The role of GATA switch in benzene metabolite hydroquinone inhibiting erythroid differentiation in K562 cells.

The phenolic metabolite of benzene, hydroquinone (HQ), has potential risks for hematological disorders and hematotoxicity in humans. Previous studies have revealed that reactive oxygen species, DNA methylation, and histone acetylation participate in benzene metabolites inhibiting erythroid differentiation in hemin-induced K562 cells. GATA1 and GATA2 are crucial erythroid-specific transcription factors that exhibit dynamic expression patterns during erythroid differentiation. We investigated the role of GATA factors in HQ-inhibited erythroid differentiation in K562 cells. When K562 cells were induced with 40 µM hemin for 0-120 h, the mRNA and protein levels of GATA1 and GATA2 changed dynamically. After exposure to 40 µM HQ for 72 h, K562 cells were induced with 40 µM hemin for 48 h. HQ considerably reduced the percentage of hemin-induced Hb-positive cells, decreased the GATA1 mRNA, protein, and occupancy levels at α-globin and ß-globin gene clusters, and increased the GATA2 mRNA and protein levels significantly. ChIP-seq analysis revealed that HQ reduced GATA1 occupancy, and increased GATA2 occupancy at most gene loci in hemin-induced K562 cells. And GATA1 and GATA2 might play essential roles in the erythroid differentiation protein interaction network. These results elucidate that HQ decreases GATA1 occupancy and increases GATA2 occupancy at the erythroid gene loci, thereby downregulating GATA1 and upregulating GATA2 expression, which in turn modulates the expression of erythroid genes and inhibits erythroid differentiation. This partially explains the mechanism of benzene hematotoxicity.

Identification of potential pathways and microRNA-mRNA networks associated with benzene metabolite hydroquinone-induced hematotoxicity in human leukemia K562 cells.

BACKGROUND: Hydroquinone (HQ) is a phenolic metabolite of benzene with a potential risk for hematological disorders and hematotoxicity in humans. In the present study, an integrative analysis of microRNA (miRNA) and mRNA expressions was performed to identify potential pathways and miRNA-mRNA network associated with benzene metabolite hydroquinone-induced hematotoxicity. METHODS: K562 cells were treated with 40 µM HQ for 72 h, mRNA and miRNA expression changes were examined using transcriptomic profiles and miRNA microarray, and then bioinformatics analysis was performed. RESULTS: Out of all the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) induced by HQ, 1482 DEGs and 10 DEMs were up-regulated, and 1594 DEGs and 42 DEMs were down-regulated. HQ-induced DEGs were involved in oxidative stress, apoptosis, DNA methylation, histone acetylation and cellular response to leukemia inhibitory factor GO terms, as well as metabolic, Wnt/ß-catenin, NF-κB, and leukemia-related pathways. The regulatory network of mRNAs and miRNAs includes 23 miRNAs, 1108 target genes, and 2304 potential miRNAs-mRNAs pairs. MiR-1246 and miR-224 had the potential to be major regulators in HQ-exposed K562 cells based on the miRNAs-mRNAs network. CONCLUSIONS: This study reinforces the use of in vitro model of HQ exposure and bioinformatic approaches to advance our knowledge on molecular mechanisms of benzene hematotoxicity at the RNA level.

Huanglian decoction suppresses the growth of hepatocellular carcinoma cells by reducing CCNB1 expression.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in human populations worldwide. Huanglian decoction is one of the most important Chinese medicine formulas, with the potential to treat cancer. AIM: To investigate the role and mechanism of Huanglian decoction on HCC cells. METHODS: To identify differentially expressed genes (DEGs), we downloaded gene expression profile data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma and Gene Expression Omnibus (GSE45436) databases. We obtained phytochemicals of the four herbs of Huanglian decoction from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. We also established a regulatory network of DEGs and drug target genes and subsequently analyzed key genes using bioinformatics approaches. Furthermore, we conducted in vitro experiments to explore the effect of Huanglian decoction and to verify the predictions. In particular, the CCNB1 gene was knocked down to verify the primary target of this decoction. Through the identification of the expression levels of key proteins, we determined the primary mechanism of Huanglian decoction in HCC. RESULTS: Based on the results of the network pharmacological analysis, we revealed 5 bioactive compounds in Huanglian decoction that act on HCC. In addition, a protein-protein interaction network analysis of the target genes of these five compounds as well as expression and prognosis analyses were performed in tumors. CCNB1 was confirmed to be the primary gene that may be highly expressed in tumors and was significantly associated with a worse prognosis. We also noted that CCNB1 may serve as an independent prognostic indicator in HCC. Moreover, in vitro experiments demonstrated that Huanglian decoction significantly inhibited the growth, migration, and invasiveness of HCC cells and induced cell apoptosis and G2/M phase arrest. Further analysis showed that the decoction may inhibit the growth of HCC cells by downregulating the CCNB1 expression level. After Huanglian decoction treatment, the expression levels of Bax, caspase 3, caspase 9, p21 and p53 in HCC cells were increased, while the expression of CDK1 and CCNB1 was significantly decreased. The p53 signaling pathway was also found to play an important role in this process. CONCLUSION: Huanglian decoction has a significant inhibitory effect on HCC cells. CCNB1 is a potential therapeutic target in HCC. Further analysis showed that Huanglian decoction can inhibit HCC cell growth by downregulating the expression of CCNB1 to activate the p53 signaling pathway.

Benzene metabolite 1,2,4-benzenetriol changes DNA methylation and histone acetylation of erythroid-specific genes in K562 cells.

1,2,4-Benzenetriol (BT) is one of the phenolic metabolites of benzene, a general occupational hazard and ubiquitous environmental air pollutant with leukemogenic potential in humans. Previous studies have revealed that the benzene metabolites phenol and hydroquinone can inhibit hemin-induced erythroid differentiation in K562 cells. We investigated the roles of DNA methylation and histone acetylation in BT-inhibited erythroid differentiation in K562 cells. When K562 cells were treated with 0, 5, 10, 15 or 20 µM BT for 72 h, hemin-induced hemoglobin synthesis decreased in a concentration-dependent manner. Both 5-aza-2'-deoxycytidine (5-aza-CdR, DNA methyltransferase inhibitor) and trichostatin A (TSA, histone deacetylases inhibitor) could prevent 20 µM BT from inhibiting hemin-induced hemoglobin synthesis and the mRNA expression of erythroid genes. Exposure to BT changed DNA methylation levels at several CpG sites of erythroid-specific genes, as well as the acetylation of histone H3 and H4, chromatin occupancy of GATA-1 and recruitment of RNA polymerase II at α-globin and ß-globin gene clusters after hemin induction. These results demonstrated that BT could inhibit hemin-induced erythroid differentiation, where DNA methylation and histone acetylation also played important roles by down-regulating erythroid-specific genes. This partly explained the mechanisms of benzene hematotoxicity.