Effects of thalidomide on the expressions of basic fibroblast growth factor in human lung adenocarcinoma cell line A_(549) and cisplatin-resistant cell line A_(549) DDP
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Objective To elucidate the effects of thalidomide on the expressions of basic fibroblast growth factor(bFGF) in parental and cisplatin resistant human lung adenocarcinoma cell line A 549 and A 549 DDP Methods RT PCR and lmmunohistochemistry were used to detect the bFGF mRNA and protein expression in A 549 and A 549 DDP Results bFGF mRNA expression levels and protein expressions in A 549 and A 549 DDP were all significant reduced after treatment of thalidomide (6 μg/ml)(all P 0 001).There existed significant differences in A 549 and A 549 DDP among different concentrations of thalidomide on the 5 th day The bFGF protein expression level was consistent with the mRNA expression levels in A 549 and A 549 DDP Conclusion Thalidomide down regulated bFGF expression significantly in A 549 and A 549 DDP cell lines in a dose dependent manner It was suggested that thalidomide not only inhibit tumor angiogenesis but also induce apoptosisCite
To explore the effects of thalidomide on the expressions of vasc ular growth factors in parental and cisplatin_resistant human lung adenocarcinom a cell line A549 and A549 .Methods:RT_PCR and Immunohistochemistry were used to detect mRNA and protein expression of VEGF in A549 and A549 .Results:From t he 1st to 5 th day after treatment with physiological concentration of thalidomi de(6 mg/L),VEGF mRNA expression levels in A549 were significant higher than that before treatment.VEGF mRNA expression levels in A549 were significant lower. There were significant differences in A549 and A549 among different concentra tion of thalidomide on the 5 th day.The protein expressions were significant coi ncided with the relative VEGF mRNA expression levels in A549 and A549 .Conclus ion:Physiological concentration of thalidomide up_regulated VEGF mRNA expression significantly in A549.But large dose of thalidomide inhibits VEGF expression si gnificantly.Moreover,thalidomide down_regulated VEGF mRNA expression significant ly in A549 .It is dose_independent.The protein expression is significantly coi ncided with the relative VEGF mRNA.
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Objective To investigate the role of Ku70 in the chemoresistance of lung cancer and the potential of Ku70 small-interfering RNA(siRNA) as a therapy for reversal of cisplatin resistance in lung adenocarcinoma.Methods The Ku70 mRNA and protein expression levels were dectected by RT-PCR and Western blotting methods in human lung adenocarcinoma cells A549 and their cisplatin-resistant variant A549/DDP.The A549/DDP cells were divided into 3 groups:blank control,negative control group in which the cells were tranfected with nonspecific si-RNA(si-Scramble) and experiment group in which the cells were transfected with Ku70-specific siRNA(si-Ku70).The cells in negative control group and experiment group were transfected 48 h prior to treatment with cisplatin.The cell survival was assessed by MTT assay.The apoptotic rate was determined by flow cytometry.The Caspase-3 activity was detected by spectrophotometer.Results The Ku70 mRNA and protein expressions in A549/DDP cells were significantly elevated compared with the parental A549 cells(P0.01).The si-Ku70 down-regulated the mRNA and protein expressions of Ku70 in A549/DDP cells.After treated with different concentrations of cisplatin for 24 h,the cell viabilities of si-Ku70 cells were decreased significantly compared with control group(P0.01).After treatment with 6 mg·L-1 cisplatin for 24 h,the apoptotic rates and the activites of Caspase-3 in experiment group were increased significantly compared with control group(P0.01).Conclusion The overexpression of Ku70 in cisplatin-resistant human lung adenocarcinoma cells suggests that Ku70 might be involved in the chemoresistance of lung cancer.Ku70-specific siRNA can down-regulate the expression of Ku70,promte apoptosis and reverse resistance to cisplatin in cisplatin-resistant A549/DDP cells.Ku70 may be a new target for reversal of chemoresistance of lung cancer.
