Reversal Effect Of Iso-suillin On Cisplatin Resistant Human Lung Adenocarcinoma Cell Line A549/DDP And Its Mechanism

Objective:To study the reversal effect of iso-suillin on cisplatin resistant human lung adenocarcinoma cell line A549/DDP in vitro,and to explore the mechanism of drug resistance from cell cycle,cell apoptosis,multidrug resistance protein?MDR1?and Akt/NF-?B signaling pathway.Methods:1 Cell culture:A549,A549/DDP and HBE cells were cultured in RPMI-1640 medium containing 10%calf serum.2 MTT assay was used to detect the inhibitory effect of different concentrations of iso-suillin on A549,A549/DDP and HBE cells The IC₅₀value was calculated according to the results.3 MTT assay was used to detect the inhibitory effect of different concentrations of cisplain on A549 and A549/DDP.According to the results,the IC₅₀ value and the drug resistance multiple of A549/DDP cells to cisplatin were calculated.4 MTT assay was used to detect the inhibitory effect of nontoxic iso-suillin combined with cisplatin on A549/DDPs.According to the results,the IC₅₀ value and the reversal times were calculated.5 The change of cell morphology in the control group,the single iso-suillin group,the single cisplatin group and the combination of iso-suillin and cisplatin group were observed by contrast microscope.6 Fluorescence dye DAPI staining,Hoechst 33258 Annexin V-FITC and PI triple staining and the assay of TUNELwere used to observe the apoptosis characteristics of the cells in each group.7 Apoptosis rate in each group detected by flow cytometry.8 The differences of cell cycle distribution in each group were detected by flow cytometry.9 Immunocytochemistry detect the difference of NF-?B expression and its cellular localization in each experimental group10 The mRNA expressions of MDR1,NF-?B were detected by real-time quantity-PCR method.11 Proteins expression of cyclin A/D1,CDK-2/4,caspase-3,MDR1,NF-?B,p-Akt were detected by western blot12 The experimental results were described by mean±standard deviation and evaluated with SPSS 13.0 by analysis of variance?One-Way ANOVA?.P<0.05 was considered statistically significant.Results:1.The IC₅₀ values of iso-suillin on A549,A549/DDP,HBE cells for 24 h were 7.78 uM,7.9uM,6.97uM respectively;1.5uM iso-suillin was selected as the using concentration in the following experiment for its low toxic.2.The IC₅₀ values of DDP on A549,A549/DDP cells for 24 h was11.14 ug/mL,33.51ug/mL respectively.The drug resistance index is 3.008times.The results of western blot showed that the expression of MDR1 of A549 was significantly higher than that of A 549/DDP.3.The IC₅₀ values of 1.5uM iso-suillin combined with DDP on A549/DDP cells for 24 h was 17.8ug/mL,and the reversal factor is 1.88.In order to avoid excessive cell death during the experiment,6 ug/mL DDP was selected as the experimental concentration.The results showed that most of the cells were round and small,and the number of floating dead cells increased in the group of 1.5 uM iso-suillin combined with 6 ug/mL DDP.4.Drug resistance reversal and apoptosis induced by iso-suillin in A549/DDP.The results of DAPI staining,Annexin V-FITC and PI triple staining and TUNEL staining showed fluorescence in combination group increased sharply;The results of flow cytometry showed that the apoptosis rate of the combined treatment group was significantly increased to 48.55%;Western blot results showed that the amount of pro-caspase3 in the combination group decreased significantly,while the amount of cleaved-caspase increased significantly.5.Drug resistance reversal and cell cycle induced by iso-suillin in A549/DDP.The results of flow cytometry showed that compared with the control group,the proportion of G0/G₁ phase increased in the iso-suillin group,and the proportion of S phase increased in the DDP group;compared with the DDP group the proportion of G0/G₁ phase increased in the combination group.Western blot results:the cyclin D₁ CDK2/4 decreased significantly in the combination group comparing with DDP group alone.6.The relationship of drug resistance reversal with the Akt/NF-?B/MDR1 signaling pathway.The results of immunofluorescence showed that the intensity of NF-?B in combination group was lower than that in cisplatin group,and the phenomenon of"empty nucleus"was obvious.The results of Real-time RT-PCR test showed that the mRNA levels of NF-?B,MDR1 in combination group were significantly lower than that in DDP group;Western blot results:the protein expression of p-Akt,NF-?B,MDR1 in combination group was significantly lower than that in cisplatin group.Conclusions:1.Iso-suillin inhibited the proliferation of A549,HBE and A549/DDP cells in a concentration-dependent manner.2.A 549/DDP is resistant to cisplatin.3.Low concentration of iso-suillin can partially reverse the resistance to cisplatin in 549/DDP cells and enhance the drug resistance of cisplatin.4.The effect of low dose iso-suillin on enhancing cisplatin may be partly through the cell cycle and apoptosis pathway to reverse the drug resistance of A549/DDP cells to cisplatin.Its mechanism is related to decreasing the expression of cyclin D1 CDK2/4 and increasing the activity of caspase3.5.Iso-suillin reversed cisplatin resistance in lung adenocarcinoma by inhibiting the expression of MDR1 in A549/DDP cells.This reversal may be related to the Akt/NF-?B signaling pathway.