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
Nephrotoxicity
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Objective
To observe the effects of JinShuiXian capsule combined with radiotherapy on the expression of basic fibroblast growth factor (bFGF) and hypoxia inducible factor-1α (HIF-1α) in lung adenocarcinoma A549 cells.
Methods
A549 cells were cultured in vitro. Cells were divided into 4 groups using simple random method: control group, JinShuiXian group, radiation group and JinShuiXian combined with radiation group. The cell growth inhibition rate of each group at different time sets was monitored by methyl thiazol tetrazolium (MTT), the number of apoptotic cells in different groups was observed by TdT-mediated dUTP nick end labeling (TUNEL), and the expression levels of mRNA and protein of HIF-1α and bFGF were detected by real-time quantitative polymerase chain reaction (Real-time PCR) and enzyme linked immunosorbent assay (ELISA).
Results
The proliferation of A549 cells in different groups at different time points showed different degrees of inhibition. Especially at 48 h, the inhibitory rate of combination group was 56.3%, and the JinShuiXian group had the lowest rate (15.8%). As compared with control group, JinShuiXian group and radiation group, the inhibitory rate of cell proliferation in combined group at 48 h was significantly increased (P=0.023). Apoptosis experiments showed that the mean number of apoptotic cells in combined group at 48 h was 32.8, which was significantly increased as compared with other groups (P=0.038). The expression levels of bFGF [(157.408 0±2.830 3) pg/ml, P=0.045] and HIF-1α [(1.455 7±0.000 7) pg/ml, P=0.021] proteins showed a decline trend over time in each group, and those in the combined group were reduced most significantly as compared with other groups. As compared with the control group, bFGF (0.285 4±0.057 9, P=0.005) and HIF-1α mRNA (0.277 1±0.022 1, P=0.011) expression levels in the treated groups showed a downward trend, and at 48 h those in the combined group was the lowest.
Conclusion
JinShuiXian in combination with radiation could significantly inhibit tumor cell proliferation and expression of bFGF and HIF-1α in lung adenocarcinoma A549 cells. JinShuiXian may enhance the inhibitory effect of radiotherapy on lung adenocarcinoma A549 cells through inhibition of bFGF and HIF-1α expression.
Key words:
JinShuiXian; Radiotherapy; Lung adenocarcinoma; Basic fibroblast growth factor; Hypoxia inducible factor-1α
Hypoxia
MTT assay
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[Objective] This study was designed to explore the role of apoptosis- associated genes and proteins on the chemo-resistance in COC1/DDP. [Methods] The apoptotic ratios of COC1 and COC1/DDP were measured with flow cytometry after treated with different concentration of cisplatin for 48 hours. The expressions of apoptosis- associated genes and proteins Bcl-2, Caspase-3, Smac, Survivin were studied by reverse trans-cription polymerase chain reaction (RT-PCR) and Western blot in COC1/ DDP and COC1. [Results] Using DDP to stimulate COC1/DDP and COC1 for 48 hours, the apoptic ratios were gradually increased with the increase of concentration of DDP. The mRNA and protein expressions of Bcl-2 and Survivin in COC1/DDP cells were significantly higher than those in COC1 cells (P 0.05), contrastly Smac and Caspase-3 were significantly lower than those in COC1 cells. [Conclusion] The cisplatin-resistance in human ovarian carcinoma cell lines may be associated with the overexpression of anti-apoptic gene and protein of Bcl-2, Survivin and the downregulation of Caspase-3 and Smac.
Survivin
Inhibitor of apoptosis
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Objective:To investigate the effect of basic fibroblast growth factor(FGF-2) on pim-3 in humanl lung cancer cell A459. Methods:Lung cancer cell A459 was treated by different concentrations (0、25、50、100 ng/mL) of FGF-2,pim-3 mRNA was measured by reverse transcription polymerase chain reaction (RT-PCR),and western blotting was used to detect the expression of PIM-3 protein. Results:The expressions of pim-3 mRNA and protein increased dramaticly in the groups treated with FGF-2 compared with conorol, and there was a significantly difference between each two experiment groups (P 0.01). Conclusion:FGF-2 can significantly upregulate the expression of pim-3 in dosage-dependent manner.
