| Objective:Colorectal cancer(CRC)is one of the digestive tract malignant tumor.Its high morbidity and mortality are serious threats to human life and health.At present,the treatment of colorectal cancer is mainly based on surgery,supplemented by comprehensive treatments such as radiotherapy and chemotherapy,but the the effect of radiotherapy and chemotherapy is restricted seriously by the side efects such as low immune function,bone marrow suppression,organ damage,and drug resistance.Traditional Chinese medicine has complex components and diverse targets,so it can act on different aspects of tumor pathogenesis.At the same time,it has low side effects and difficult to form drug resistance,It has become an important component of adjuvant treatment for colorectal cancer.Ampelopsin(AMP)is a small molecule flavonoid extracted from Chinese medicinal herb Ampelopsis,which has shown a broad spectrum of anti-tumor effect and cycotoxicity on many kinds of cancers.In recent years,it has been found that autophagy also plays an important role in the occurrence and development of tumor cells,and the relationship between autophagy and apoptosis is very important on the progression and treatment of tumors.However,the mechanism of AMP-induced apoptosis and autophagy in human colorectal cancer cell lines are not deeply investigated.In the present study,we investigated the mechanism of AMP-induced apoptosis and autophagy in human colorectal cancer cell lines using the techniques of metabolomics,cell biology and molecular biology.Methods:(1)Human colorectal cancer HCT-116,SW480,SW620,LOVO,Colo 205,LS174 T cells were treated with various concentrations of AMP(6.25,12.5,25,50 and 100 μg/ml)for 24,48 and 96 h,MTT assay was used to detect the effect of AMP on the proliferation inhibition rate of cells.The effect of AMP on cell clone formation rate was observed by colony formation assay,and the effect of AMP on cell migration ability was observed by wound healing test.(2)The therapeutic efficacy and safety profile of AMP was further tested in vivo using HCT-116 xenograft model.(3)Based on untarged metabnomics of UPLC-TOFMS,we investigated the tissue sample of HCT-116 xenograft model for screening the different metabolites and metabolic pathways.(4)Human colorectal cancer HCT-116 and SW480 cells were treated with AMP(50,100 μg/ml)for 48 h.The effects of AMP on cell cycle and apoptosis were observed by flow cytometry.The morphology of the cells was observed by inverted microscope.The changes of cellular autophagy vesicles were observed under fluorescence microscope by staining with Acridine orange(AO).The effect of AMP on autophagy was observed by electron microscopy.Western blotting was used to detect the expression of BCl-2,Bax,Cleaved-Caspase-3,LC3,Beclin-1 and Atg5 in cells or HCT-116 xenografts in nude mice.(5)Human colorectal cancer HCT-116 and SW480 cells were treated with AMP comnbined with autophagy inhibitor 3-methyladenine(3-MA)or autophagy promoter Rapamycin(Rapa).MTT assay was used to test effect of AMP combined with 3-MA or Rapa on the proliferation inhibition rate of cells.The apoptosis rate was tested with flow cytometry,and the autophagy vesicles were detected by fluorescence microscope.Western-blotting was used to detect the expression of BCl-2,Bax,Cleaved-Caspase-3,LC3,Beclin-1 and Atg5.(6)Western blotting was used to detect the expression of PI3 K,p-mTOR,Total-mTOR,p-Akt,Total-Akt,p-P70S6 K and Total-P70S6 K.Results:(1)AMP inhibited human colorectal cancer HCT-116,SW480,Colo205 cells in dose and time-dependent manner,and IC50 gradually decreased with time.However,the inhibition of proliferation was the strongest at 72 h,and then the inhibition decreased with time for human colorectal cancer SW620,LOVO and LS174 T cells.The colony formation rate and cell migration area was significantly reduced.(2)AMP 200 mg/kg inhibited the growth of HCT-116 xenografts in nude mice,There was no significant difference in organ index between control group and administration group.In addition,HE staining results showed that there were no obvious abnormalities in the vital organs of the animals.(3)Based on metabnomics,we found that AMP could inhibit the growth of Colorectal cancer by regulating tricarboxylic acid cycle,amino acid metabolism,nucleotides Metabolism and aminoacyl tRNA biosynthesis.(4)Flow cytometry analysis showed that the population of AMP-treated cell was significantly increased,with a corresponding decrease in G1 phase,and the apoptosis rate increased significantly.Typical apoptosis was observed under light microscope and electron microscope.The fluorescence microscope analysis showed that the proportion of red fluorescence was significantly increased in AMP treated cells.In addition,autophagosomes and autophagic lysosome with a double-layer or single-layer membrane were observed in the cytoplasm.Western-blotting analysis showed that AMP down-regulated the expression of BCl-2,up-regulated the expression of Bax and Cleaved-Caspase-3,and the expression of LC3-II,Beclin-1 and Atg5 were increased significangtly.(5)Treatment combined with 3-MA increased the growth of HCT-116 and SW480 cells,decreased the ratio of red fluorescence and expression of LC3 II,Beclin-1 and Atg5 in the cells,whereas combined with Rapa inhibited the growth of cells,increased the ratio of red fluorescence and expression of LC3 II,Beclin-1 and Atg5 in the cells.Flow cytometry analysis showed that the apoptosis rate was significantly lower treatment combined with 3-MA than that of AMP treatment group,the situation of treatment combined with Rapa was opposie.Western-blotting analysis showed that the ratio of Bcl-2/Bax did not change significantly treatment combined with AMP,but the expression of Cleaved-Caspase-3 was significantly lower than that of AMP treatment group.The proportion of Bcl-2/Bax was significantly decreased,and the expression of Cleaved-Caspase-3 was significantly up-regulated treatment combined with RAPA.(6)Western-blotting analysis showed that the expression of PI3 K,p-Akt,p-mTOR and p-P70S6 K were decreased in human colorectal cancer HCT-116,SW480 cells and HCT-116 xenografts.Conclusion:(1)AMP inhibits the proliferation of CRC cells,meanwhile inhibits the migration and clonegenic of HCT-116 and SW480 cells.(2)AMP exhibits significant anti-tumor activity in vivo,and there is not significantly toxic to the major organs.(3)AMP mainly affects abnormal energy metabolism to inhibit the growth of colorectal,especially by regulating amino acid metabolism.(4)AMP increases cells in G1 phase,meanwhile induces apoptosis and autophagy in HCT-116 and SW480 cells.(5)Autophagy promotes AMP-induced apoptosis.(6)AMP induces CRC cells apoptosis and autophagy mainly through inhibiting the activity of PI3K/Akt/mTOR signal pathway. |
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