Ku70
MTT assay
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Objective: To study the effect of 8-Br-cAMP with cisplatin(DDP) on human pulmonary adenocarcinoma cell A549 growth and the expression level of EGFR in vitro.Methods: Proliferation ability and cell cycle changes of A549 cells were detected by MTT method and flow cytometry.The mRNA levels of EGFR were determined by RT-PCR.The protein expression of EGFR were valued by Western blot.Results: Both DDP and 8-Br-cAMP could inhibit the proliferation of A549 cells in vitro by a time-and dose-dependent manner.Compared with either 8-Br-cAMP or DDP alone,combining 8-Br-cAMP at 15 μmol·L-1 with DDP at 0.5 μg·ml-1,1 μg·ml-1,2 μg·ml-1,4 μg·ml-1 respectively increased the growth inhibition rate of A549 cells significantly(P0.05).These results suggested additive actions of the two drugs.A549 cells showed G2/M stage arrest after 8-Br-cAMP administration(P0.05).Compared with the control group,expression of EGFR mRNA and EGFR protein significantly decreased in the 8-Br-cAMP group and combining 8-Br-cAMP with DDP group.Conclusions: Both DDP and 8-Br-cAMP could inhibit the proliferation of lung cancer cells in vitro.8-Br-cAMP has also shown synergistic antitumor effects when combined with DDP.8-Br-cAMP can decrease the expression of EGFR and block the cell cycle at G2/M stage.
Human lung
MTT assay
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Objective To study the effect of inhibitor of cyclooxygenase-2 on the expression of vascular endothelial growth factor(VEGF)in A549 cell lines of non-sman cell lung cancers. Methods The proliferation of A549 lung carcinoma cells was measured by MTT assay After A549 cells were treated with different dose celecoxib; After A549 cells were treated with celecoxib 50μmol/L for 48 h, the level of VEGF m RNA was detected RT-PCR, and the expressions of VEGF protein was detected by western blot method. Results Celecoxib could inhibit the proliferation of A549 cell by a time-and dose-dependent manner. Compared with control group, the level of VEGF m RNA was decreased significantly detected by RT-PCR after A549 cells were trealed with celecoxib 50μmol·L-1for 48 h, P0.01 and the expression of VEGF protein was decreased significantly detected by western blot method, P0.01.Conclusion celecoxib might inhibite the proliferation of A549 lung carcinoma cells by regulating the expressions of VEGF.
Celecoxib
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Objective To observe the inhibition of cilengitide on tumor growth in a nude mouse model with lung adenocarcinoma cell line A549 and to explore its possible mechanism, in addition, to observe the therapeutic effect of cilengitide combined with cisplatin. Methods A549 cells were inoculated subcutaneously into the hind flank region of nude mice to establish xenograft models. The nude mice were randomly divided into 6 groups, control group(sodium chloride), cisplatin alone group, cilengitide alone(100 μg/d) group, cilengitide alone(200 μg/d) group, cilengitide(100 μg/d) plus cisplatin group and cilengitide(200 μg/d) plus cisplatin group. The tumor growth was observed. The expression of integrin β3 and β5 were determined by Western blot, and the expression levels of osteopontin(OPN), phosphorylated extra-cellular signal-regulated protein kinase 1(p-ERK1), vascular endothelial growth factor(VEGF) mRNA and protein were detected by reverse transcription polymerase chain reaction(RT-PCR) and Western blot, respectively. After cultured in vitro with cilengitide, VEGF agonist basic fibroblast growth factor(bFGF), vascular endothelial growth factor inhibtor(SU5416), ERK1 agonist epidermal growth factor(EGF) and ERK inhibitor PD98059, the phosphorylated ratio of ERK1 and VEGF protein expression in A549 cells were detected. Results Compared with the control group, OPN mRNA and protein expression in cilengitide groups were not significantly changed, while the expression of p-ERK1, VEGF mRNA and protein were significantly decreased in cilengitide groups. Moreover, tumor growth and the expression of p-ERK1, VEGF mRNA and protein were significantly decreased in cilengitide plus cisplatin groups compared with those in cisplatin alone groups. Conclusion Cilengitide could inhibit the tumor growth and enhance the effect of cisplatin in a nude mouse model with lung adenocarcinoma cell line A549, which may be involved in inhibiting ERK1 activation and VEGF expression.
