大剂量骨化三醇通过抑制 mTOR 激活抑制肿瘤微环境中的糖酵解和 M2 巨噬细胞极化：体外和分子对接研究。

A High Dose of Calcitriol Inhibits Glycolysis and M2 Macrophage Polarization in the Tumor Microenvironment by Repressing mTOR Activation: in vitro and Molecular Docking Studies.

机构信息

Nutritional Sciences, Texas Tech University, Lubbock, TX, USA.

Obesity and Cancer Biology Lab, Natural and Medical Sciences Research Center, University of Nizwa, Nizwa, Sultanate of Oman.

出版信息

Cell Physiol Biochem. 2023 Apr 12;57(2):105-122. doi: 10.33594/000000618.

DOI:10.33594/000000618

PMID:37052042

Abstract

BACKGROUND/AIMS: Macrophages interact with tumor cells within the tumor microenvironment (TME), which plays a crucial role in tumor progression. Cancer cells also can instruct macrophages to facilitate the spread of cancer and the growth of tumors. Thus, modulating macrophages-cancer cells interaction in the TME may be therapeutically beneficial. Although calcitriol (an active form of vitamin D) has anticancer properties, its role in TME is unclear. This study examined the role of calcitriol in the regulation of macrophages and cancer cells in the TME and its influence on the proliferation of breast cancer cells.

METHODS

We modeled the TME, in vitro, by collecting conditioned medium from cancer cells (CCM) and macrophages (MCM) and culturing each cell type separately with and without (control) a high-dose (0.5 µM) calcitriol (an active form of vitamin D). An MTT assay was used to examine cell viability. Apoptosis was detected using FITC (fluorescein isothiocyanate) annexin V apoptosis detection kit. Western blotting was used to separate and identify proteins. Quantitative real-time PCR was used to analyze gene expression. Molecular docking studies were performed to evaluate the binding type and interactions of calcitriol to the GLUT1 and mTORC1 ligand-binding sites.

RESULTS

Calcitriol treatment suppressed the expression of genes and proteins implicated in glycolysis (GLUT1, HKII, LDHA), promoted cancer cell apoptosis, and reduced viability and Cyclin D1gene expression in MCM-induced breast cancer cells. Additionally, calcitriol treatment suppressed mTOR activation in MCM-induced breast cancer cells. Molecular docking studies further showed efficient binding of calcitriol with GLUT1 and mTORC1. Calcitriol also inhibited CCM-mediated induction of CD206 and increased TNFα gene expression in THP1-derived macrophages.

CONCLUSION

The results suggest that calcitriol may impact breast cancer progression by inhibiting glycolysis and M2 macrophage polarization via regulating mTOR activation in the TME and warrants further investigation in vivo.

摘要

背景/目的：巨噬细胞与肿瘤微环境（TME）中的肿瘤细胞相互作用，在肿瘤进展中起着关键作用。癌细胞还可以指示巨噬细胞促进癌症的扩散和肿瘤的生长。因此，调节 TME 中的巨噬细胞-癌细胞相互作用可能具有治疗益处。虽然钙三醇（维生素 D 的一种活性形式）具有抗癌特性，但它在 TME 中的作用尚不清楚。本研究探讨了钙三醇在调节 TME 中的巨噬细胞和癌细胞及其对乳腺癌细胞增殖的影响中的作用。

方法

我们通过收集癌细胞（CCM）和巨噬细胞（MCM）的条件培养基，并在有无（对照）高剂量（0.5 µM）钙三醇（维生素 D 的一种活性形式）的情况下分别培养每种细胞类型，在体外模拟 TME。MTT 测定法用于检测细胞活力。使用 FITC（异硫氰酸荧光素）annexin V 凋亡检测试剂盒检测细胞凋亡。使用 Western blot 分离和鉴定蛋白质。使用定量实时 PCR 分析基因表达。进行分子对接研究以评估钙三醇与 GLUT1 和 mTORC1 配体结合位点的结合类型和相互作用。

结果

钙三醇处理抑制了糖酵解（GLUT1、HKII、LDHA）相关基因和蛋白的表达，促进了癌细胞凋亡，并降低了 MCM 诱导的乳腺癌细胞的活力和 Cyclin D1 基因表达。此外，钙三醇处理抑制了 MCM 诱导的乳腺癌细胞中 mTOR 的激活。分子对接研究进一步表明钙三醇与 GLUT1 和 mTORC1 的有效结合。钙三醇还抑制了 CCM 介导的 THP1 衍生巨噬细胞中 CD206 的诱导，并增加了 TNFα 基因的表达。