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Objective To investigate the effect of the SMAC gene on paclitaxel sensitivity and cellular activity in lung adenocarcinoma cells based on the caspase-3/Bcl-2/Bax signaling pathway. Methods A paclitaxel-resistant cell line A549/Taxol was established for lung adenocarcinoma, and the cells were divided into four following groups: pcDNA-NC (transfected with pcDNA-NC blank vector), pcDNA-SMAC (transfected with pcDNA-SMAC vector), siRNA-NC (transfected with siRNA-NC empty virus vector), and siRNA-SMAC groups (transfected with siRNA-SMAC lentiviral vector). The SMAC mRNA expression in cells was detected by qRT-PCR; cell sensitivity was detected by MTT; cell proliferation ability was detected by cloning assay; cell invasion ability was detected by Transwell; apoptosis ability was detected by flow cytometry assay; and caspase-3, Bcl-2 and Bax protein expression in cells were detected by Western blot analysis. Results The SMAC mRNA expression was significantly lower in A549 cells compared with BEAS-2B cells (P < 0.05). The SMAC mRNA expression was significantly higher in the pcDNA-SMAC group than that in the pcDNA-NC group cells (P < 0.05). The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than that in the siRNA-NC group. The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than in the siRNA-NC group. Compared with the pcDNA-NC group, the cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly lower in the pcDNA-SMAC group, the cell resistance index reversal was 2.51-fold, and the apoptosis ability and caspase-3, as well as Bax protein expression, were significantly higher (P < 0.05). Compared with the siRNA-NC group, cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly higher in the siRNA-SMAC group, and apoptosis ability and caspase-3 and Bax protein expression were significantly lower (P < 0.05). Conclusion High expression of SMAC increases paclitaxel sensitivity, inhibits cell growth and invasion, promotes apoptosis in lung adenocarcinoma cells, and has a regulatory effect on the caspase-3/Bcl-2/Bax signaling pathway.
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Objective To explore the expression of the receptor for advanced glycosylation end products(RAGE) and its intracellular signaling molecules DIAPH1 in lung adenocarcinoma A549 cells and the effect of RAGE ligands on cell migration and apoptosis. Methods The expressions of RAGE and DIAPH1 in lung adenocarcinoma A549 cells and human bronchial epithelial cells BEAS-2B were tested by qRT-PCR and Western blot. A549 cells was treated with 10,100 μg /ml RAGE ligands CML-AGE and 1,10,100 μg /ml S100B,and wound healing test was used to identify the effect of migration ability. A549 cells was treated with 25,50,100 μg /ml RAGE ligands CMLAGE, the gene expression of BCL-2 and BAX were tested by using qRT-PCR. Results The results of qRT-PCR and Western blot showed,compared with human bronchial epithelium cells BEAS-2B,the expression of RAGE and DIAPH1 were both significantly down-regulated in lung adenocarcinoma A549 cells (P < 0. 001). After treated with 10,100 μg /ml RAGE ligands CML-AGE and 1,10,100 μg /ml S100B ,both groups showed the ligands inhibit lung adenocarcinoma A549 cells migration in concentration-depend manners (P < 0. 01). After treated with 25, 50,100 μg /ml RAGE ligands CML-AGE,the expression of anti-apoptotic gene BCL-2 was down-regulated and proapoptotic gene BAX was upregulated in the experimental group in concentration-depend manners(P < 0. 05),the difference was significant. Conclusion The expression levels of RAGE and DIAPH1 in lung adenocarcinoma A549 cells are both significantly lower than human bronchial epithelium cells BEAS-2B. RAGE ligands can inhibit cells migration and promote cell apoptosis in lung adenocarcinoma A549 cells and may provide a new target for the therapy of lung adenocarcinoma cells.
