Study On The Inhibitory Effect And Mechanism Of Quinones From Blaps Rynchopetera Defense Secretion On Colorectal Tumor Cells

Objective:To investigate the antineoplastic effect and mechanism of quinones,the main active components from defense secretion of Blaps rynchopetera on colorectal tumor cells.Methods:1.MTT assay was used to analyze the inhibitory effect of the samples on the proliferation of human colorectal cancer cell HT-29 and human normal colon epithelial cell CCD841,detecting the proliferation inhibitory activity of the sample on tumor cells,and investigating whether it has a selective inhibitory effect.2.Enzyme-linked immunosorbent assay was used to detect the influence of samples on the expression of HT-29 tumor-related factors TNF-α,IL-10 and IL-6.3.The effects on apoptosis of normal and tumor cells was tested by flow cytometry.4.Flow cytometry was used to investigate the changes of cell cycle in HT-29.5.The expression of Wnt/β-catenin pathway related genes was detected by RT-PCR.6.The expression of Wnt/β-catenin pathway related proteins was detected by Western Blot.Results:1.Methyl p-benzoquinone,ethyl p-benzoquinone and methyl hydroquinone inhibited the proliferation of HT-29 cells and CCD841 cells in a dose-dependent manner after 24h treatment,but there was no selective inhibition effect.2.Since methyl p-benzoquinone and ethyl p-benzoquinone have similar structures and the IC₅₀ of methyl p-benzoquinone is smaller,methyl p-benzoquinone and methyl hydroquinone were selected for further study.The results showed that both p-benzoquinone and methyl hydroquinone could inhibit the expression of TNF-αand IL-6 in a dose-dependent manner（P<0.01 or P<0.05）,IL-10 expression was inhibited in low and high dose groups of methyl p-benzoquinone and medium and high dose groups of methyl hydroquinone（P<0.01 or P<0.05）.3.Methyl-p-benzoquinone and methyl-hydroquinone can both increase the apoptosis rate of HT-29 and CCD841,while which of HT-29 cells was higher than CCD841.4.Cell cycle detecting results showed that Methyl p-benzoquinone and methyl hydroquinone could decreased the proportion of HT-29 cells in G1 phase,increased the proportion of cells in S phase,and the cells were blocked up before G2 phase.5.RT PCR and Western Blot results showed that methylp-benzoquinone and methyl-hydroquinone up-regulated GSK-3βand APC m RNA and protein expression in Wnt/β-catenin pathway,and decreased which ofβ-catenin,Frizzled 1,c-Myc and Cyclin D1（P<0.01 or P<0.05）.Conclusions:The results showed that quinones（Methyl p-benzoquinone,ethyl p-benzoquinone and methyl hydroquinone）in the defense secretion from B.rynchopetera can inhibited the proliferation of colorectal tumor HT-29 cells,decreased the expression of tumor-related factors（TNF-α,IL-6,IL-10）,selectively promoted the apoptosis of HT-29 cells,and regulated the cycle distribution of HT-29 cells through the Wnt/β-catenin pathway.