The Investigation Of Grape Seed Proanthocyanidins For Its Inhibitory Effect And Mechanism On Colon Cancer Cells

Aims:The morbidity and mortality of colon cancer(colorectal,cancer,CRC)are very high.It is found that dietary structure is closely related to the occurrence of cancer,and anti-oxidants play an important role in inhibiting the process of tumorigenesis.As an important antioxidant,procyanidins occupy a higher proportion in people’s daily diet.Procyanidins is a typical representative of the homology of medicine and food,and it is a kind of common pol-yphenolic compounds.Researches indicated that polyphenols could prevent and treat cancers with low toxic and side effects to human body,so it could be used as potential drugs to pre-vent and treat cancers.In this study,we treated the colorectal cancer cells SW480 and SW620 with Grape Seed Proanthocyanidins(GSPs)and the changes of cells’ biological characteristics were observed,e.g.:cellular morphology,cell proliferation,cell cycle,cell apoptosis,epithe-lial to mesenchymal transition(EMT)and exosome.The mechanism was investigated by identifying some genes with alterative expression using second generation sequencing tech-nology following by enrichment analysis of KEGG Pathway.Among them Akt pathway was investigated further since many identified genes concerns this pathway.So,this study may shed light on understanding colorect cancer prevention and open a new avenue for alternative medicine to treat other cancers.Methods:1.The authenticate of biological characteristics:(1)Cell morphologic obser-vation.Observe the change of cellular morphology of the SW480 and SW620 cells which were treated GSPs;(2)Detection of cell reproductive capacity.MTT method was used to de-tect the influence of GSPs on cell proliferation;(3)Cell cycle and cell apoptosis.FCM(flow cytometry)was used to detect influence of GSPs on two CRC cells.2.The molecular biolog-ical identification:(1)Western blotting was used to detect EMT,the Quantitative Real-time Polymerase Chain Reaction(Q-PCR)was used to detect the expression of miRNA in exo-somes,assessing the influence of GSPs on EMT and characteristics of exosome secreted by two CRC cells.(2)The influence of GSPs on the gene regulation of the CRC cells.Second generation sequencing technology was employed to identify the genes with different expres-sion between control and experimental group,and candidate genes were subjected to bioin-formatics analysis,GO function and KEGG Pathway enrichment analysis will be done with those differentially expressed genes.(3)Verification of partial target gene.Combine with the result of date analysis,the Q-PCR and Western blotting were employed to detect the mRNA and protein respectively before or after GSPs treatmentResults:The characteristics of SW480 and SW620 cells after GSPs treatment were as follows:(1)The changes of cells’ morphology:e.g.:being expanding,being roundness,being coarse and irregular.(2)Cell proliferation:a growth promoting effect was detected when SW480 and SW620 cells were treated with GSPs in a short time(24 h),and followed by in-hibiting ether for long term treatment(48 h and 72 h)or for increase of GSPs concentration.FCM analysis indicated that GSPs could trigger SW480 and SW620 cells cycle arrest in G0/G1 phase consistently.In addition,GSPs could also induce cell apoptosis.(3)EMT:GSPs inhibited the EMT on SW480 and SW620 cells,which is consistent with either the increase of E-cadherin expression in both cell body and exosome or decrease of total miRNA in exosome.With a pathway enrichment analysis,we found that more than 200 differential expres-sion genes concern Akt pathway.Further study on Akt pathway showed that GSPs downregu-lates both Akt expression and activity.In addition,some genes concerning cell proliferation and apoptosis were investigated and showed that GSPs downregulated Bcl-2,Cyclin D1 and CDK4 and upregulated Bad,Bax,caspase 3 and caspase 9.Conclusions:According to the authenticate of biological characteristics observation,GSPs could inhibit cell proliferation,EMT of CRC cells SW480 and SW620,lead to cell cy-cle arrested in GO/G1 phase and induce cell apoptosis.Consistently,GSPs could change the expression of molecules which have relation to cell proliferation,EMT and apoptosis.Gene screening showed that GSPs could induce alteration of many gene expression in SW480 and SW620 and many of them concerning Akt pathway.Further study on Akt pathway showed that GSPs could suppress both Akt expression and activation.Thus,this study may shed light on understanding CRC prevention and open a new avenue for alternative medicine to treat other cancers.