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Objective To investigate the apoptosis and toxicity of oncolytic virus H101 combined with radiation on apoptosis of A549 lung adenocarcinoma cells. Methods A549 lung adenocarcinoma cells in exponential growth phase were divided into four groups: control ( PBS) group, radiation ( IR) group, oncolytic virus (H101) group and radiation combined with oncolytic virus (IR+H101) group. The cells were double dyed with Annexin fluorescein isothiocyanate ( V-FITC/PI ) and then the apoptosis ratio of cells in every group was detected by the flow cytometry. The cytotoxic effect of cells in every group was detected by lactate dehydrogenase ( LDH) release test. The mRNA expression of oncolytic viruses H101 capsid protein Hexon was detected by real-time fluorescence PCR ( RT-PCR) to compare the oncolytic virus replication in each group. Results Cell apoptosis rate in H101 group (55. 37%) was significantly higher than that in PBS group (1.03%) (t =36.51, P <0.05). Cell apoptosis rate in IR + H101 group (93. 06%) was significantly higher than that in H101 group (55. 37%), IR group (12. 67%) and PBS group (1. 03%) (t=13. 51, 24. 14, 38. 99, P<0. 05). LDH releasing percentage in IR group and H101 group at different time after virus transfection was significantly higher than that in PBS group ( t=25. 84,39. 38, 32. 51, 78. 18, P<0. 05;t=31. 40, 2. 68, 23. 43, 60. 98, P<0. 05). LDH releasing percentage in IR+H101 group was significantly higher than that in PBS group (t=80. 71, 119. 74, 109. 80, 123. 94, P<0. 05), IR group (t=28. 80, 54. 34, 72. 34, 61. 91, P<0. 05) and H101 group (t=42. 02, 57. 45, 57. 01, 58. 83, P<0. 05). Compared with H101 group at the same time point, the mRNA expression of Hexon in IR + H101 group at 24, 48 and 72 h was increased by 16. 26, 28. 37 and 39. 58 times, respectively (t=54. 50, 33. 73, 29. 28, P<0. 05). Conclusions The oncolytic virus H101 plays a role of radiosensitization in tumor cells. Radiation also increases the replication of the oncolytic virus H101 and thereby enhances the oncolytic effect of the oncolytic virus H101. Therefore, oncolytic virus H101 combined with radiotherapy has synergistic effect on killing tumor cells.
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Objective To investigate the apoptosis and toxicity of oncolytic virus H101 combined with radiation on apoptosis of A549 lung adenocarcinoma cells. Methods A549 lung adenocarcinoma cells in exponential growth phase were divided into four groups: control ( PBS) group, radiation ( IR) group, oncolytic virus (H101) group and radiation combined with oncolytic virus (IR+H101) group. The cells were double dyed with Annexin fluorescein isothiocyanate ( V-FITC/PI ) and then the apoptosis ratio of cells in every group was detected by the flow cytometry. The cytotoxic effect of cells in every group was detected by lactate dehydrogenase ( LDH) release test. The mRNA expression of oncolytic viruses H101 capsid protein Hexon was detected by real-time fluorescence PCR ( RT-PCR) to compare the oncolytic virus replication in each group. Results Cell apoptosis rate in H101 group (55. 37%) was significantly higher than that in PBS group (1.03%) (t =36.51, P <0.05). Cell apoptosis rate in IR + H101 group (93. 06%) was significantly higher than that in H101 group (55. 37%), IR group (12. 67%) and PBS group (1. 03%) (t=13. 51, 24. 14, 38. 99, P<0. 05). LDH releasing percentage in IR group and H101 group at different time after virus transfection was significantly higher than that in PBS group ( t=25. 84,39. 38, 32. 51, 78. 18, P<0. 05;t=31. 40, 2. 68, 23. 43, 60. 98, P<0. 05). LDH releasing percentage in IR+H101 group was significantly higher than that in PBS group (t=80. 71, 119. 74, 109. 80, 123. 94, P<0. 05), IR group (t=28. 80, 54. 34, 72. 34, 61. 91, P<0. 05) and H101 group (t=42. 02, 57. 45, 57. 01, 58. 83, P<0. 05). Compared with H101 group at the same time point, the mRNA expression of Hexon in IR + H101 group at 24, 48 and 72 h was increased by 16. 26, 28. 37 and 39. 58 times, respectively (t=54. 50, 33. 73, 29. 28, P<0. 05). Conclusions The oncolytic virus H101 plays a role of radiosensitization in tumor cells. Radiation also increases the replication of the oncolytic virus H101 and thereby enhances the oncolytic effect of the oncolytic virus H101. Therefore, oncolytic virus H101 combined with radiotherapy has synergistic effect on killing tumor cells.
