Loss of Cxcr2 in Myeloid Cells Promotes Tumour Progression and T Cell Infiltration in Invasive Bladder Cancer.

BACKGROUND: CXCR2 is a chemokine receptor expressed in myeloid cells, including neutrophils and macrophages. Pharmacological inhibition of CXCR2 has been shown to sensitize tumours to immune checkpoint inhibitor immunotherapies in some cancer types. OBJECTIVE: To investigate the effects of CXCR2 loss in regulation of tumour-infiltrating myeloid cells and their relationship to lymphocytes during bladder tumorigenesis. METHODS: Urothelial pathogenesis and immune contexture was investigated in an OH-BBN model of invasive bladder cancer with Cxcr2 deleted in myeloid cells (LysMCre Cxcr2 flox/flox ). CXCR2 gene alterations and expression in human muscle invasive bladder cancer were analysed in The Cancer Genome Atlas. RESULTS: Urothelial tumour pathogenesis was significantly increased upon Cxcr2 deletion compared to wildtype mice. This was associated with a suppression of myeloid cell infiltration in Cxcr2-deleted bladders shortly after the carcinogen induction. Interestingly, following a transient increase of macrophages at the outset of tumour formation, an increase in T cell infiltration was observed in Cxcr2-deleted tumours. The increased tumour burden in the Cxcr2-deleted bladder was largely independent of T cells and the status of immune suppression. The Cxcr2-deleted mouse model reflected the low CXCR2 mRNA range in human bladder cancer, which showed poor overall survival. CONCLUSIONS: In contrast to previous reports of increased CXCR2 signalling associated with disease progression and poor prognosis, CXCR2 was protective against bladder cancer during tumour initiation. This is likely due to a suppression of acute inflammation. The strategy for sensitizing checkpoint immunotherapy by CXCR2 inhibition in bladder cancer may benefit from an examination of immune suppressive status.

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.

Changes in DNA methylation of erythroid-specific genes in K562 cells exposed to catechol in long term.

Catechol is one of phenolic metabolites of benzene that is a general occupational hazard and a ubiquitous environmental air pollutant. Catechol also occurs naturally in fruits, vegetables and cigarettes. Previous studies have revealed that 72h exposure to catechol improved hemin-induced erythroid differentiation of K562 cells accompanied with elevated methylation in erythroid specific genes. In present study, K562 cells were treated with 0, 10 or 20µM catechol for 1-4weeks, hemin-induced hemoglobin synthesis increased in a concentration- and time-dependent manner and the enhanced hemoglobin synthesis was relatively stable. The mRNA expression of α-, ß- and γ-globin genes, erythroid heme synthesis enzymes PBGD and ALAS2, transcription factor GATA-1 and NF-E2 showed a significant increase in K562 cells exposed to 20µM catechol for 3w, and catechol enhanced hemin-induced mRNA expression of these genes. Quantitative MassARRAY methylation analysis also confirmed that the exposure to catechol changed DNA methylation levels at several CpG sites in several erythroid-specific genes and their far upstream of regulatory elements. These results demonstrated that long-term exposure to low concentration of catechol enhanced the hemin-induced erythroid differentiation of K562 cells, in which DNA methylation played a role by up-regulating erythroid specific genes.

High glucose induced endothelial to mesenchymal transition in human umbilical vein endothelial cell.

BACKGROUND: Studies have shown that endothelial-to-mesenchymal transition (EndMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis. The aim of this study was to investigate the influence of high glucose and related mechanism of MAPK inhibitor or specific antioxidant on the EndMT. METHODS: In vitro human umbilical vein endothelial cells (HUVEC) were cultured with 11mM, 30mM, 60mM and 120mM glucose for 0, 24, 48, 72 and 168h. Endothelial cell morphology was observed with microscope, and RT-PCR was used to detect mRNA expression of endothelial markers VE-cadherin and CD31, mesenchymal markers α-SMA and collagen I, and transforming growth factor TGF-ß1. Immunofluorescence staining was performed to detect the expression of CD31 and α-SMA. The concentration of TGF-ß1 in the supernatant was detected by ELISA. ERK1/2 phosphorylation level was detected by Western blot analysis. RESULTS: High glucose induced EndMT and increased the TGF-ß1 level in HUVEC cells. Cells in high glucose for 7 days showed a significant decrease in mRNA expression of CD31 and VE-cadherin, and a significant increase in that of α-SMA and collagen I, while lost CD31 staining and acquired α-SMA staining. ERK signaling pathway blocker PD98059 significantly attenuated the high glucose-induced increase in the ERK1/2 phosphorylation level. PD98059 and NAC both inhibited high glucose-induced TGF-ß1 expression and attenuated EndMT marker protein synthesis. CONCLUSION: High glucose could induce HUVEC cells to undergo EndMT. NAC and ERK signaling pathway may play important role in the regulation of the TGF-ß1 biosynthesis during high glucose-induced EndMT.