Altered Protein Expression Profiles Of HBM-MSCs Induced By1,4-BQ

Benzene exposure could result in leukemia and other blood disorders. It is generally accepted that in order to exert its cytotoxicity, benzene has to be metabolised. First, it is metabolised by P4502E1in the liver to phenol, catechol, and hydroquinone. These metabolites are transported to the bone marrow and further oxidised to1,4-BQ. As a major metabolites of benzene,1,4-BQ induces the majority of the toxic damage attributed to benzene exposure. The toxic effects of benzene are affected by the complex intercellular interactions present in the bone marrow microenvironment. Human bone marrow mesenchymal stem cells (hBM-MSCs) localizing in the bone marrow are required for the marrow microenvironment maintenance.Previously, using a proteomics-based two-dimensional gel electrophoresis (2-DE) assay, we have shown that exposure of human liver cells to hydroquinone induced significant differential proteomic expression. In the present study, using a similar approach we further investigated the protein expression profiles in hBM-MSCs treated with different concentrations of1,4-BQ (10,25and50uM) for24h. We chose the differential expression spots using the criteria as following:spot intensity was at least1.5-fold greater than the corresponding spot on control gels, with a P-value of less than0.05. According to the criteria,75protein spots were found to be differentially expressed. Forty-two protein spots were chosen for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and of these,32were identified. Sixteen of the identified proteins were up-regulated, whereas16were down-regulated. Thirty-two proteins with differential expression identified by LC-MS/MS were cytoplasmic(56.25%), nuclear(18.75%), secreted protein located in mitochondrion, endoplasmic reticulum and lysosome (15.63%), or membrane-bound(9.38%). Most of them had been reported to be involved in various aspects of biological process in cells. As for their function, the32proteins maybe classified as the following groups: metabolism(25.00%), cell stress response (15.63%), cell skeleton(12.5%), transcription regulation(12.5%), membrane trafficking (6.25%), signal transduction (6.25%), and miscellaneous function (21.88%).The changed protein expression of HSP27, Vimentin, Copinel, and Copine3was further confirmed by Western blot analysis. No statistically significant difference of HSP27, Vimentin, and Copine3protein expression was observed in10and25μM1,4-BQ-treated hBM-MSCs. However, the protein expression level of HSP27and Copine3significantly decreased while the protein expression level of Vimentin significantly increased in50μM1,4-BQ-treated hBM-MSCs. These results mentioned above consisted with the results of2-DE. Results of Western blot analysis showed that no statistically significant difference of Copinel protein expression was observed in10|iM1,4-BQ-treated hBM-MSCs while the protein expression level of Copinel significantly decreased in50μM1,4-BQ-treated hBM-MSCs. It was consistent with the proteomics results. However the Copinel was also significantly down-regulated in hBM-MSCs treated by25μM1,4-BQ, which was not consistent with our2-DE analysis results.To examine whether low expression of HSP27, Copinel, Copine3and overexpression of Vimentin proteins in1,4-BQ-treated hBM-MSCs was attributable to transcriptional regulation, we conducted quantitative real-time RT-PCR analysis of mRNA expression of the four proteins verified by Western blot analysis. Significant difference of the mRNA level of HSP27and vimentin was observed between50μM1,4-BQ-treated and non-treated cells. No significant difference in10and25μM1,4-BQ-treated group was seen at mRNA level. This result indicated that the protein expression level of HSP27and Vimentin is correlated with their mRNA expression. The copinel and copine3was down-regulated at transcriptional level when cells were treated with1,4-BQ above lOμmol/L and25μmol/L respectively while the Western blot results confirmed that both copinel and copine3were also down-regulated treated with1,4-BQ above25μmol/L and50μmol/L respectively. Copine response to1,4-BQ at transcriptional level is more sensetive in comparison with the alteration of protein expression.Together, these data indicate1,4-BQ treatment leads to dramatic changes of protein expression in hBM-MSCs. The differentially expressed proteins may be useful candidate molecular markers of hematopoietic disorders of benzene and the proteome profile of hBM-MSCs treated by1,4-BQ found in this study is likely to give important further insights into the molecular mechanisms of hematopoietic toxicity of benzene and its metabolites such as1,4-BQ.