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Objective To study the effect of the inhibitor of apoptosis protein,Livin on proliferation and multi?drug resistance of lung adenocarcino?ma cells A549. Methods A549 cells were transfected with the eukaryotic expression vector pcDNA3.1?Livin. A549 cell clone with stable expres?sion of Livin was obtained through G418 screening. Expressions of Livin mRNA and protein in the transfected cells were respectively measured by re?verse transcription polymerase chain reaction(RT?PCR)and Western blot. The distribution of cell cycle phase was determined using flow cytometry. The level of P?gp mRNA and protein in A549 cells transfected with pcDNA3.1?Livin was detected by RT?PCR and Western blot. The analysis of multi?drug resistance of A549 treated with different chemotherapeutics was performed by MTT. Results The mRNA and protein expressions of Liv?in were both significantly increased in the transfected A549 cells. The flow cytometry analysis showed there was higher percentage of S phase and low?er percentage of G0/G1 phase in A549 cells transfected with pcDNA3.1?Livin. Compared with control groups,the expression of P?gp mRNA and pro?tein was increased in A549 cells transfected with pcDNA3.1?Livin,which showed a higher drug resistance and lower sensitivity to chemotherapic drugs such as ADM,MTX,CTX,and DDP(P<0.05). Conclusion Overexpression of Livin could enhance the proliferation of A549 cells,and high expression of P?gp caused by Livin could serve as one of the causes for multi?drug resistance in lung adenocarcinoma against chemotherapies.
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Objective To investigate the effect of C-erbB-2 shRNA on chemosensitivity of mouse lung adenocarcinoma cells and its mechanism,and to find new therapy method for non-small cell lung cancer,especial lung adenocarcinoma.Methods The mouse lung adenocarcinoma Lewis cells were cultured regularly and divided into non-transfected group, pGPU6/RFP/Neo-shNC group and pGPU6/RFP/Neo-erbB-2 group. The plasmids were synthesized and transfected into Lewis cells in each group by Lipofectamine 2000.The expression levels of C-erbB-2 mRNA and protein in the cells in various groups were tested by RT-PCR and Western blotting method, respectively. The Lewis cells were divided into non- transfected group, pGPU6/RFP/Neo-shNC group, carboplatin group, pGPU6/RFP/Neo-erbB-2 group, pGPU6/RFP/Neo-shNC+carboplatin group and pGPU6/RFP/Neo-erbB-2+ carboplatin group. The apoptotic rates of the cells in each group were detected by flow cytometry;the expression levels of Bcl-2 and Bax proteins in each group were determined by Western blotting method.Results The expression levels of C-erbB-2 mRNA and protein in pGPU6/RFP/Neo-erbB-2 group were lower than those in non-transfected group and pGPU6/RFP/Neo-shNC.The apoptotic rate of the cells in pGPU6/RFP/Neo-erbB-2+carboplatin group was the highest in all of the groups (P<0.01);compared with the others, the expression of Bax protein in pGPU6/RFP/Neo-erbB-2+carboplatin group was increased, while the expression level of Bcl-2 protein was decreased.Conclusion C-erbB-2 shRNA can increase the Lewis cells’sensitivity to carboplatin.The mechanism may be that it can enhance the Lewis cells’apoptosis induced by carboplatin through increasing the expression of Bax protein and decreasing the expression of Bcl-2 protein.
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[ ABSTRACT] AIM:To study the effect of rapamycin ( Rap) on the proliferation, invasion, adhesion, apoptosis and autophagy of human adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum ( DDP) .METHODS: Human adenocarcinoma A549 and resistant A549/DDP cell lines were cultured.The inhibitory effects of Rap alone or combined with DDP on A549 and resistant A549/DDP cells were detected by MTT assay.The in vitro invasion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by Transwell methods. The in vitro adhesion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by adhesion experiments.The apoptosis of A549 and resistant A549/DDP cells induced by Rap alone or combined with DDP was ana-lyzed by flow cytometry.The cell autophagy marker proteins beclin-1 and LC3 in A549 and resistant A549/DDP cells trea-ted with Rap alone or combined with DDP were detected by Western blotting.RESULTS:Compared with Rap or DDP a-lone group, the combination of Rap and DDP significantly inhibited the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells in vitro, and promoted the cell apoptosis and autophagy marker proteins beclin-1 and LC3 expres-sion ( all P<0.05) .CONCLUSION:Rap enhances the effect of DDP through promoting the cell autophagy, thereby in-hibiting the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells and inducing the cell apoptosis with a synergistic effect.