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Objective To investigate the effect of thalidomide on vitro liver cancer cell growth,invasion,metastasis and its osteopontin expression. Methods Human hepatoma cell line Hep-G2 was cultured by conventional culture methods. The cell proliferation of Hep-G2 by CCK-8 at different drug concentrations and times was assayed. Cells scratch test was used to verify the impact of thalidomide on the invasion and metastasis ability of Hep-G2. The level of osteopontin expression of Hep-G2 cells was detected by the method of immunocytochemistry and Western blot after the action of different drugs and different reaction time. Result 400 μg / ml of thalidomide could inhibit cell proliferation,compared with the control group in 24,48,72 h( P 0. 01). And the effects positively correlated with reaction time. At the point of 24、48、72 h,compared with the control group,thalidomide significantly inhibited the cell migration( P 0. 01). The immunocytochemistry score was 1. 728 ± 0. 051 in ThD group and 2. 328 ± 0. 245 in control group( P 0. 01). Thalidomide could inhibit the osteopontin expression( P 0. 01) at 24、48、72 h time point,and the effects positively correlated with reaction time. Conclusions Thalidomide can inhibit the proliferation of hepatoma cells and the expression of OPN. And the effects positively correlate with the drug concentration and reaction time.
Osteopontin
Hep G2
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Objective To study the expressions of NF-κB and VEGF in human ovarian cancer Caov-3 cells after using proteasome inhibitor MG132 combined with DDP.Methods Immunohistochemical assessment was performed for the changes in expressions of NF-κB and VEGF.Results The expression levels of NF-κB and VEGF decreased in Caov-3 cells after being treated with MG132 combined with DDP for 24 hours.The differences were statistically significant(P0.01).Conclusions MG132 could significantly induce the apoptosis of Caov-3 cells.by inhibiting the activation of NF-κB,and thus inhibite the growth of tumor vessel.
MG132
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OBJECTIVE:To investigate molecule mechanism on the Wnt/β-catenin signal transduction pathway mediating drug resistanc in lung adenocarcinoma.METHODS:Human lung adenocarcinoma A549cells and its cisplatin resistance A549/DDP cells were cultured in vitro and the cells in logarithm growth period were used.MTT assay was used to determine IC50values of cisplatin in A549and A549/DDP cells.AnnexinⅤ analysis using flow cytometry and Hoechst33342staining were used to observe apoptosis of A549and A549/DDP cells treated with cisplatin compared to untreated cells.Western blot analysis was used to detect the expressions of β-catenin and survivin in A549and A549/DDP cells.Transient interference of β-catenin by siRNA was used to measure the IC50values,apoptosis and the expressions of β-catenin and survivin in A549/DDP cells.RESULTS:IC50values of cisplatin in A549/DDP cells(28.984±1.404)were higher than those in A549cells(5.888±0.338),t=27.696,P0.001.The 20μmol/L cisplatin induced apoptosis in A549/DDP cells(21.75±0.96)% was significantly lower than that of A549cells(39.38±0.88)%,t=23.474,P0.001.The expression ofβ-catenin protein(1.890±0.060)in A549/DDP cells was significantly higher than that in A549cells(1.063±0.035),t=20.595,P0.001.The expression of survivin protein(1.107±0.061)in A549/DDP cells was significantly higher than that in A549cells(0.503±0.025),t=15.814,P0.001.In A549/DDP cells,the IC50of cisplatin after knockdown of β-catenin was(14.615±0.939)μmol/L,significantly lower than that before knockdown of β-catenin(28.984±1.404)μmol/L,t=14.732,P0.001.The proportion of early apoptotic cells after knockdown of β-catenin was(37.57±0.64)%significantly higher than that before knockdown of β-catenin(21.75±0.96)%,t=23.699,P0.001.The expression of survivin protein after knockdown of β-catenin was(0.527±0.065)remarkably lower than that before knockdown of β-catenin(1.027±0.025)and that of positive group of transient transfection(1.033±0.040),F=116.944,P0.001.CONCLUSION:Inhibition of Wnt/β-catenin signal transduction pathway can reduce the resistance to cisplatin in A549/DDP cells.
Survivin
MTT assay
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