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Angiogenesis, the growth of new blood vessels, plays an important role in tumor growth and metastasis. Both cetuximab and endostatin have been found to reduce the expression of endothelial-stimulating growth factors such as vascular endothelial growth factor (VEGF) and interleukin (IL)-8. However, the effects of cetuximab alone or in combination with endostatin on human lung adenocarcinoma cell growth remain unclear.The aim of this study was to evaluate the cellular and molecular effects of cetuximab alone and in combination with endostatin on human lung adenocarcinoma cell lines HI 299, SPC-A1, and H460 in vitro. Methods The epidermal growth factor receptor (EGFR) status of a panel of human lung adenocarcinoma cell lines was characterized using Western blot analysis. We used a modified tetrazolium salt assay to evaluate the growth-inhibitory effects of cetuximab and endostatin alone and in combination on the cell lines. We also determined the effects of these 2 drugs on VEGF and IL-8 expression using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Cells were treated for 4 days with cetuximab 12.5 μ/mL, endostatin 25 μ/mL, or cetuximab 12.5 μg/mL + endostatin 25 μg/mL. Untreated cells cultured for 4 days served as controls.EGFR expression in the H1299 cells was higher than in the SPC-A1 and H460 cells. Varying concentrations of cetuximab alone were associated with a significant growth-inhibitory effect on all 3 cell lines in a dose-dependent manner after 4 days of exposure compared with controls (all, P < 0.05). Compared with controls, varying concentrations of endostatin alone were not associated with significant inhibition of cell growth in any of the 3 cell lines. The inhibitory ratio of cetuximab + endostatin at varying concentrations was significantly greater than that of cetuximab alone (all, P < 0.05). On ELISA, either drug alone was associated with significant reductions in secreted VEGF and IL-8 in the HI 299, SPC-A1, and H460 cell lines (all, P < 0.05), with the exception of IL-8 concentration in the H460 cells. Mean (SD) VEGF expression with combination treatment in the H1299 and SPC-A1 cell lines (687 [21] and 629 [23] pg/mL, respectively) was significantly lower than with cetuxi-mab alone (878 [31] and 708 [20] pg/mL; both, P < 0.001); in the H460 cell line, combination treatment was not associated with a significant further reduction in VEGF expression. IL-8 concentrations with cetuximab in the H1299, SPC-A1, and H460 cell lines were 628 (20), 484 (29), and 532 (28) pg/mL, respectively, while the IL-8 concentrations with the combination treatment were 516 (20), 480 (18), and 467 (30) pg/mL. An enhanced effect of endostatin on IL-8 was observed in the H1299 and H460 cell lines (P < 0.001 and P = 0.018, respectively); however, no enhanced effect in the SPC-A1 line was observed. Similar results for VEGF and IL-8 expression were found using Western blot analysis.The results from this in vitro study suggest that cetuximab treatment might both inhibit human lung adenocarcinoma cell line growth and reduce the expression of VEGF and IL-8, which are the biomarkers of angiogenesis. Endostatin was not associated with inhibition of human lung adenocarcinoma cell line growth directly. Findings with the combination of cetuximab + endostatin suggest that endostatin might enhance the antiangiogenic and antitumor activity of cetuximab through an apparent effect on VEGF expression and, to a lesser degree, on IL-8 expression.
Endostatin
Interleukin 8
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OBJECTIVE:To study the effects of chemical drugs Vp-16,DDP and TXT on the gene expression of death receptors DR4 and DR5 in A549 cells. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was applied to semi-quantitatively assay mRNA expression of death receptors DR4 and DR5 in human lung adenocarcinoma cells A549 before and after the treatment of sub-toxic doses of Vp-16,DDP and TXT for different hours. RESULTS: 1)Sub-toxic dose of Vp-16 up-regulated the expression of DR4 and DR5 mRNA in A549 cells. The ratios of DR4/β-actin and DR5/β-actin in A549 cells treated by Vp-16(7.5 μg/mL) for 8 h were 1.13±0.13 and 1.06±0.25, and the ratios treated for 12 h 1.18±0.20 and 1.02±0.02. There were significantly statistical differences between the above two groups and normal control group, P 0.05( P =0.04,0.02,0.00 and 0.02).2)Sub-toxic doses of DDP or TXT had no significant effects on the expression of DR4 and DR5 mRNA in A549 cells. The expression of DR4 and DR5 mRNA in A549 cells treated by DDP(5 μg/mL) or TXT(0.005 μmol/L) for 4 h and 8 h was not significantly higher than that of normal control groups, P 0.05. CONCLUSIONS: The effects of reversal by drugs on TRAIL apoptosis pathway and mechanisms of resistance to TRAIL are diverse. Some of chemical drugs can up-regulate the expression of death receptors DR4 and DR5 mRNA in tumor cells. At the same time, the possibility of existence of other mechanisms can not be excluded simply.
