Study on effects of oridonin on glutathione metabolism network in esophageal squamous cell carcinoma cells

WANG Mindan; LIU Li; CUI Xialian; WEI Yixin; ZHANG Wenwen; CHEN Xiuping; ZHANG Fang; FENG Chenguo

doi:10.16305/j.1007-1334.2024.2403056

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Study on effects of oridonin on glutathione metabolism network in esophageal squamous cell carcinoma cells

更新时间：2024-07-04

- Study on effects of oridonin on glutathione metabolism network in esophageal squamous cell carcinoma cells
- Shanghai Journal of Traditional Chinese Medicine Vol. 58, Issue 7, Pages: 77-82(2024)
- 作者机构：
  
  上海中医药大学创新中药研究院（上海 201203）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2024.2403056
  CLC：
- Published：10 July 2024，
  
  Received：13 March 2024，
- 稿件说明：
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王敏丹,刘莉,崔夏莲,等.冬凌草甲素对食管鳞状细胞癌细胞内谷胱甘肽代谢网络影响的研究[J].上海中医药杂志,2024,58(7):77-82.

WANG Mindan,LIU Li,CUI Xialian,et al.Study on effects of oridonin on glutathione metabolism network in esophageal squamous cell carcinoma cells[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(7):77-82.
王敏丹,刘莉,崔夏莲,等.冬凌草甲素对食管鳞状细胞癌细胞内谷胱甘肽代谢网络影响的研究[J].上海中医药杂志,2024,58(7):77-82. DOI： 10.16305/j.1007-1334.2024.2403056.

WANG Mindan,LIU Li,CUI Xialian,et al.Study on effects of oridonin on glutathione metabolism network in esophageal squamous cell carcinoma cells[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(7):77-82. DOI： 10.16305/j.1007-1334.2024.2403056.

摘要

目的

探究冬凌草甲素（ORI）对人食管鳞状细胞癌（ESCC）细胞KYSE-150内谷胱甘肽代谢网络（GMN）中13个主要代谢物的影响，包括半胱氨酸（Cys）、高半胱氨酸（HCys）、谷胱甘肽（GSH）、甲硫氨酸（Met）、

-腺苷高半胱氨酸（SAH）、胱硫醚（Cysta）、甘氨酸（Gly）、谷氨酸（Glu）、谷氨酰胺（Gln）、谷氨酰半胱氨酸（GC）、半胱氨酰甘氨酸（CG）、

-乙酰丝氨酸（OAS）和丝氨酸（Ser）。

方法

首先，采用细胞计数试剂盒（CCK-8）法检测ORI对人ESCC细胞KYSE-150活性的影响，以半抑制浓度（IC

）作为后续细胞培养的干预浓度。将KYSE-150细胞按照不同的培养时间分为0 h、12 h、24 h、36 h组，以0 h组作为对照（空白培养液），12 h、24 h、36 h组采用含ORI的培养液（12.5 μmol/L）对细胞进行相应时间的干预。然后，采用超声提取法提取细胞中的内容物，

利用液相色谱-质谱（LC-MS）法直接检测ORI与GMN中巯基代谢物结合形成的缀合物，利用LC-MS结合化学衍生的方法检测13个目标GMN代谢物。最后，通过活性氧（ROS）检测试剂盒检测细胞提取物中ROS水平。

结果

①ORI对人ESCC细胞KYSE-150有明显的抑制作用，IC

值为12.5 μmol/L。②与0 h组相比，ORI干预后的3组KYSE-150细胞内GMN中的大部分代谢物（CG、GSH、GC、Cly、Glu、SAH、Cys、OAS和Cysta），尤其是含巯基的代谢物（CG、GSH、GC和Cys），浓度呈现先升高后下降的趋势。③在细胞提取物中检测到ORI与Cys、HCys、GSH这3个巯基内源性代谢物反应生成的缀合物。④ORI干预后KYSE-150细胞内ROS水平持续升高。

结论

ORI可能通过影响KYSE-150细胞内GMN中代谢物水平引起ROS的蓄积，进而降低细胞对抗脂质过氧化和氧化应激的能力。

Abstract

Objective

To investigate the effects of oridonin （ORI） on the 13 metabolites within the glutathione metabolic network （GMN） in human esophageal squamous cell carcinoma （ESCC） KYSE-150 cells， including cysteine （Cys）， homocysteine （HCys）， glutathione （GSH）， methionine （Met），

-adenylyl homocysteine （SAH）， cystathionine（Cysta）， glycine （Gly）， glutamic acid （Glu）， glutamine （Gln）， glutamylcysteine （GC）， cysteinylglycine （CG），

-acetyl-serine （OAS） and serine （Ser）.

Methods

Firstly， the Cell Counting Kit-8 （CCK-8） assay was utilized to assess the effect of ORI on the viability of KYSE-150 cells， and the half-maximal inhibition concentration （IC

） was used as the intervention concentration for subsequent cell culture. KYSE-150 cells were then divided into 0 h， 12 h， 24 h， and 36 h groups based on different culture time. The 0 h group was set as control （blank culture medium）， and the 12 h， 24 h and 36 h groups were treated with culture medium containing ORI （12.5 μmol/L） for corresponding time. Subsequently， cell contents from each group were extracted via ultrasonication. Liquid chromatography-mass spectrometry （LC‑MS） method was employed for the identification of ORI and GMN thiol adducts， while LC-MS combined with chemical derivatization method was utilized for detecting the 13 target metabolites. Finally， reactive oxygen species （ROS） levels of cellular extracts w

ere quantified using a ROS assay kit.

Results

①ORI had a significant inhibitory effect on human ESCC KYSE-150 cells with an IC

value of 12.5 μmol/L. ②Compared with the 0 h group， the concentrations of most metabolites within the GMN of KYSE-150 cells （CG， GSH， GC， Cly， Glu， SAH， Cys， OAS and Cysta）， especially thiol-containing metabolites （CG， GSH， GC and Cys）， showed a trend of initial increase followed by decrease after intervention with ORI. ③Three conjugates resulting from the binding of ORI to thiol-containing metabolites （Cys， HCys and GSH） were identified in cellular extracts. ④The level of ROS in KYSE-150 cells continued to increase after ORI intervention.

Conclusion

ORI may induce ROS accumulation by modulating metabolite levels within the GMN of KYSE-150 cells， consequently impairing the cells' capacity to counteract lipid peroxidation and oxidative stress.

关键词

食管鳞状细胞癌冬凌草甲素谷胱甘肽活性氧代谢组学中药研究

Keywords

esophageal squamous cell carcinomaoridoninglutathionereactive oxygen speciesmetabolomicstraditional Chinese medicine research

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