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[ ABSTRACT] AIM:To investigate the role of canonical transient receptor potential channel 1 ( TRPC1 ) in the epithelial-mesenchymal transition ( EMT) of human bronchial epithelial ( HBE) cells induced by transforming growth fac-tor-β1 (TGF-β1).METHODS:EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluores-cence and Western blotting.Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells.The influence of SKF96365 (a TRPC1 blocker) and siRNA-me-diated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting.RESULTS:Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells.Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells.Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive.The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 ( P<0.05).The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silen-cing compared with TGF-β1 group.The protein expression of E-cadherin andα-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05).CONCLUSION:TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.
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Objective To investigate the mechanism of malignant transformation in human bronchial epithelial cell line BEP2D exposed to α-particles.Methods The levels of intracellular ROS and malonaldehyde (MDA) in BEP2D,RH22 (passage 22 of α-particle-irradiated BEP2D cells) and BERP35T-1 cells (derived from nude mice bearing malignant transformed cells generated from the passage 35 of α-particle-irradiated BEP2D cells) were assayed with DCFH-DA and MDA kit,respectively.The expressions of 8-OH-dG and γ-H2AX in BEP2D,RH23 (passage 23 of α-particle-irradiated BEP2D cells)and BERP35T-1 cells were also measured with immunocytochemistry and immunofluorescence staining.Results Compared to BEP2D cells,the levels of ROS ( t =4.30 and 3.94,P ＜ 0.05 ) and MDA ( t =4.89 and 15.10,P ＜0.05) increased in RH22 and BERP35T-1 cells.The expressions of 8-OH-dG (t =3.80 and 2.92,P ＜ 0.05 ) and γ-H2AX ( t =7.61 and 12.67,P ＜ 0.05 ) in RH23 and BERP35T-1 cells were also higher than those in BEP2D cells.Conclusions Oxidative stress induces lipid peroxidation and DNA damage leading to genomic instability,which could contribute to cellular malignant transforming process in the human bronchial epithelial cell line BEP2D with α-particle exposure.
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Objective To investigate the antioxidant ability and radiosensitivity in the malignant transformed human bronchial epithelial cell line BEP2D induced by α-particle exposure.Methods Glutathione Peroxidase (GPX),Catalase (CAT) and Glutathione (GSH) assay kits were employed to detect GPX and CAT enzyme abilities and the levels of GSH in BEP2D,RH21 ( passage 21 of α-particle-irradiated BEP2D cells),and BERP35T-1 cells (derived from nude mice bearing malignant transformed cells generated from cells of passage 35 of α-particle-irradiated BEP2D cells).MTT assay were used to test the growth rate of BEP2D,RH21 and BERP35T-1 cells treated with 0,30,60,90,120,and 150 μmoL/L H2O2.Colony-forming test and MTT assay were used to examine the cell survival fraction and the growth rate of BEP2D,RH21 and BERP35T-1 cells exposed to 0,2,4,and 8 Gy of γ-rays,respectively.Results GPX and CAT enzyme activities in RH21 and BERP35T-1 cells were obviously lower than in BEP2D( t = 5.75-67.92 ,P ＜ 0.05 ).CAT enzyme activity in BERP35T-1 was lower than that in RH21 cells (t =22.25 ,P ＜0.01 ).Compared to BEP2D cells,decreased level of GSH was detected in BERP35T-1 cells(t = 7.76,P ＜ 0.05 ),but there was no change in RH21.After treatment with 30,60,90,120,and 150 μmol/L H2O2,the growth rates of BEP2D were all higher than those of BERP35T-1 cells(t = 10.37-58.36,P ＜0.01 ).Meanwhile,the growth rates of BEP2D were all higher than those of RH21 cells after treatment with 60,90,120,and 150 μ mol/L H2O2 (t = 29.90-84.68,P ＜ 0.01 ).In addition,compared to BEP2D cells,decreased cell survival fraction and growth rate of BERP35T-1 cells were observed after irradiation with 2,4,and 8 Gy of y-rays ( t = 5.87-34.17,P ＜ 0.05 ).The cell survival fraction and growth rate of RH21 were all lower than those of BEP2D cells at 4 and 8 Gy post-irradition( t =6.33- 45.00,P ＜ 0.05 ).Conclusion The function of antioxidant system decreased in the α-particleinduced transformed cells,which could contribute to the acceleration of cellular malignant transforming process and radiosensitivity.
