超高含量的α-酮戊二酸和谷胱甘肽促成HeLa细胞耐受纳米银

Silver nanoparticles-resistance of HeLa cell associated with its unusually high concentration of α-ketoglutarate and glutathione

摘要:

纳米银（silver nanoparticles，AgNPs）兼有优良的抗菌和抗癌作用，但病原体或癌细胞对其的耐受作用将影响其临床应用，前者耐受纳米银已见报道。本研究以HeLa细胞为模型，探讨癌细胞耐受纳米银的可能性及耐受机制。将HeLa细胞代谢物与纳米银混合后测定其抗菌活性和细胞毒性变化，并用紫外-可见（ultraviolet visible，UV-Vis）分光光度计、粒度仪和透射电镜等检测混合体系中纳米银的理化特征。用非靶向代谢组学分析纳米银结合的代谢物种类，经腹腔注射HeLa细胞建立荷瘤小鼠，并分析血清对纳米银稳定性的影响。结果表明HeLa细胞代谢物可抑制纳米银的抗癌和抗菌作用，这种抑制作用表现出剂量依赖性，对纳米银生物学活性产生抑制作用的效应代谢物耐热、不溶于氯仿、含硫元素，分子量小于1 kDa。抑制作用的实质是使纳米银发生了聚集，筛选得到有115种代谢物能结合纳米银。进一步探究发现仅当α-酮戊二酸（α-ketoglutarate，AKG）和谷胱甘肽（glutathione，GSH）的浓度共同达到一定阈值才导致纳米银聚集，而HeLa细胞代谢物中两者的浓度分别是正常宫颈上皮细胞的10倍和6倍，达到了该阈值。动物实验结果显示荷瘤小鼠血清导致纳米银聚集率显著高于健康鼠血清（P<0.05）。本研究揭示HeLa细胞中具有超常含量的α-酮戊二酸和谷胱甘肽，两者协同破坏纳米银的胶体稳定性，从而实现逃逸纳米银的抗癌作用。

Abstract:

Silver nanoparticles (AgNPs) is known as one of the most valuable metal nanoparticles in antibacterial and anticancer application. AgNPs-resistant bacteria has been documented, but it is unclear whether cancer cells can also escape the anti-cancer effect of AgNPs. In this study, we aimed to investigate this phenomenon and its underlying mechanism. The antibacterial activity and cytotoxicity of AgNPs were measured in the presence of HeLa cell metabolites. The status of AgNPs in the system associated with metabolites were characterized by UV-Vis, Zetasizer Nano ZS, and transmission electron microscopy. Non-targeted metabolomics was used to reveal the metabolites components that bind with AgNPs. HeLa cells were injected intraperitoneally to establish the tumor-bearing mice model, and the stability of AgNPs in mice serum was analyzed. The results manifested that HeLa cell metabolites inhibited the anticancer and antibacterial effects of AgNPs in a dose-dependent manner by causing AgNPs aggregation. Effective metabolites that inhibited the biological activity of AgNPs were stable in 100℃, insoluble in chloroform, containing sulfur elements, and had a molecular weight less than 1 kDa in molecular weight. There were 115 compounds bound with AgNPs. In vitro experiments showed that AgNPs aggregation occurred only when the concentration of α-ketoglutarate (AKG) and glutathione (GSH) together reached a certain threshold. Interestingly, the concentration of AKG and GSH in HeLa cellular metabolites was 10 and 6 times higher than that in normal cervical epithelial cells, respectively, which explained why the threshold was reached. Furthermore, the stability of AgNPs in the serum of tumor-bearing mice decreased by 20% (P<0.05) compared with the healthy mice. In conclusion, our study demonstrates that HeLa cells escaped the anti-cancer effect of AgNPs through the synergistic effect of AKG and GSH, suggesting the need to develop strategies to overcome this limitation.