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Objective:To investigate the effect and mechanism of saikosaponin A （SSA） on the reversal of cisplatin （DDP） resistance in human lung cancer cell line A549/DDP. Methods:The resistance of A549 and A549/DDP cells to DDP and the inhibitory effects of SSA against the proliferation of A549 and A549/DDP cells were detected using cell counting kit-8 （CCK-8）. The apoptosis rates of A549/DDP cells treated with SSA or DDP or SSA combined with DDP and the changes in reactive oxygen species （ROS） were determined by flow cytometry. The mRNA expression levels of C-myc， B-cell lymphoma 2 （Bcl-2） and cysteinyl aspartate-specific protease-3 （Caspase-3） were detected by real-time polymerase chain reaction （Real-time PCR）， followed by the determination of <italic>β</italic>-catenin transcriptional activity using the TopFish dual-luciferase reporter assay system and the measurement of <italic>β</italic>-catenin protein expression in A549/DDP cells by Western blot. Results:The results of CCK-8 assay showed that the DDP resistance of A549/DDP cells was 12.82 times that of A549 cells （<italic>P</italic><0.05）. SSA inhibited the viability of A549 cells with the half maximal inhibitory concentration （IC<sub>50</sub>） being 34.9 μmol·L<sup>-1</sup>， and also suppressed the viability of A549/DDP cells in a concentration-dependent manner. Since the inhibition rate of 20 μmol/L SSA against A549/DDP cells was less than 10%， the reversal concentration was set at 20 μmol/L. Flow cytometry revealed that compared with the control， DDP alone increased the apoptosis rate of A549/DDP cells （<italic>P</italic><0.05）， stimulated the accumulation of intracellular ROS （<italic>P</italic><0.05）， down-regulated the mRNA expression levels of C-myc and Bcl-2 in A549/DDP cells， up-regulated Caspase-3 mRNA expression， and reduced the transcriptional activity of <italic>β</italic>-catenin （<italic>P</italic><0.05）. Compared with the DDP group， the SSA+DDP group exhibited obviously increased apoptosis of A549/DDP cells， enhanced accumulation of intracellular ROS， down-regulated C-myc and Bcl-2 mRNA expression， up-regulated Caspase-3 mRNA expression （<italic>P</italic><0.05）， and weakened <italic>β</italic>-catenin transcription （<italic>P</italic><0.05）. DDP combined with SSA better decreased the <italic>β</italic>-catenin protein expression in contrast to that of control or DDP （<italic>P</italic><0.05）. Conclusions:SSA enhances the sensitivity of A549/DDP cells to DDP possibly by inhibiting the activation of Wnt/<italic>β</italic>-catenin pathway.
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Objective: To investigate the reversal effects in A549/DDP by standard substances (SS) from Traditional Chinese Medicine. Methods: Cell proliferation assays were performed to investigate the Resistant Index of A549/DDP and its tolerance to selected SS. The working concentrations of SS, IC5 calculated by nonlinear regressions, were applied as reversal doses to investigate the effects. Results: The resistant index of A549/DDP was 31.79. Tetramethylpyrazine and Matrine were well tolerated. Berberine hydrochloride, Dauricine, Oridomin exhibited dose-depend inhibitory effects on A549/DDP cell line. The working concentrations of them could effectively reverse the resistance of A549/DDP cell line to DDP. (P＜0.01) . Conclusion: The selected standard substances from Traditional Chinese Medicine were capable to reverse the resistance of A549/DDP cell line to DDP.
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This study was designed to reverse multidrug resistance of lung cancer cells by downregulating MDR1 genes through RNA interference (RNAi) technology. A novel biodegradable cationic polymer (PEG-bPLG-g-PEIs, GGI) was synthesized and characterized by 1H NMR. The particle size and zeta potential were measured by dynamic light scattering (DLS). The cell viability profile of GGI was tested by MTT method with both A549 and A549/DDP cell lines. Flow cytometry (FCM) technology was used to investigate the efficiency and intensity of delivering siRNA to cells by GGI polymer. RT-PCR and Western blot were used to detect the mRNA and P-gp expression after GGI/MDR1 siRNA transfection assay. The sensitivity of cisplatin administration after transfecting GGI/MDR1 siRNA polyplexs was performed with MTT and Annexin V-FITC/PI methods. The results suggest that the particle size and zeta potential of GGI/siRNA were 150-200 nm and 16-28 mV. GGI exhibited a lower cell cytotoxity than PEI 25K and higher efficiency of delivering siRNA, which dramatically decreased the expression of MDR1 mRNA and P-gp of A549/DDP cells and increased much sensitivity to cisplatin in A549/DDP cells. GGI holds a great potential in gene delivery as a novel cationic polymer for further investigation.
