Nuclear Proteome Analysis Of Benzo(A)Pyrene Treated HeLa Cells

[Background] Polycyclic aromatic hydrocarbons (PAHs) are common pollutants found in our daily living environments, which is mainly produced by the incomplete combustion of organic materials, such as residential heating, refuse incineration and vehicle exhausts. As immunotoxic and carcinogenic environmental contaminants, PAHs exert various toxic effects toward human health, including carcinogenic, immunosuppressive, atherogenic, and inflammatory effects. BaP is a powerful carcinogen and has been used as a model chemical for PAHs. It is thought to cause cancer through covalent binding of its reactive metabolite(BaP-7,8-diol-9,10-epoxides, BPDE) to DNA, forming DNA adducts. Presently, the BaP-induced DNA damage response (DDR) has been extensively studied, in an effort to understand the mechanisms of BaP-induced mutagenesis and carcinogenesis [5].However, most of these studies focus on whole-cell proteomic measures, one disadvantage of which is their limited ability to detect,’low-abundance’proteins. Therefore, it is necessary to use subcellular fractionation, or organelle proteomics to identify changes in the expression levels of those lower-abundance proteins, such as nuclear proteins, which play pivotal roles in controlling cellular processes, including mutagenesis and carcinogenesis. In addition, DNA damage response initiates in cell nuclei, which will cause changes in expression of many nuclear proteins. Thus, in the present study, we mainly examined the nuclear proteosome response of HeLa cells to different concentrations of BaP.[Objective] In order to further understand the mechanisms of BaP-induced DNA damage response, and to identify the underlying novel signal pathway, we used high-throughput technologies to analyze the nuclear proteomic response of HeLa cells to different concentrations of BaP.[Methods]1. HeLa cells were treated by different concentrations of BaP for6h, nuclear proteins were extracted by the Nuclear Protein Extract Kit, and protein concentration was determined by Bradford method.2. Nuclear proteins were subjected to two-dimensional Polyacrylamide Gel Electrophoresis(2D-PAGE) combined with mass spectrometry(MS) identification3. Proteins identified successfully by MS were searched in Swiss-Prot database, and classified according to their functions.4. The functions of some of the identified proteins were evaluated by siRNA interference, Western Blot, etc.[Results]1. Compared with control group,125protein spots in experimental groups showed changed expression after BaP exposure for6h by2D-PAGE analysis (79proteins were upregulated, and46proteins were downregulated).39proteins were identified by MS successfully.2. The results of Western blot were consistent with that of2D-PAGE:the expression of ANXA1increased significantly in HeLa cells after BaP (1μM) treatment.3. After siRNA interference, the expression of ANXA1was reduced significantly, while the DNA damage induced by BaP increased significantly (as indicated by the formation of γH2AX foci). However, no significant effect on cell apoptosis was observed.4. The expression of NF-κB in cell nucleus was increased while its expression in the cytoplasm was decreased as a consequence of the decreased expression of P28GANK [Conclusion]1. BaP treatment led to changes in HeLa cell nuclear protein expression profiles.2. ANXA1can protect cells from BaP-induced DNA damage.3. NF-κB pathway might be involved in BaP-induced DNA damage response.