| The Traditional Chinese Medicine garlic is the bulbs of Allium sativum L.the Allium species of the Liliaceae family,In the Compendium of Materia Medica,garlic was recorded to smell pungent and warm with slight toxicity.It is mainly used for the treatment of febrile illnesses,dry cholera,chronic heartache,malaria,malignant nuclear swelling,alopecia alopecia in children and snake or scorpion stinging.Volatile oils extracted from garlic garlic oil has significant anticancer activity.Its main active components against tumors are organosulfur species.Studies have shown that allitricin(Diallyl Trisulfide,DATS)is the most potent antitumor substance from garlic.Some studies have found that allitricin has significant proliferation inhibitory effect on human gastric cancer cells and can arrest human gastric cancer cells in G2/M phase,but the mechanism is still unclear.Here,we investigate the mechanism of cell cycle arrest induced by DATS in human gastric cancer MKN45 cells by adopting network pharmacology method and experimental validation,which will provide scientific basis for its application on prevention and treatment of gastric cancer.In this paper,MTT assay was first applied to observe the in vitro inhibitory effect of DATS on human gastric cancer cells MKN45,and the results showed that after treatment for 48 h,the IC50 of DATS was 186.41 μmol·L-1,Then,PI staining and flow cytometry were used to determine the effect of DATS on the cell cycle distribution of MKN45 cells.It was found that DATS treatment could increase the ratios of cells in the G2/M phase significantly.Meanwhile,compared with control group,the cell ratios in the G0/G1 and S phases were significantly decreased after DATS treatment(P<0.01).Western blot was subsequently applied to determine the effect of DATS on cycle related proteins in MKN45 cells.Here,we found that after treatment with 50,100,and 200μmol·L-1 of DATS for 48h,the expressions of CDK1 and Cdc25C were markedly decreased(P<0.01),and the expression of p21 was notably increased(P<0.01),Whereas the expression level of p-CDK1 in the MKN45 was significantly increased and the expression level of cyclinB1 was dramatically decreased after treated with 100 and 200μmol·L-1 DATS for 48h(P<0.01).In order to further explore whether DATS mainly blocks gastric cancer MKN45 cells in M phase or G2 phase,the phosphorylation of histone H3,a marker protein of M phase,was detected by Western blot.The results showed that the phosphorylation of histone H3 increased with the increase of DATS concentration.Combined with the follow-up study,it demonstrated that allicin could induce a significant increase of cyclinB1 protein at 12 h(P<0.01),and with the prologation of DATS administration,a notable decrease of the expression of cyclinB1 was observed,indicating that DATS could block gastric cancer MKN45 cells in M phase.Next,the effects of mitotic spindle assembly checkpoint related proteins were examined by Western blot,and the results showed that DATS decreased the expression of PLK1,Aurora A,Bub1,and BubR1 proteins(P<0.01)and increased the expression of securin proteins significantly(P<0.01),affecting the mitotic process of MKN45 cells.Effect of DATS on cytoskeletal microfilaments of gastric cancer MKN45 cells was observed by using immunofluorescence,which revealed a disrupted microfilament structure with increasing concentrations of DATS.Next,network pharmacology was used to predict the core targets and mechanisms of DATS.128 DATS targets were obtained from databases of Stitch,Swiss target prediction,PharmMapper and literature mining.Using "gastric cancer " as the key word,289 targets from Genecards database,53 targets from GAD database,5 targets from TTD data were obtained respectively.After removing the duplicate targets,a total of 318 gastric cancer-related targets were collected.Then,Venny 2.1 online tool was used to draw the Venn diagram of DATS and gastric cancer targets,and 29 intersection targets were obtained.Key targets were imported into String database to construct the PPI network model,and the results were imported into cytoscape3.2.1,involving a total of 29 nodes,252 edges,among which the key proteins were obtained as TP53>Jun>MAKP3>VEGFA.Finally,the core targets were inputed into David database for GO and KEGG analysis of targets in DATS-acting gastric cancer,the top biological processes or pathways were screened and visualized with Omicshare,and a total of 20 pathways were involved,including 12 disease pathways,8 signaling pathways,among which the key pathways related to cell cycle were MAPK signaling pathway,Ras signaling pathway,PI3K/AKT signaling pathway,VEGF signaling pathway.To verify the results of network pharmacology prediction,Western blot was employed to determine the effect of allitricin on cell cycle related proteins in MKN45 cells.The result showed that after treated with 50,100,and 200μmol·L-1 DATSfor 48 h there were no significant changes on the expression of ERK and PDGFRβ in human gastric cancer MKN45 cells.However,the expression of p-ERK,p-FOXO3a,p-PDGFRβ were significantly decreased(P<0.01).100 and 200 μmol·L-1 DATS treatment could also increase the expression of FOXO3a significantly(P<0.01).To investigate the mechanism of MKN45 cell death induced by DATS in gastric cancer,inverted microscope was used to observe the morphological changes of MKN45 cells treated with DATS.It was found that growth density of MKN45 cells was reduced,and the apoptotic bodies appeared.Flow cytometry was used to detect the effect of allitricin on cells apoptosis rate.The result showed that the cell ratio in the sub-G0/G1 phase of MKN45 cells increased obviously with the increasing concentrations of DATS.We next assessed the mechanism of allitricin by detecting apoptosis related proteins by Western blot.The result showed that different concentrations of DATS could significantly increase the death receptor apoptosis related proteins TRAIL,caspase-8 and caspase-3(P<0.01).100 and 200 μmol·L-1 DATS treatment could also increase the expression of DR5 significantly(P<0.01).In conclusion,DATS could induce M phase arrest of human gastric cancer MKN45 cells by decreasing the expression of PLK1,Aurora A,Bub1,BubR1 and securin through PDGFRβ/ERK/FOXO3a pathway.Finally,the death receptor pathway was activated to induce cell apoptosis of MKN45 cells,However,the relevant mechanism remains to be further studied. |
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