Kong Yun, Guo Li
Department of pharmacy, The Second Affiliated Hospital of Jiaxing University, Jiaxing, China.
J Biochem Mol Toxicol. 2023 Jun;37(6):e23333. doi: 10.1002/jbt.23333. Epub 2023 Feb 16.
This work aimed to investigate the role and mechanism of Sunitinib (Sun) in suppressing M2 polarization of macrophages in tumor microenvironment (TME). IL-4 was applied to induce the M2 polarization of RAW264.7 cells, followed by treatment with Sun at 50 and 100 nM. Flow cytometry (FCM) was conducted to detect the proportion of F4/80 + CD206 + cells. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of IL-10, Arg-1 and VEGF. Immunofluorescence (IF) staining was carried out to detect the expression of CD206 and Arg-1. Besides, western-Blot (WB) assay was performed to measure the levels of p-JAK1 and p-STAT6 proteins. After polarization, the macrophage culture medium was employed to culture hepatocellular carcinoma (HCC) Hca-F cells. Thereafter, Transwell assays were conducted to examine cell invasion and migration, whereas plate clone formation assay was carried out to detect the clone forming capacity. In further experiments, cells were treated with the STAT6 inhibitor, or STAT6 inhibitor + Sun. Then, the polarization levels of RAW264.7 cells were detected. Moreover, this study established the xenograft tumor mouse model. Later, CD206 and Ki67 expression, IL-10, Arg-1 and VEGF expression levels in tissues, and p-JAK1 and p-STAT6 protein levels were detected by histochemical staining. Sun suppressed the M2 polarization of RAW264.7 cells. Compared with IL-4 treatment, the proportion of F4/80 + CD206 + cells decreased. Meanwhile, the levels of IL-10, Arg-1 and VEGF were downregulated, and the phosphorylation level of JAK1-STAT6 signaling was suppressed. After being cocultured with Hca-F, the malignant behaviors of HCC cells were suppressed after Sun treatment. Similarly, STAT6 inhibitor treatment suppressed the M2 polarization, while the combined application of Sun did not further restrain the polarization level. In the mouse model, Sun suppressed the expression of CD206 and Ki67, simultaneously inhibiting the polarization of JAK1-STAT6 signaling. Sunitinib can suppress the M2 polarization of macrophages to exert the anti-HCC effect, which is its another anticancer mechanism.
本研究旨在探讨舒尼替尼（Sun）在抑制肿瘤微环境（TME）中巨噬细胞M2极化的作用及机制。应用白细胞介素-4（IL-4）诱导RAW264.7细胞发生M2极化，随后分别用50和100 nM的舒尼替尼进行处理。采用流式细胞术（FCM）检测F4/80+CD206+细胞的比例。通过酶联免疫吸附测定（ELISA）检测白细胞介素-10（IL-10）、精氨酸酶-1（Arg-1）和血管内皮生长因子（VEGF）的水平。进行免疫荧光（IF）染色以检测CD206和Arg-1的表达。此外，采用蛋白质免疫印迹法（WB）检测磷酸化JAK1和磷酸化STAT6蛋白的水平。极化后，用巨噬细胞培养基培养肝癌（HCC）Hca-F细胞。此后，进行Transwell实验检测细胞侵袭和迁移能力，同时进行平板克隆形成实验检测克隆形成能力。在进一步实验中，细胞分别用STAT6抑制剂或STAT6抑制剂+舒尼替尼处理。然后，检测RAW264.7细胞的极化水平。此外，本研究建立了异种移植肿瘤小鼠模型。之后，通过组织化学染色检测组织中CD206和Ki67的表达、IL-10、Arg-，并检测组织中IL-10、Arg-1和VEGF的表达水平以及p-JAK1和p-STAT6蛋白水平。舒尼替尼抑制RAW264.7细胞的M2极化。与IL-4处理组相比，F4/80+CD206+细胞的比例降低。同时，IL-10、Arg-1和VEGF的水平下调，JAK1-STAT6信号通路的磷酸化水平受到抑制。与Hca-F细胞共培养后，舒尼替尼处理后肝癌细胞恶性行为受到抑制。同样，STAT6抑制剂处理抑制了M2极化，而舒尼替尼联合应用并未进一步抑制极化水平。在小鼠模型中，舒尼替尼抑制CD206和Ki67的表达，同时抑制JAK1-STAT6信号通路的极化。舒尼替尼可抑制巨噬细胞的M2极化以发挥抗肝癌作用，这是其另一种抗癌机制。
