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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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Aim To investigate the effects of cryptotanshinone on cell viability Aim To investigate the effects of cryptotanshinone on cell viability and ferroptosis-related gene expression in A549 and A549/DDP cells, and to explore its possible mechanisms. Methods A549 and A549/DDP cells were treated with different concentrations of CTS. Cell viability was measured by CCK-8 assay. The mRNA levels of TFR1, DMT1, IREB2, HSPB1, VDAC2, VDAC3 and GPX4 were measured by qPCR, and the protein levels of TFR1 were measured by Western blot. Results The cell viability was down-regulated by CTS in A549 and A549/DDP cells, while the cisplatin-resistant A549/DDP cells were more susceptible to CTS. After CTS treatment, the mRNA levels of ferroptosis-related genes showed different degrees of change. The mRNA levels of HSPB1 and GPX4 increased in A 5 4 9 cells , of which the mRNA levels of IREB2, VDAC2 and VDAC3 were reduced and the mRNA levels of TFR1 and DMT1 exhibited no significant change. The mRNA levels of TFR1 and IREB2 increased in A549/DDP cells, while the mRNA levels of VDAC3 decreased, and the expression levels of DMT1, HSPB1, VDAC2 and GPX4 did not changesignificantly. Conclusions Cryptotanshinone may inhibit the proliferation of lung cancer A549 and A549/DDP cells, and affect the expression of ferroptosis-re-lated genes.
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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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Objective Studies show that the ERCC1 gene may be involved in secondary cisplatin resistance.This article aims to investigate the effects of shRNA targeting silencing excision repair cross-complementation group 1 (ERCC1-shRNA) on the proliferation and apoptosis of lung cancer A549/DDP cells treated with different concentrations of cisplatin.Methods Lung cancer A549/DDP cells were divided into a negative control, a blank control, an ERCC1-shRNA1, and an ERCC1-shRNA2 group.Human interfering RNA (RNAi) targeting the human ERCC1 gene was constructed and transfected into the A549/DDP cells using Lipofectamine 2000.The mRNA and protein expressions of ERCC1 in the A549/DDP cells were detected by real-time PCR and Western blot respectively, the proliferation-inhibition rate was assessed by MTT, and their cell cycle and apoptosis were determined by flow cytometry.Results ERCC1-shRNA was successfully constructed and transfected into the A549/DDP cells.Both the mRNA and protein expressions of ERCC1 were significantly lower in the ERCC1-shRNA1 (0.20±0.04 and 0.24±0.10) and ERCC1-shRNA2 (0.47±0.28 and 0.37±0.11) than in the negative control (0.96±0.12 and 1.32±0.13) and blank control groups (0.84±0.07 and 1.45±0.23) (P<0.01).Compared with the negative and blank control groups, the ERCC1-shRNA1 group showed a significantly decreased IC50 value (16.71±2.33 and 16.69±1.69 vs 7.78±0.54, P<0.01) and an increased proportion of G0/G1 phase cells ([72.87±3.23] and [71.75±4.56] vs [82.99±4.23]%, P<0.05), with the cell cycle arrested in the G0/G1 phase.The apoptosis rate of the cells in the ERCC1-shRNA1 group was remarkably lower after treated with cisplatin at the concentrations of 6.25 and 12.5 μg/mL than at 0 μg/mL ([8.17±0.65] and [11.91±1.41] vs [29.97±3.14]%, P<0.05).Conclusion ERCC1-shRNA can inhibit the proliferation and enhance the apoptosis of A549/DDP cells by silencing the expression of the ERCC1 gene.
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This study was aimed to explore the effects of Buzhong Yiqi decoction on the expression levels of Bad, NF-κB, caspase-9, Survivin, and mTOR in nude mice with A549/DDP transplantation tumors.Sixty BALB/C mice were randomly divided into blank control group, tumor-bearing control group, cisplatin group and Buzhong Yiqi decoction of high, medium and low doses+cisplatin groups (hereinafter referred to as the high,medium and low combined groups). A549/DDP cells (concentration of 5×106 cells/mL)were cultured and inoculated in various groups, then the tumor-forming situations were observed. Corresponding treatment was given in all groups. Fourteen days later, immunohistochemistry and Real-time PCR methods were used to detect the expression levels of Bad, NF-κB, caspase-9, Survivin, mTOR protein and mRNA in tumors.Results showed that Buzhong Yiqi decoction combined with cisplatin could reduce the volume of transplanted tumors, and there was significant difference between medium combined group and high combined group(P<0.05). As compared with the tumor-bearing control group, the expression levels of Bad, NF-κB, Survivin and mTOR were significantly reduced in medium and high combined groups(P<0.05); the protein and mRNA expression levels of caspase-9 were gradually increased in medium combined and high combined groups(P<0.05), with statistical difference with tumor-bearing control group(P<0.05). There were statistical difference in mRNA expression of Bad, NF-κB and caspase-9 between medium combined group, high combined group and cisplatin group, low-combined group, tumor-bearing control group(P<0.05), but there was no statistical difference between cisplatin group, low-combined group, and tumor-bearing control group. In addition, there was no statistical difference between medium combined group and high combined group in protein and mRNA expression levels of various factors. Experimental results showed that Buzhong Yiqi decoction combined with cisplatin can inhibit the growth of A549/DDP transplanted tumors, and the mechanism may be associated with regulating Bad, NF-κB, caspase-9, Survivin, and mTOR levels as well as promoting apoptosis.
