关键词: 黑水虻, 抗菌肽, 人结肠癌, HCT-8细胞, 细胞代谢组学, 谷氨酰胺, 谷氨酸, 代谢通路

Abstract:

【Aim】 To enrich the knowledge of the anticancer mechanism of antimicrobial peptide HI-3 extracted from Hermetia illucens by studying the effects of HI-3 on the amino acid metabolism of human colon carcinoma HCT-8 cells. 【Methods】 CCK-8 assay was used to determine the inhibition rates of different concentrations (80, 160, and 320 μg/mL) of antimicrobial peptide HI-3 on HCT-8 cells. The metabolites in HCT-8 cells were determined by GS-MS, and then the metabolic pathway with the most significant difference in amino acid contents and the target enzyme in this pathway were screened by pathway analysis based on R software. After the HCT-8 cells were treated with 320 μg/mL HI-3, the activity of the selected target enzyme glutaminase (GLS) was assayed by enzyme activity kit, its mRNA and protein expression levels were detected by RT-qPCR and Western blot, respectively, and the changes in the contents of important metabolites including glutamine (Gln), glutamic acid (Glu), glutathione (GSH), α-ketoglutaric acid (α-KG) and ATP involved in the glutamine and glutamic acid metabolic pathway in HCT-8 cells were detected by biochemical kits and ELISA kit. 【Results】 The inhibition rates of HCT-8 cells treated by HI-3 at the concentrations of 80, 160, and 320 μg/mL were 33.85%  ±3.50%, 46.26%±0.90%, and 55.53%±1.70%, respectively, and the inhibition rates increased with the increase of the HI-3 concentration. HI-3 at the concentration of 320 μg/mL showed the greatest effects on the glutamine and glutamic acid pathway with the contents of amino acid metabolites in this pathway most significantly different from those in the negative control group (0 μg/mL HI-3). The activity of GLS (the target enzyme in this pathway) and the mRNA and protein expression levels of its gene GLS in the 320 μg/mL HI-3 treatment group were extremely significantly lower than those in the negative control group. Moreover, the contents of important metabolites in this pathway, including Gln, Glu, GSH, α-KG and ATP, in the 320 μg/mL HI-3 treatment group were also significantly reduced as compared with those in the negative control group. 【Conclusion】 Antimicrobial peptide HI-3 from H. illucens at the concentration of 320 μg/mL has the most significant effect on the glutamine and glutamic acid metabolic pathway in HCT-8 cells, and can significantly inhibit the proliferation of HCT-8 cells by blocking this pathway.

Key words: Hermetia illucens, antimicrobial peptide, human colon carcinoma, HCT-8 cell, cell metabolomics, glutamine, glutamic acid, metabolic pathway