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AIM:To study the expression of microRNA (miRNA)-181a in different human lung adenocarcinoma cell lines, and to investigate the effect of miRNA-181a on cell function and its mechanism in human lung adenocarcinoma drug resistant cell A549/DDP.METHODS:Real-time PCR was used to detect the expression of miRNA-181a in BEAS-2B cells, A549 cells and A549/DDP cells.The A549/DDP cells were transfected with pGenesil-miRNA-181a eukaryotic ex-pression plasmid.At the same time, the untransfection group and negative transfection group were also set up .The expres-sion of miRNA-181a, cell viability, cell growth inhibition and apoptosis rate during cis-diamminedichloroplatinum ( DDP) treatment, cell cycle, cell invasion, the protein expression of miRNA-181a target genes bcl-2 and p53 in the A549/DDP cells were determined by real-time fluorescence quantitative PCR , MTT assay, flow cytometry, Transwell method and West-ern blot, respectivly.RESULTS:The expression of miRNA-181a in A549 cells and A549/DDP cells was significantly lower than that in BEAS-2B cells, and the lowest expression level was observed in A 549/DDP cells (P<0.05).The expression of miRNA-181a in A549/DDP cells was significantly increased after transfection with pGenesil-miRNA-181a (P<0.05).The cell viability, cell cycle and invasion ability of the A549/DDP cells were inhibited after miRNA-181a transfection (P <0.05).The cell growth inhibition rate and apoptotic rate of the A 549/DDP cells were increased (P<0.05).The expression of Bcl-2 was reduced, but the expression of P53 was increased after transfection with miRNA-181a in A549/DDP cells (P<0.05).CONCLUSION: miRNA-181a may be correlated with the development of human lung adenocarcinoma .miRNA-181a can serve as a new target for treatment of lung cancer .
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Objective To study the effect of Buzhong Yiqi Decoction serum on cell cycle of A549/DDP cell. Methods A549/DDP cells and splenocytes cells were cultured in vitro and divided randomly into three groups:control group, Buzhong Yiqi Decoction group and splenocyte group. A 549/DDP cells were stimulated by different concentrations of Buzhong Yiqi Decoction serum and spleen cells, the direct killing effect on cells were measured by MTT method, cell apoptosis were detected by lfow cytometric analysis and the apoptotic body were observed by immunolfuorescence method. Results MTT results showed that cell growth were inhibited by Buzhong Yiqi Decoction serum in contrast with control groups, and the effect was same as spleen cells, and both treated groups had similar effects. Marked apoptotic peak and the apoptotic body were seen by lfow cytometric analysis and lfuorescence microscope in both two groups. Conclusion Buzhong Yiqi Decoction could inhibit the proliferation of A549/DDP cells in vitro and induced cell apoptosis, which is the similar as spleen cells.
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Objective To evaluate reverse effect of recombinant human Endostatin on drug-resistance of A549/DDP cells to cisplatin (DDP). Methods Lung adenocarcinoma cell line A549 and its DDP-resistant cell line A549/DDP were treated with DDP and recombinant human Endostatin. Difference in drug resistance was analyzed between different regimens ( DDP, Endostatin and combination) and between different cell lines ( human lung adenocarcinoma A549 and drug resistant A549/DDP), after a 72h-treatment in vitro. Reverse effect of recombinant human Endostatin on drug-resistance of A549/DDP to DDP was tested by MTT assay. Results The observed 50% inhibitory concentration ( IC50 ) was (0.72 ± 0.05 ) ug/ml against A549 and ( 11.54 ± 0.64)against A549/DDP in DDP, and (2.0 ± 0.1 ) μg/ml against A549/DDP in rh-Endostatin- DDP combination respectively, with a reversal fold (RF) of 5.77 and a relative reversal rate of 88. 2%. Conclusion rh-Endostatin may reverse drug-resistance of A549/DDP cells to DDP.