05).5. The treatment with concentrations of 10 ng/mL Curumin and 8μmol/L Gefitinib alone and combination on NCI-H1975 cells after 24 hours, and then P-glycoproein was examined by flow cytometry with mouse anti human P-gp FITC, set up without medicine for blank control group. The results indicating that:Curumin can significantly induce expression of P-glycoproein on NCI-H1975 (p<0.05).Conclusions:1. Curcumin can significantly reduce the Gefitinib IC50 of NCI-H1975, Curcumin reduce drug resistance of NCI-H1975 may by the way of decrease expression of P-glycoproein of tumor cell.2. Co-treatment with Curcumin and Gefitinib can significantly improved the ability of Gefitinib to inhibit cell proliferation, suppress the clonogenic capacity, and enhance apoptosis of NCI-H1975 cell.3. The mechanism of co-treatment with Curcumin improve Gefitinib’s pharmacological effects on the NCI-H1975, may mediated via the decrease in phosphorylation of proteins in pathways MAPK and PI3K/AKT which are engaged downstream of the epidermal growth factor receptor.