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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 observe the effect of Buzhong Yiqi Decoction medicated serum on PI3K、AKT、survivin in lung adenocarcinoma A549/DDP cells,and explore its functional mechanism.Methods Buzhong Yiqi Decoction medicated serum of middle dosage (0.576 g/mL)were prepared,and A549/DDP cells were divided into four groups:blank serum group,the best containing medicine serum group and LY294002 group (blockers group), the best containing medicine serum +LY294002 group (combination group).The protein expression of PI3K,AKT and Survivin were detected by Immunocytochemistry and Western blot.Results Compare with blank serum group,the protein expression of PI3K、AKT and Survivin in best containing medicine serum and combination group were significantly lower(P<0.05 ),and the later was more lower,but there was no significant differences between best containing medicine serum and combination group.The expression of survivin in LY294002 group were significantly lower than blank serum group(P<0.05 ).Conclusion Buzhong Yiqi Decoction can reduce the protein expression of PI3K,AKT and surviving,increase the sensitivity of A549/DDP to DDP through PI3K/AKT signal transduction pathway.
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Objective:To observe the effects of genistein on proliferation and apoptosis of human non -small cell lung cancer cell line A549/DDP.Methods:①MTT assay was applied to evaluate the resistance index of A 549/DDP cell line to cisplatin and half in-hibitory concentration ( IC50 ) .②Inhibition rate of A549/DDP cell proliferation and IC 50 value were evaluated by MTT assay after treat-ment with 0, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80 μg/ml genistein for 48 hour respectively.③A549/DDP cell cycle and apoptosis were evaluated by flow cytometry after treatment with 6.25, 12.5, 25 μg/ml genistein for 24 hours respectively.Results:①In expo-sing to cisplatin, the IC50 of A549 and A549/DDP was 33.6 μmol/L and 76.9 μmol/L respectively.The resistance index was 2.3. Cell growth inhibition rate increased following the cisplatin concentration increasing gradually .②A549/DDP growth inhibition rate in-creased at first and later decreased gradually following treatment with the genistein dose increased .The IC50 of A549 and A549/DDP was about 85.1 μg/ml and 80.2μg/ml respectively.③After treatment with 6.25, 12.5, 25μg/ml genistein for 24 hours, there were more A549/DDP cells arresting and showing apoptosis along with the genistein dose increased .Conclusion: Genistein can inhibit A549/DDP proliferation, cause A549/DDP arresting in G2/M phase and induce A549/DDP cell apoptosis with dose dependently .
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Objective: To investigate the effect of elemene on the drug resistance of cisplatin (DDP)-resistant human lung adenocarcinoma A549/DDP cells, and its possible mechanism. Methods: The growth inhibition of A549/DDP cells treated with elemene alone or combined with different concentrations of DDP was detected by MTT assay. The morphological changes of apoptosis of A549/DDP cells treated with different concentrations of elemene (20 and 40 μg/mL) for 24 h were observed by Hoechst 33342 staing under fluorescence a microscope. The apoptosis rate of A549/DDP cells treated with different concentrations of elemene (20 and 40 μg/mL) after 24 h were detected by FCM (flow cytometry). The expressions of cytochrome C, pro-caspase-3, caspase-3 and the Bcl-2 family proteins were measured by Western blotting analysis. Results: MTT result showed that different concentrations elemene could inhibit the proliferation of A549/DDP cells in a time- and dose- dependent manner. Intriguingly, elemene plus DDP enhanced the sensitivity of A549/DDP cells to DDP and reversed the resistance of A549/DDP cells. Elemene was also a strong inducer of apoptosis in this model system, and a synergistic effect on induction of cell death was observed when the tumor cells were treated with both agents. The result showed that elemene could enhance A549/DDP cell apoptosis. Furthermore, the combination of elemene and DDP also enhanced the protein expressions of cytochrome C, caspase-3 and Bad, and reduced the protein levels of Bcl-2 and pro-caspase-3 in the A549/DDP cells. Conclusion: The reversal of the multi-drug resistance of A549/DDP cell line by elemene may result from its effect on induction of apoptosis. Elemene treatment can impaire mitochondrial membrane, release cytochrome C into cytoplasm, activate caspase-3,up-regulate pro-apoptotic protein Bad and down-regulate anti-apoptotic protein Bcl-2, and finally caused apoptosis. Copyright © 2013 by TUMOR.
