二甲双胍增加 U87MG 人胶质母细胞瘤细胞中外泌体的生物发生和分泌：一种治疗抵抗的可能机制。

Metformin Increases Exosome Biogenesis and Secretion in U87 MG Human Glioblastoma Cells: A Possible Mechanism of Therapeutic Resistance.

机构信息

Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran.

Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran; Department of Medical Physics and Imaging, Urmia University of Medical Sciences, Urmia, Iran.

出版信息

Arch Med Res. 2021 Feb;52(2):151-162. doi: 10.1016/j.arcmed.2020.10.007. Epub 2020 Oct 13.

DOI:10.1016/j.arcmed.2020.10.007

PMID:33059952

Abstract

BACKGROUND

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Metformin, an anti-diabetic drug, can suppress tumor cells. Exosomes from GBM cells contribute to intercellular communication, tumor aggressiveness, and therapeutic resistance. We studied the effect of metformin on the exosomal secretory pathway in U87 MG cells.

METHODS

Cell survival against metformin was investigated using MTT assay. Expression of miRNA-21, miRNA-155, and miRNA-182, as well as the genes involved in exosome biogenesis and secretion such as Rab27a, Rab27b, Rab11, CD63, and Alix were calculated by real time-PCR. The expression of CD63 protein was analyzed by western blotting, while the subcellular distribution of CD63 protein was monitored by flow cytometry. Exosomes were characterized by transmission and scanning electron microscopes, and flow cytometry. Amount of exosomes was assayed using acetylcholinesterase activity assay and ELISA. The expression of autophagic markers LC3 and P62 were assessed using ELISA.

RESULTS

Data showed that metformin decreased cell survival and expression of miRNA-21, miRNA-155, and miRNA-182 (p <0.05). Expression of Rab27a, Rab27b, Rab11, CD63, and Alix as well as protein level of CD63 up-regulated in treated cells (p <0.05). Concurrently, flow cytometry analysis showed that surface CD63/total CD63 ratio was increased in treated cells (p <0.05). We found that acetylcholinesterase activity and CD63 protein of exosomes from treated cells increased (p <0.05). The expression of LC3 and P62 was not affected by metformin (p >0.05).

CONCLUSION

Data indicates metformin could promote exosome biogenesis and secretion in U87 MG cells, proposing the therapeutic response against metformin.

摘要

背景

胶质母细胞瘤（GBM）是最常见和侵袭性最强的脑肿瘤。二甲双胍是一种抗糖尿病药物，可抑制肿瘤细胞。GBM 细胞来源的外泌体有助于细胞间通讯、肿瘤侵袭性和治疗抵抗。我们研究了二甲双胍对 U87 MG 细胞中外泌体分泌途径的影响。

方法

用 MTT 法检测细胞对二甲双胍的存活率。通过实时 PCR 计算 miRNA-21、miRNA-155 和 miRNA-182 的表达，以及参与外泌体生物发生和分泌的基因，如 Rab27a、Rab27b、Rab11、CD63 和 Alix。用 Western blot 分析 CD63 蛋白的表达，用流式细胞术监测 CD63 蛋白的亚细胞分布。通过透射和扫描电子显微镜以及流式细胞术对 exosomes 进行表征。通过乙酰胆碱酯酶活性测定和 ELISA 测定 exosomes 的量。用 ELISA 评估自噬标记物 LC3 和 P62 的表达。

结果

数据表明，二甲双胍降低了细胞存活率和 miRNA-21、miRNA-155 和 miRNA-182 的表达（p<0.05）。处理细胞中 Rab27a、Rab27b、Rab11、CD63 和 Alix 的表达以及 CD63 蛋白水平上调（p<0.05）。同时，流式细胞术分析显示处理细胞表面 CD63/总 CD63 比值增加（p<0.05）。我们发现，处理细胞中外泌体的乙酰胆碱酯酶活性和 CD63 蛋白增加（p<0.05）。二甲双胍对 LC3 和 P62 的表达没有影响（p>0.05）。