Human lung
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Effect of 4-HPR and DDP on proliferation and expression of Akt /Bax in human ovarian cell line SKOV3
Objective To study the effect of 4-HPR and DDP on proliferation and expression of Akt/Bax in human ovarian cell line SKOV3. Methods The effect of 4-HPR and DDP on SKOV3 cell proliferation was measured by MTT assay. The cell cycle was analyzed by flow cytometry ( FCM) . The Akt / Bax expressions of SKOV3 cells were detected by FCM. Results DDP combined with 4-HPR ( 1,5 and 10 μmol / L ) significantly inhibited the proliferation of ovarian cancer SKOV3 cells,which had a statistical difference compared with the only-used DDP group ( P 0. 05) . 4-HPR combined with DDP significantly arrested SKOV3 cell at G2-M and S stage compared with only-used DDP group( P 0. 01) . Apoptosis rate of 4-HPR combined with DDP group was significantly higher than only-used DDP group( P 0. 01) . 4-HPR combined with DDP affected the expressions of Akt / Bax compared with only-used DDP group( P 0. 01 ) . Conclusion To use 4-HPR combined with DDP can significantly inhibit the proliferation and block cell cycle of ovarian cancer SKOV3 cells,which is associated with Akt / Bax expressions.
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Lung cancer is one of the most malignant cancers which is hazarding the people's health and life in the world. At present, it is a highlight to exploit antitumor drug from plant at home and abroad. The aim of this study is to observe the effects of polysaccharid (PS-T) on expression of angiogenic-related gene mRNA in human high-metastatic large cell lung cancer cell line L9981, and to explore its possible molecular mechanism.L9981 in vitro was cultured, and the growth data were obtained by trypan blue staining. The mRNA transcript expression of β-catenin, E-cadherin, TIMP-1, CD44V6, MMP-2, endostatin, VEGF was detected in L9981 by RT-PCR before and after treating with PS-T. The ability of invasion of L9981 was determined by Boyden chamber method.(1)PS-T had remarkably inhibitive effects on the growth of L9981 in vitro. The inhibitive rate of PS-T on L9981 was concentration-dependent. No significant difference of inhibitive rate was found among the PS-T (1g/L), cisplatin (3mg/L) and PS-T (0.05g/L) + cisplatin (1.5mg/L)(P > 0.05). (2)The mRNA expression level of β-catenin, E-cadherin, TIMP-1, endostatin and MMP-2 was upregulated, while that of VEGF and CD44V6 was downregulated. Out of them the mRNA expression level of TIMP-1 and endostatin was remarkably upregulated, the expression level of CD44V6 was significanyly downregulated. (3)The in vitro invasive abilities of L9981 was significantly decreased in the PS-T, DDP and PS-T+DDP groups compared with that in blank control group.(1)PT-S could inhibit the growth of human high-metastatic large cell lung cancer cell line L9981 in vitro, the effect is dose-dependent. (2)PS-T can down- or up-regulate the mRNA transcript expression of some angiogenic-related gene mRNA. (3)PS-T has remarkably coordinating effects with cisplatin in the L9981 lung cancer cell line.
Endostatin
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Objective:This study was designed to compare the effects of STI 571 on proliferation,cell phases,and apoptosis between A549 and cisplatin-resistant A549 cells(A549/DDP)and detect the expressions of STI 571-related receptors in non-small cell lung cancer(NSCLC) tissues.Methods:A549 and cisplatin-resistant A549 cells(A549/DDP)were cultured in vitro.The cell proliferation was measured by MTT assay.Cell cycle distribution and apoptosis induced by STI 571,cisplatin and their combination were detected by flow cytometry in NSCLC cell lines.The expressions of platelet-derived growth factor receptor(PDGFRs)-α and-β and c-KIT in the non-small cell lung cancer cell lines and tissues were investigated by immunocytochemistry and immunohistochemistry,respectively.Kaplan-Meier survival curves were used to compare the correlation of PDGFR-α and-β expression with total survival of NSCLC patients.Results: STI 571 inhibited proliferation of A549/DDP cells,sensitized them to cisplatin chemotherapy,increased the cell number in G2/M and S phase,and induced apoptosis.However,STI 571 at the concentration below 10 μmol/L had no effect on the proliferation of A549 cells.Both A549 and A549/DDP cells expressed PDGFR-α and-β,but c-KIT expression was only observed in A549/DDP cells.The expression rates of PDGFR-α and-β were 65.5% and 69% in pulmonary adenocarcinoma,similar to those in squamous carcinoma(70.4% and 74.1%).Only one case of squamous carcinoma expressed c-Kit(3.7%).The PDGFR-α or β-positive patients had similar 3-year survival rates and overall survival time with the PDGFRα or β-negative patients.Conclusion: STI 571 could suppress proliferation of A549/DDP cells,induce apoptosis and increase the sensitivity of A549/DDP to cisplatin.The levels of PDGFRα and β expression did not correlate with the prognosis of NSCLC patients.
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