The Function Of Tnf-α And Nf-κB In Malignant Transformation Of BEAS-2B Cells Induced By Coal Tar Pitch Smoke Extract Promoting By THP-1Cells

Background:A number of animal experiments and epidemiological data revealed that there was a causal relationship between chronic inflammation and tumorigenesis. Tumor-associated macrophages (TAM) were inflammatory cells which existed in the tumor microenvironment and played a crucial role in the development, survival and growth of tumor cells. Nuclear factor-kappa B (NF-κB) was a key connection between inflammation and cancer, and regulated cell proliferation and apoptosis. Tumor necrosis factor-a (TNF-a) which was secreted by tumor-associated macrophages was a tumor promoting cytokine. Coal tar pitch was widely present in industrial production. The World Health Organization and International Agency for Research on Cancer has recognized it as a definite carcinogen for lung cancer. There were lots of studies focus on the mechanism how the coal tar pitch caused lung cancer. But there were few reports about the mechanism how inflammation performed in the process of lung cancer caused by Coal tar pitch. Therefore, further research was needed.In this study, human bronchial epithelial cells (BEAS-2B) was exposed to mid-temperature coal tar pitch fume extract.The co-cultured model of BEAS-2B and THP-1cells was established to explore the function and effect of TAM in the malignant transformation of the BEAS-2B cells, in order to provide early diagnosis biomarkers of lung cancer and a theoretical basis for the prevention of occupational lung cancer caused by CTP, the functions of NF-κB signaling pathway and TNF-a in the process of carcinogenesis were explored by studying the changes of NF-κB gene and protein expression.Methods:1Detection of two kinds of culture supernatant of cells with TNF-a expression levels:The BEAS-2B and THP-1cells co-cultured model were used as objects, Cultivate than as a10:1, in the exposure of coal tar pitch fume extract. The BEAS-2B cell was in the exposure of coal tar pitch fume extract, too. Collection of cell culture metabolism supernatant, ELISA method was used to detect the expression levels of cytokines TNF-α.2There were three groups, BEAS-2B and THP-1co-culture (Co-culture group) group, PDTC groups and TNF-a Neutralizing antibody group. The malignant of cells was estimated by soft agar colony formation assay, chromosome aberrations and cell cycle tests.3The expressions of NF-κB p65, TRAF2, CyclinDl mRNA were determined by RT-PCR and the expression of TRAF2, CyclinDl, NF-κB p65protein were determined by Western Blot.Result:1TNF-a expression level:The concentration of TNF-a in the co-culture group was more than CTP group, the difference was statistically significant (P<0.05).2The malignant transformation of cells:2.1Cell cycle tests:Cell cycle analysed by Flow cytometry showed that control groups in20th generations compared with its10th generations of cells displayed a reduction of G1phase cells and an increase of S phase cells. The S phase cells of the PDTC group and TNF-a Neutralizing antibody group in the20th generations were less than control group (P<0.05).2.2Aberrations of chromosome number:The number of chromosome abnormalities could be observed in the10th generations control group, In the20th cells, control group compared with its10th generations of cells, there was an increase of the ratio of aneuploid and polyploid. compared to the PDTC group and TNF-a Neutralizing antibody group, Low rate of chromosomal aberrations and the difference was statistically significant (P<0.0083).2.3Soft agar colony formation:The number of the colonies was less than20th in the10th generation of each group. In the20th generation cells, compared with control group, the number of colonies formation in the PDTC group and TNF-a Neutralizing antibody group was significantly less and the difference were statistically significant(/’<0.05).3The expressions of Gene and protein:3.1mRNA expressions of TRAF2, CyclinD1and NF-κB p65:Compared with control group, the mRNA expressions of TRAF2, Cyclin D1and NF-κB p65of the PDTC group and TNF-a Neutralizing antibody group were less. And the difference was statistically significant (P<0.05).3.2Protein expressions of TRAF2, CyclinDl and NF-kB p65:Compared with the control group, the Nuclear proteins expressions of NF-κB p65of the PDTC group and TNF-a Neutralizing antibody group was less; compared with control group, the total protein expressions of TRAF2, CyclinD1of the PDTC group and TNF-a Neutralizing antibody group were less, and the difference was statistically significant (P<0.05).Conclusion:Malignant transformation of human bronchial epithelial cells induced by coal tar pitch extracts, Tumor-associated macrophages may promote this process through the secretion of cytokines TNF-a which may activate, TNF-a may play the role of promoting tumor through non-classical signal transduction pathway to activate NF-κB.