Abstract:

Objective To discuss the effect of histone deacetylase 6 （HDAC6） inhibitor ACY-241 on macrophage polarization and the proliferation， migration and invasion of esophageal squamous cell carcinoma cells， and to clarify its mechanism. Methods Phorbol ester was used to induce human monocytic leukemia cell line THP-1 to differentiate into M0 macrophages. The M0 macrophages were co-cultured with human esophageal squamous cell carcinoma cells KYSE150 and KYSE150 cells treated with HDAC6 inhibitor ACY-241， respectively. Flow cytometry was used to detect macrophage polarization after co-culture； enzyme-linked immunosorbent assay （ELISA） was used to determine the level of CC chemokine ligand 2 （CCL2） in the supernatant of KYSE150 cells after co-culture； Western blotting method was used to detect the expression level of CC chemokine receptor 2 （CCR2） protein in macrophages in two groups after co-culture； pLVX-CCL2 lentivirus and its negative control pLVX-NC lentivirus were used to transfect KYSE150 cells， and real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the transfection efficiency. The M0 macrophages were co-cultured with KYSE150 cells transfected with lentivirus and treated with ACY-241， and divided into control group， ACY-241 group， pLVX-NC+ACY-241 group and pLVX-CCL2+ACY-241 group； ELISA method was used to determine the levels of CCL2 in supernatant of KYSE150 cells in various groups； Western blotting method was used to detect the expression levels of CCR2 protein in macrophages in various groups； flow cytometry was used to detect macrophage polarization in various groups； RT-qPCR method was used to detect the expression levels of inducible nitric oxide synthase （iNOS）， interleukin-6 （IL-6）， interleukin-10 （IL-10） and arginase 1 （Arg-1） mRNA in macrophages in various groups； CCK-8 method， colony formation assay and Transwell chamber assay were used to detect the proliferation activities， colony formation numbers， number of migration cells and number of invasion cells in the KYSE150 cells in various groups， respectively. Results Compared with control group， the percentage of M1 macrophages in ACY-241 group was increased （P<0.05）， the percentage of M2 macrophages was decreased （P<0.05）， the level of CCL2 in the supernatant of KYSE150 cells was decreased （P<0.05）， and the expression level of CCR2 protein in macrophages was decreased （P<0.05）. After transfection with pLVX-CCL2 lentivirus， the expression levels of CCL2 mRNA and protein in KYSE150 cells were significantly increased （P<0.05）. Compared with control group， the expression levels of iNOS and IL-6 mRNA in macrophages in ACY-241 group were significantly increased （P<0.05）， the expression levels of IL-10 and Arg-1 mRNA were significantly decreased （P<0.05）， the proliferation activity of KYSE150 cells was decreased （P<0.05）， and the colony formation number， number of migration cells and number of invasion cells were decreased （P<0.05）. Compared with pLVX-NC+ACY-241 group， the level of CCL2 in the supernatant of KYSE150 cells in pLVX-CCL2+ACY-241 group was increased （P<0.05）， the expression level of CCR2 protein in macrophages was increased （P<0.05）， the percentage of M1 macrophages was decreased （P<0.05）， the percentage of M2 macrophages was increased （P<0.05）， the expression levels of iNOS and IL-6 mRNA in macrophages were decreased （P<0.05） and the expression levels of IL-10 and Arg-1 mRNA were increased （P<0.05）， the proliferation activity of KYSE150 cells was increased （P<0.05）， and the colony formation number， number of migration cells and number of invasion cells in KYSE150 cells were increased （P<0.05）. Conclusion HDAC6 inhibitor ACY-241 can promote macrophage polarization towards M1 type and reduce its polarization towards M2 type， and inhibit the proliferation， migration and invasion of esophageal squamous cell carcinoma KYSE150 cells； its mechanism may be related to suppressing the CCL2/CCR2 axis.

Key words: Esophageal squamous cell carcinoma, Histone deacetylase 6, Macrophage polarization, CC chemokine ligand 2, CC chemokine receptor 2