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Objective: To investigate the inhibitory effects of RNA silencing via adenovirus-mediated vascular endothelial growth factor receptor (VEGFR) shRNA on proliferation of lung adenocarcinoma cells in vitro and in vivo. Methods: Ad-VEGFRshRNA adenovirus containing enhanced green fluorescent protein (EGFP) gene and VEGFRshRNA was constructed and was used to infect A549 cells; fluorescent microscopy was used to observe the infection efficiency. Western blotting assay was used to examine the expression of VEGFR protein in A549 cells. MTT method was used to examine the cell viability and the cell growth curve was drawn. The inhibition of cell growth was examined by cell cycle and colony-forming test. Meanwhile, nude mice were transplanted with A549 cells to establish tumor-bearing model, and the long term growth of tumor was observed. Results: Western blotting revealed that the expression of VEGFR was obviously decreased in the RNA interference group. The cell growth curve indicated that the cell growth was obviously inhibited after RNA interference. Cell cycle and colony-forming test indicated that the tumor growth was obviously inhibited after RNA interference. In vivo study with nude mice also indicated that RNA interference obviously inhibited tumor growth. Conclusion: The constructed VEGFR-targeted shRNA can effectively inhibit VEGFR expression in A549 cells and can suppress the growth of A549 cells.
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Objective To investigate the changes of telomerase activity after knocking down endogenous expression of T-STAR (testes-signal transduction and activator of RNA) gene in human lung adenocarcinoma cell line A549 by antisense strategy. Methods The mRNA and protein expression of T-STAR gene were determined by RT-PCR and Western blotting, and the telomerase activity was measured by PCR-ELISA, after transfection of T-STAR antisense gene into A549 cells with lipofectamine. Sense pcDNA-STAR and blank pcDNA3.1 transfection served as control. Results The expression of T-STAR gene was significantly inhibited at mRNA and protein level, and the telomerase activity was significantly decreased. Conclusion The down-regulation of telomerase activity may result from inhibition of T-STAR gene expression in lung adenocarcinoma A549 cells.
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Objective To explore the molecular mechanism of Qingjin Desheng Tablets on inhibiting the growth of lung cancer cells and to supply experimental evidences for Chinese herbal medicine preventing and curing lung cancer in clinical application .Methods Immunohistochemistry staining was used to detect the activity of telomerase and the expression of bcl-2 and bax in human lung adenocarcinoma cells after treated with serum containing Qingjin Desheng Tablets.Results The activity of telomerase was inhibited significantly and the positive rate of bcl-2 protein reduced,and the positive rate of bax2 increased in human lung adenocarcinoma cells after 72 hour-treatment with the serum containing Qingjin Desheng Tablets.Conlusion Qingjin Desheng Tablets exert the anti-tumor action through inhibiting the activity of telomerace and modulating the expression of bcl-2 and bax protein.
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Aim To construct the eukaryotic expression vectors of short hairpin RNA targeting survivin and observe its effect on biologic behavior of A549 cells and sensitivity of A549 cells to paclitaxel.Methods The DNA fragment targeting human survivin was inserted into the plasmid,and the recombinant plasmid was constructed.The recombinant plasmids cells were transfected into A549 cells by FuGENE transfection reagent.The expression levels of survivin gene were detected with RT-PCR and Western blot before and after transfection,respectively.Cell apoptosis was detected by TUNEL method.The sensitivity of A549 cells to paclitaxel was detected by MTT after transfection.Results The recombinant plasmid was successfully constructed.RNAi group cells showed lower expression of survivin than control group.The apoptosis rate of A549 cells increased after transfection.The IC50 of paclitaxel inhibiting A549 cells was 11.9 fold before transfection compared with those after transfection.There was significant difference between the two groups(P
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Objective To explore the sensitization effect of insulin to chemotherapy as a metabolic promoter on human lung adenocarcinoma cells A549.Methods The sensitivity of human adenocarcinoma cells A549 to DDP was observed by MTT methods,and IC30 values of the cells were calculated.The optimal concentration of insulin to promoting A549 cells' growth was determined by MTT assay.The A549 cells were pretreated with insulin of various concentrations for different time period,and then the cell sensitivity to DDP was evaluated with MTT assay.Flow cytometry was used to study the cell cycle distribution of the A549 cells treated with insulin.Results The IC30 value of DDP was about 36.87?g/ml.It was found that the cell growth was significantly promoted with pretreatment of insulin at the concentration of 4~16mU/ml for 8~16 hours.Insulin enhanced the inhibitory effect of DDP(36.87?g/ml)on cell proliferation when human lung adenocarcinoma cells A549 were treated with insulin(2.0~16.0mU/ml;8~16h)as indicated by MTT assay(P