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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2022 Vol.23 No.8 P.682-698
Investigation and experimental validation of curcumin-related mechanisms against hepatocellular carcinoma based on network pharmacology
Abstract: ObjectiveTo determine the potential molecular mechanisms underlying the therapeutic effect of curcumin on hepatocellular carcinoma (HCC) by network pharmacology and experimental in vitro validation.
MethodsThe predictive targets of curcumin or HCC were collected from several databases. the identified overlapping targets were crossed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. Two of the candidate pathways were selected to conduct an experimental verification. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay was used to determine the effect of curcumin on the viability of HepG2 and LO2 cells. The apoptosis and autophagy of HepG2 cells were respectively detected by flow cytometry and transmission electron microscopy. Besides, western blot and real-time polymerase chain reaction (PCR) were employed to verify the p53 apoptotic pathway and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) autophagy pathway. HepG2 cells were pretreated with pifithrin-α(PFT-α) and GSK690693 for further investigation.
ResultsThe 167 pathways analyzed by KEGG included apoptosis, autophagy, p53, and AMPK pathways. The GO enrichment analysis demonstrated that curcumin was involved in cellular response to drug, regulation of apoptotic pathway, and so on. The in vitro experiments also confirmed that curcumin can inhibit the growth of HepG2 cells by promoting the apoptosis of p53 pathway and autophagy through the AMPK pathway. Furthermore, the protein and messenger RNA (mRNA) of the two pathways were downregulated in the inhibitor-pretreated group compared with the experimental group. The damage-regulated autophagy modulator (DRAM) in the PFT-α-pretreated group was downregulated, and p62 in the GSK690693-pretreated group was upregulated.
ConclusionsCurcumin can treat HCC through the p53 apoptotic pathway and the AMPK/Unc-51-like kinase 1 (ULK1) autophagy pathway, in which the mutual transformation of autophagy and apoptosis may occur through DRAM and p62.
Key words: Curcumin; Network pharmacology; p53; Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK); Apoptosis; Autophagy
1陕西中医药大学基础医学院，陕西咸阳，712046
2陕西中医药大学药学院，陕西咸阳，712046
3陕西中医药大学医学科研实验中心，陕西咸阳，712046
4陕西省中医体质与疾病防治研究重点实验室，陕西咸阳，712046
目的：运用网络药理学和体外细胞实验探究姜黄素防治肝癌的分子机制。
创新点：运用网络药理学预测姜黄素作用与肝癌防治间的相关性及作用靶点。以此为基础，探索姜黄素是否通过能量代谢感受器AMPK诱导AMPK/ULK1自噬通路并触发自噬性凋亡减轻肝癌；并对姜黄素诱导肝癌细胞凋亡时，是否存在p53/DRAM凋亡促自噬通路进行验证；最后研究在姜黄素治疗肝癌中，细胞凋亡和细胞自噬是否通过DRAM和p62相互转移。
方法：从多个数据库收集姜黄素作用和肝癌治疗潜在靶点，利用DAVID平台将交集靶点进行GO和KEGG分析，预测姜黄素防治肝癌的相关信号通路。结合预测结果，采用MTT法检测姜黄素对HepG2和LO2细胞活力的影响；运用流式细胞术和透射电镜检测姜黄素处理后，HepG2细胞的凋亡和自噬情况；并经western blot和real-time PCR验证p53凋亡及AMPK自噬通路在姜黄素治疗肝癌中的作用。最后，通过p53抑制剂pifithrin-α和AMPK抑制剂GSK690693的使用进一步探索姜黄素治疗肝癌潜在的分子机制。
结论：姜黄素可通过p53凋亡和AMPK/ULK1自噬途径治疗肝癌，其中自噬和凋亡可能通过DRAM和p62相互转化。
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DOI:
10.1631/jzus.B2200038
CLC number:
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On-line Access:
2022-08-12
Received:
2022-01-22
Revision Accepted:
2022-04-27
Crosschecked:
2022-08-12