The Expression Of NF-κB In Malignant Transformation Of BEAS-2B Cells Induced By Coal Tar Pitch Smoke Extract Promoting By Tumor Associated Macrophages

Background Epidemiological and genetic data revealed that chronic inflammation could promote tumorigenesis. Tumor-associated macrophages (TAM) was major regulators between inflammation and cancer. TAM which was created in the tumor microenvironment displayed the function to promote tumorigenesis. Nuclear factor kB (NF-kB) was a key connection between inflammation and cancer. Lung cancer induced by coal tar was a main component of occupational tumors in coke oven workers. There were few reports about the mechanism how inflammation performed in the process of carcinogenesis induced by the mixture of coal tar pitch.In this study, middle temperature coal tar pitch smoke extract was used to induce the malignant transformation of immortalized human bronchial epithelial cells (Human Bronchial Epithelial Cells, BEAS-2B). The BEAS-2B and THP-1 cells co-cultured model 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 to provide a theoretical basis for the prevention of occupational lung cancer caused by CTP, the functions of NF-kB signaling pathway in the process of carcinogenesis were explored by studying the changes of NF-kB gene and protein expression.Methods BEAS-2B cells were used as objects. Four groups were established, the extracts of coal tar pitch exposure group (CTP group), benzo(a)pyrene (B(a)P) group, dimethyl sulfoxide (DMSO) group, BEAS-2B and THP-1 co-culture (Co-culture group) group. Carcinogenesis model was established. The malignant of cells was estimated by soft agar colony formation assay, chromosome aberrations and cell cycle tests. The expression of NF-kB p65 mRNA was detected by RT-PCR and the expression of NF-kB p65 protein was detected by Western Blot.Results:1 Cell cycle tests:Cell cycle analysed by Flow cytometry showed that compared with DMSO group, the extract of coal tar pitch exposure group, benzo (a) pyrene group and co-culture group in 10th generation,20th generation and 30th generation displayed a reduction of Gl phase cells and an increase of S phase cells. That indicated the three groups of exposed cells showed tendency of accelerated growth. The S phase cells of the co-culture group in the 20th generations were more than extraction of coal tar pitch smoke exposure group and benzo(a)pyrene group, the difference was statistically significant(P=0.001, P=0.001). To the 30th generations, the difference of proportion of S phase cells were more obviously.2 Aberrations of chromosome number: The number of chromosome abnormalities could be observed in early stage of the experiment, the performance was an increase of the ratio of aneuploid and polyploid, and a decrease in the number of diploid..3 Soft agar colony formation:The number of the colonies was less than 20 in the 10th generation of each group. To the 20th generation cells, compared with the co-cultured group, the number of colonies formation in the extraction of coal tar pitch smoke exposure group and benzo(a)pyrene group and the difference were statistically significant(P=0.001, P=0.001). The colonies formation of the co-culture group was more efficiency than the other two groups. To the 30th generation cells, compared with benzo(a)pyrene group and extracts of coal tar pitch smoke exposure group, there were more colonies in the co-culture group and the difference was statistically significant(P=0.001, P=0.001). Colonies of the extracts of coal tar pitch smoke exposure group were higher than the benzo(a)pyrene group and the difference was statistically significant(P=0.032).4 NF-kB p65 mRNA and protein expression:In the 10th generation cells, compared with DMSO group, the expression of NF-kB p65 of coal tar pitch group, benzo(a)pyrene group and co-culture group was increased. However, there was no difference among benzo(a)pyrene group, the coal tar pitch group and the co-culture group. In the 20th generation cells, analysis showed exposed group expressed high levels of NF-kB p65 than DMSO group, and co-culture group expressed high levels of NF-kB p65 than the others exposed group. In the 30th generation cells, co-culture group expressed high levels of NF-kB p65 than the others exposed group.The expression of NF-kB p65 in benzo(a)pyrene group was statistically lower than the coal tar pitch group.Conclusion:The extracts of coal tar pitch smoke could induce malignant transformation of human bronchial epithelial cells. NF-kB signaling pathway involved in the malignant transformation process of BEAS-2B which was accelerated by the present of THP-1 cells.