| Objective:To observe the effect and the mechanism of cytokine-induced killer cells (CIK) on reversal of cisplatin (DDP) resistance of DDP-resistant human lung adenocarcinoma cell line A549/DDP. And to explore the possible roles of the cytokines secreted by CIK on reversing DDP resistance of A549/DDP.Methods:DDP sensitivity of A549and A549/DDP was detected using MTT, and IC50sof these two cell lines were calculated. Multiple differentially expressed resistance associated genes between A549and A549/DDP were screened out using semi-quantity RT-PCR and real time-PCR. A549/DDP co-cultured with CIK in different time ranging from6h to20h, and untreated A549and A549/DDP were used as negative controls. The change of resistance, the expression of resistance associate genes and proteins and the DDP concentrations were detected by MTT, RT-PCR and Western blot and high performance liquid chromatograph(HPLC), respectively. Supernatants of co-cultured systems were collected to detect the levels of IFN-y, TNF-a, IL-2using ELISA. The correlations between cytokines and resistance reversal indexs were analyzed at different co-cultured time points. Specific neutralizing antibodies against IFN-y, TNF-a and IL-2were added into co-cultured system to evaluat the effects of above cytokines on reversal of DDP resistance in A549/DDP, as well as expressions of genes.Results:With a strong resistance to DDP, the resistance index of A549/DDP was14.5compared to A549(IC50:72.6±4.65μmol/L vs.5.0±1.25μmol/L, P<0.05). The DDP consentration in A549/DDP and A549were0.29±0.02μg/ml and1.5±0.03μg/ml, respectively (P<0.05). the differently expressed resistance associated genes were filtered out, including glutathione S-transferase (GST-n) gene, the human copper transporter protein (hCTRl) gene, multidrug resistance-associated protein1(MRP1) gene. The GST-π and MRP1expression was significantly increased in A549/DDP than in A549, but hCTRl expression was contrary to the former two genes. After co-cultured with CIK, the DDP resistance of A549/DDP decreased significantly in a time-dependent manner, in which the IC50of A549/DDP co-cultured with CIK for6h significantly decreased compared to A549/DDP cultured alone (25.88±4.35μM vs.72.6±4.65μM, P<0.05), and the IC50of A549/DDP co-cultured with CIK for12h also significantly decreased compared to A549/DDP co-cultured with CIK for6h (18.33±2.98μM vs.25.88±4.35μM, P<0.05), but there were no significant differents among the IC50s of A549/DDP co-cultured with CIK for12h,16h and20h (18.33±2.98μM vs.15.65±3.74μM vs.14.70±2.12μM, P>0.05). The resistant reversal index of A549/DDP co-cultured with CIK for20h was4.93times. The DDP concentration in A549/DDP co-cultured with CIK for20h was0.99±0.02μg/ml.The expression of GST-π at mRNA and protein levels significantly decreased (P<0.05), the expression of MRP1at mRNA and protein levels increased. ELISA and correlation analysis showed that IFN-y was related to the reversal of DDP resistance of A549/DDP.After neutralized by anti-IFN-y Antibody, the DDP resistance of A549/DDP co-culture with CIK for20h recovered significantly (P<0.05), the DDP concentration also recovered significantly, and the expression of GST-π at mRNA level significantly recovered compared to A549/DDP co-cultured without anti-IFN-y antibody (P<0.05).Conclusion:CIK could partly reverse the DDP resistance of cisplatin-resistant lung cancer cell A549/DDP. The expression of GST-π changed significantly in A549/DDP co-cultured with CIK, which prompted that the expression change of GST-π may be one mechnism of reversal to DDP resisatance of A549/DDP by CIK. IFN-y secreted by CIK might decrease the expression of GST-π in A549/DDP to accomplish the reversal process. |
PDF Full Text Request