Department of Bone Oncology, the People's Hospital of Liaoning Province, Shenyang, Liaoning Province 110016, China.
Department of Immunology, College of Basic Medical Science, China Medical University, Shenyang, Liaoning Province 110122, China.
Int Immunopharmacol. 2023 Dec;125(Pt B):111226. doi: 10.1016/j.intimp.2023.111226. Epub 2023 Nov 15.
This study aimed to investigate the underlying regulatory effects of methionine enkephalin (MENK) on osteosarcoma.
The Cell Counting Kit-8 assay, clone formation, wound healing, transwell assay, and flow cytometry were performed to measure the effects of MENK on the proliferation, migration, invasion, and apoptosis of MG-63 and Saos-2 cells. Opiate growth factor receptor expression (OGFr) in cells was stably knocked down using siRNA. A tumor model was established by inoculating MG-63 cells into mice. Flow cytometry was performed to identify alterations in mice bone marrow, spleen, and tumor tissue immune cells. The phenotype of tumor-associated macrophages was determined using immunohistochemistry. After OGFr knockdown or/and treatment with MENK, Bax, Bcl-2, caspase 3, caspase 9, and PARP expression levels were characterized using qRT-PCR, western blot, and WES, respectively.
MENK could significantly inhibit the proliferation, invasion, and migration of MG-63 and Saos-2, arrest the cell cycle in the G0/G1 phase, upregulate Bax, caspase 3, caspase 9, and PARP expression, and downregulate Bcl-2 expression. Tumor size and weight were lower in the MENK group than those in the control group. MENK-treated mice exhibited a reduced ratio of CD11b + Gr-1 + myeloid-derived suppressor cells. MENK increased the ratio of M1-type macrophages and decreased the proportion of M2-type macrophages in tumor tissue. Furthermore, the level of TNF-α significantly increased while that of IL-10 decreased in MENK-treated mice. The effect of MENK could be partly reversed by OGFr knockdown.
MENK reduces the abundance of myeloid-derived suppressor cells, induces M1 polarization of macrophages, and exhibits an inhibitory effect on osteosarcoma.
本研究旨在探讨甲硫氨酸脑啡肽（MENK）对骨肉瘤的潜在调节作用。
采用细胞计数试剂盒-8 检测、克隆形成、划痕愈合、Transwell 检测和流式细胞术检测 MENK 对 MG-63 和 Saos-2 细胞增殖、迁移、侵袭和凋亡的影响。用 siRNA 稳定敲低阿片生长因子受体表达（OGFr）。将 MG-63 细胞接种到小鼠体内建立肿瘤模型。采用流式细胞术检测小鼠骨髓、脾脏和肿瘤组织免疫细胞的变化。采用免疫组织化学法检测肿瘤相关巨噬细胞的表型。OGFr 敲低或/和 MENK 处理后，采用 qRT-PCR、western blot 和 WES 分别检测 Bax、Bcl-2、caspase 3、caspase 9 和 PARP 的表达水平。
MENK 可显著抑制 MG-63 和 Saos-2 的增殖、侵袭和迁移，将细胞周期阻滞在 G0/G1 期，上调 Bax、caspase 3、caspase 9 和 PARP 的表达，下调 Bcl-2 的表达。MENK 组肿瘤体积和重量均低于对照组。MENK 处理组小鼠 CD11b+Gr-1+髓系来源抑制细胞的比例降低。MENK 增加了肿瘤组织中 M1 型巨噬细胞的比例，降低了 M2 型巨噬细胞的比例。此外，MENK 处理组小鼠肿瘤组织中 TNF-α 水平显著升高，IL-10 水平降低。OGFr 敲低可部分逆转 MENK 的作用。
MENK 减少髓系来源抑制细胞的丰度，诱导巨噬细胞 M1 极化，并对骨肉瘤具有抑制作用。