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Objective To identify the changes of DNA methylation profile in the process of malignant transformation of BEP2D cell induced by α particles.Methods The genomic DNAs were isolated from the malignant transformation BERP35T4 cells and immortalized human bronchial epithelial cell line BEP2D.Genomic DNAs were digested by MseI and ligated of PCR linkers.Methylated DNAs were digested by BstUI and amplified by PCR.The methylated DNA probes were prepared by labeling with Cy3 and Cy5 fluorescence dyes individually and hybridized to the methylation CpG-Island microarray.The hybridization results were scanned and analyzed.Intensity values were quality controlled and normalized.The normalized data were used to identify the differentially expressed genes based on a 1.5 fold difference of the expression level.Results There were 16 genes which showed changes of methylation level in malignant transformation BERP35T4 cells, 9 of them were hypermethylation and 7 were hypomethylation.These genes were including the SKIP gene, PPP3CC gene, MAP2K6 gene, KIR2DL1 gene, KIR2DL4 gene, KIR3DP1 gene, ZNF493 gene, ZNF100 gene, NKX2-5 gene, TFAP2D gene, DR1 gene, KCNJ16 gene, CCDC18 gene, FNBP1L gene, IRX4 gene, EPB41L3 gene, TCP10 gene and so on.Conclusions The DNA methylation might have effects on ionizing radiation drived tumorigenesis.
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Objective To investigate the role of Rho in SiO2 induced α-SMA expression in human bronchial epithelial cells (HBECs). Methods HBECs were cultured and stimulated with SiO2. Immunocytochemistry and Western blot were used to detect the expression of α-SMA. The activity of Rho was determined by GST pull down assay. In the prevention experiment,SiO2-stimulated HBECs were incubated with Rho inhibitor Y27632,and the expression of α-SMA was examined by Western blot. Results With SiO2 (0-300 μg/mL) treatment,the expression of α-SMA increased gradually,and 200 μg/mL of SiO2 led to the highest expression of α-SMA which was (5.09±1.98) times of the expression of α-SMA in the control group(P＜0.01). HBECs treated with SiO2 (200 μg/mL) for indicated time (1,2,6,12,and 24 h)showed an obvious increase of Rho activity(P＜0.01). Y27632 inhibited SiO2-induced α-SMA expression significantly,and the inhibition rate of 20 and 30 μmol/L Y27632 was 68% and 75%,respectively (P＜0.01).Conclusion Rho signaling pathway may mediate SiO2 induced α-SMA expression in HBECs.
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Objective To explore the effect of BPDE on the expression of N-Ras in the human bronchial epithelial cell line. Methods The levels of mRNA and protein expression in BPDE transformed 16HBE cells(16HBE-T) and untransformed control 16HBE cells(16HBE-N) were examined by using RT-PCR and Western blot. Locations and expression levels of protein in both kinds of cells were analyzed by immunocytochemical method. Results Compared with 16HBE-N, the levels of mRNA and protein of N-Ras significantly increased to 3.616 and 1.600 times in 16HBE-T. Immunocytochemical method showed N-Ras protein in 16HBE-T and 16HBE-N expressed in the cytomembrane and cytoplasm, but the expression level of protein in 16HBE-T was significantly higher than that in 16HBE-N. Conclusion The up-regulated expression of N-Ras oncogene may play an important role in the malignant transformation of 16HBE induced by BPDE
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Objective To explore the mechanisms of chemical carcinogenesis and the relationship between malignant transformation and genomic/chromosomal instability of human bronchial epithelial cell line by formaldehyde.Methods BEAS-2B cell line was toxicated by formaldehyde and the LC_(50) of formaldehyde was tested by MTT assay.BEAS-2B cells were induced by formaldehyde at 20% LC_(50),and the transformation of BEAS-2B cells was identified and induced clones were selected.By G-banding technique,the metaphase modal chromosome number,karyotypes and nonrandom structural change of induced cell strains were analyzed.Results The growth rate of BEAS-2B cells induced by formaldehyde was significantly increased.The induced cells showed a trend of infinite proliferation and the anchorage independent growth was seen in 15~(th) generation cells.As compared with the relatively stable pseudodiploid karyotype of BEAS-2B cells,most of induced cells were triploid and/or tetraploid karyotypes and their chromosome 14 had lost two copies of chromosome replaced by the other two.They also had high frequency of unstable aberration(39%),including chromosome loss,endoduplication,translocation,breakage,two and/or three centromeres etc..Conclusion The formaldehyde could lead to progression of malignant transformation and result in genomic instability of human bronchial epithelial cells in vitro.
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Purpose:To detect the abnormalities of WWOX(WW domain containing oxidoreductase) gene in human lung adenocarcinoma cell line A549. Methods:Deletion of WWOX exons 6-8 transcript was analyzed by reverse transcriptase-PCR technology; loss of heterozygosity (LOH) of WWOX gene was analyzed by PCR-based assays for dinucleotide repeat polymorphisms technology. Aberrant expression of WWOX protein was analyzed by western blot. Results:A549 cells samples showed loss of WWOX exons 6-8 transcript.This deletion was not detected in normal primary cultured human bronchial epithelial cells samples.Three microsatellites(D16S3029、D16S3096、D16S504)did not have LOH in the normal primary cultured human bronchial epithelial cells samples, but D16S2029 and D16S3096 were all found to have LOH in A549 Cells samples. We further observed that expression of WWOX protein was significantly lower in A549 cell samples compared to the normal primary cultured human bronchial epithelial cells samples. Conclusions:WWOX gene may be important during tumorigenesis in lung adenocarcinoma cancer.Deletion of exons 6-8,LOH and aberrant expression of protein are all modes of WWOX gene inactivity in lung adenocarcinoma cancer.