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Objective: To investigate the effects of gene-associated with retinoid-interferon-induced mortality 19 (GRIM -19) on chemotherapeutic sensitivity of cisplatin (DDP)-resistant human lung caner cell A549/DDP and the expressions of related genes. Methods: The recombinant plasmid PIRES-Puro2-GRIM-19-Myc and the empty vector PIRES-Puro2-Myc were transfected into A549/DDP cells by liposome transfection reagent, respectively. The expression levels of GRIM-19 protein in the A459 parental cells, A459/DDP cells, A549/DDP cells transfected with GRIM -19 gene and A549/DDP cells transfected with an empty vector were detected by Western blotting. The changes of chemotherapeutic sensitivities to multi-drugs in A549/DDP cells stably transfected with GRIM -19 gene were detected by MTT assay. The expression levels of GRIM-19, signal transducers and activators of transcription 3 (STAT3), vascular endothelial growth factor (VEGF) and P-glycoprotein (P-gp) mRNAs in the A549/DDP cells stably transfected with GRIM -19 gene were examined by real-time fluorescence quantitative-PCR (RFQ-PCR). Results: The Western blotting result showed that the expression level of GRIM-19 protein was increased in the A549/DDP cells after transfection with GRIM -19 gene. The resistance index of A549/DDP cells was 16.86±1.32 folds higher than that of the A549 parental cells. The resistance index of the A549/DDP cells after transfection with GRIM -19 gene was decreased by 3.70±0.91 folds as compared with that of the A549/DDP cells transfected with an empty vector. The expression levels of STAT3, VEGF and P-gp mRNAs in the A549/DDP cells transfected with GRIM -19 gene were decreased. Conclusion: GRIM-19 can increase the chemotherapeutic sensitivity of A549/DDP cells. This effect may be associated with the down-regulations of STAT3, VEGF and P-gp. GRIM-19 may become a potential therapeutic agent to reverse drug-resistance in A549/DDP cells. Copyright © 2012 by TUMOR.
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Objective To evaluate the effects of hyperbaric oxygen (HBO) and cisplatin (DDP) on inhibition of A549/DDP cells in vitro and to explore the impacts of HBO on cell cycle and apoptosis.Methods The cytotoxicity of HBO and DDP was determined by Methyl thiazolyl tetrazolium (MTF) assay; flow cytometry (FCM) was used to investigate the impacts on cell cycle and apoptosis of cell line A549/DDP.Results Hyperbaric oxygen alone significantly suppressed A549/DDP cells proliferation (P ＜ 0.05),compared with control group of normal oxygen,and the inhibition ratio was 2.90％.DDP at concentrations ranging from 3 μg/ml to 24 μg/ml significantly inhibited the proliferation of A549/DDP cells in a dose dependent manner,that was concentrations of 3,6,12 and 24 μg/ml brought inhibition ratio of 1.87％,4.62％,11.15％ and 30.45％ ; Hyperbaric oxygen can enhance the inhibition effect of DDP to A549/DDP cells,when hyperbaric oxygen affiliated with DDP of concentrations above,the inhibition ratios were 4.37％,8.92％,21.88％ and 48.71％,respectively,which has significant difference with the single DDP groups.FCM analyses showed that HBO arrested A549/DDP cells at the S phase,that was cell number of G0/G1 phase reduced significantly and of S phase raised significantly,compared with normal oxygen group(P ＜0.01),that was the percentages of cells in S phase of control group were 9.5％ and HBO group were 20.9％,while the percentages of cells in G0/G1phasc were 82.2％ and 66.3％,respectively.In addition,there were more apoptotic cells in the HBO group with the percentage of 7.9％,while the percentage was 2.8％ in the normal oxygen group.Conclusion Hyperbaric oxygen could play together with DDP to inhibit A549/DDP cells proliferation.HBO could block the cell cycle at S period and induce apoptosis.
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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.
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Background and purpose: Multidrug resistance (MDR) of tumor cell is the main reason for clinical chemotherapy failure as well as cancer recurrence and metastasis. This study was to construct a lentiveral vector of RNA interference of MDR1 gene and observe its inhibitive role on the expression of MDR1 in A549 and A549/DDP cells. Methods: Oligos of base pairs for hairpin RNA targeting MDR1 were chemically synthesized. Via annealing and inserting them into the down-stream of H1 promoter of linearized pSUPER, we obtained the siRNA constructs for MDR1, which were afterwards transfected into A549, a human lung cancer cell line expressing high level MDR1, the impact of constructs was observed on the expression and interference efficiency of siRNA against MDR1. The effective sequence of siRNA targeting MDR1 gene was confirmed. Both sense and antisence oligo DNA of the targeting sequence was designed, synthesized and cloned into the PTM vector, containing a promoter and a green fluorescent protein (GFP). The resulting lentiviral vector containing MDR1 siRNA was named PTM-siMDR1 and then transfected into A549 and A549/DDP cells after being confirmed by PCR and sequencing. Results: Restriction digestion and DNA sequencing showed that the siRNA constructs for MDR1 were successfully produced and the expressed siRNA could effectively down-regnlate the expression of MDRI. PCR demonstrated that the lentivirus RNAi vector of MDR1 producing PTM-siMDR1 was constructed successfully. The chemosensitivity of A549/DDP cells to cisplatin were enhanced obviously after trartsfection. Conclusion: The lentivirus RNAi vector of MDR1 can significantly revise the resistance ofA549/ DDP cells with eisplatin after infection.