| Colon cancer has the third and second highest incidence and death rates,respectively,of all types of cancer worldwide.The first-line treatment for colon cancer is usually drug therapy,surgical excision,and chemotherapy in combination with monoclonal antibodies or proteins that resist vascular endothelial growth factor and epidermal growth receptor.In addition to traditional chemotherapy,naturally derived plant compounds are currently receiving much attention as adjunctive therapies for colon cancer treatment,which can improve the therapeutic effect,reduce side effects and prolong the survival of patients.Cafestol,a diterpenoid compound extracted from coffee beans,has a wide range of activities,including anti-inflammatory,anti-angiogenic and anti-tumor properties.Our previous in vitro cell experiments showed that cafeol had good antitumor activity on human colon cancer HCT116 cells,and inhibited the proliferation of colon cancer cells in vitro through the LKB1/AMPK/ULK1 signaling pathway.However,after LKB1 gene was knocked out,the corresponding tumor inhibition effect would not occur.In order to further explore the influence of cafestol on animals,we established a nude mouse transplanted tumor model and applied different concentrations of cafestol to observe its anti-tumor effect on colon cancer and explore the possible mechanism of action.This paper tries to find out more safe and effective chemotherapeutic drugs to provide more theoretical basis for the clinical application of cafestol in the treatment of colon cancer.Objectives:1.This study was designed to establish an ectopic colon cancer model of nude mice based on the level of animal experiments in vivo,and explore whether cafestol can inhibit the proliferation of colon cancer tumor tissues in nude mice by detecting the intervention of different concentrations of cafestol.2.By detecting the levels of autophagosomes and autophagy marker proteins in nude mice,the preliminary conclusion was that cafestol could induce autophagic death of tumor cells and inhibit the proliferation of colon cancer tissues in nude mice.To determine the key pathway and molecular mechanism of cafestol induced autophagy in tumor tissue cells.3.Fecal microflora of nude mice in different treatment groups were detected to explore the effects of cafestol intervention on intestinal microflora microecology and species relative abundance and diversity of nude mice with colon cancer tumors.Methods:1.Routine culture of human colon cancer HCT116 cells was performed by using serumless medium to make a single cell suspension with a concentration of 2.5×10~7cells/m L.0.15 m L/tumor cells were inoculated subcutaneously into the right armpit of nude mice,and the growth of transplanted tumors was observed daily.During animal modeling,the inhibition of weight,tumor volume and tumor weight of nude mice were preliminarily observed.2.HE staining was used to analyze the tumor and colon tissues of nude mice with different concentrations of cafestol.3.Autophagosomes were observed by transmission electron microscopy to study whether cafestol can promote autophagic death of tumor cells in vivo.4.Western Blot was used to detect the expression levels of autophagy marker protein and LKB1/AMPK/ULK1 pathway protein in tumor tissue of nude mice after cafestol intervention,so as to explore the mechanism of cafestol inhibiting the growth of colon cancer in vivo.5.The 16s r DNA high-throughput sequencing technology was used to analyze the effects of cafestol intervention on intestinal microecology,relative species abundance and diversity in nude mice with colon cancer tumors.6.x±s description was used for those with normal distribution of measurement data;t test was used for comparison between two groups;one-way analysis of variance was used for comparison between multiple groups;LSD method was used for pduo comparison;pindicated statistically significant difference.Spearman correlation analysis was used to explore the association between intestinal flora level species and autophagy and pathway-related proteins in nude mice.Results:1.During animal modeling,it was found that the body weight of nude mice in the model group decreased rapidly,while the body weight of nude mice in the cafestol intervention group remained unchanged;During animal anatomy,tumor volume and tumor weight were observed,and it was found that the tumor volume and tumor weight of nude mice in the cafestol intervention group were significantly lower than those in the model group and the difference was significant.It indicates that cafestol intervention can inhibit the growth of tumor tissue.2.HE stained sections were made for pathological analysis of tumor and colon tissues of nude mice,and it was found that tumor tissues in the model group showed more mitotic phases and multiple necrosis,while the mucosal epithelial cells in the cafestol intervention group had clear and complete structure and normal morphology.In the model group,there was mild edema of colonic mucosa and occasional parasites in the intestinal cavity,while in the cafestol intervention group,the intestinal mucosa epithelium was intact and the cell morphology was normal.The results showed that cafestol can improve the lesion of tumor tissue and intestinal tissue cells caused by ectopic colon cancer.3.Electron microscopy showed that the autophagosomes in the tumor tissues of the cafestol intervention group were significantly increased.The subsequent Western Blot detection of the autophagy signature proteins LC3B-II and Beclin-1 showed that the protein expression levels increased with the increase of cafestol intervention concentration.It is suggested that cafestol may induce autophagy in tumor cells.4.The expression level of LKB1 protein was detected by immunohistochemistry,and it was found that it increased significantly with the increase of cafestol intervention concentration.The protein expression levels of colon cancer autophagy pathway LKB1/AMPK/ULK1 were detected by Western Blot analysis.With the increase of cafestol dose,the expression levels of LKB1,AMPK and ULK1 were significantly up-regulated.It is suggested that cafestol may induce autophagy of tumor cells by upregulating LKB1 and activating AMPK/ULK1.5.The 16s high-throughput sequencing analysis of fecal flora of mice in different treatment groups showed that the species richness of inferior bacteria,such as Firmicutes and Burkholderia,was higher in the model group.Alloprevotella and Muribaculaceae were relatively abundant in the cafestol intervention group.It is suggested that cafestol can improve the microecology of intestinal flora in nude mice and play a beneficial effect on colon cancer.6.Spearman correlation analysis was performed on autophagy and pathway-related proteins of the top 12 species with relative abundance of intestinal flora in nude mice at genus level.It was found that the three beneficial bacteria genera with the highest content of Muribaculaceae,Bacteroides and Prevotellacece were positively correlated with LC3B-II and Beclin-1 autophagy signature proteins and LKB1,AMPK and ULK1 pathway proteins Shut.And the harmful bacteria,For example,Lachnospiraceae,Helicobacter and Alistipes were significantly negatively correlated with LC3B-II and Beclin-1 autophagy marker proteins and LKB1,AMPK and ULK1 pathway proteins.These results suggest that the changes in autophagy of tumor cells in ectopic carcinoma of nude mice may lead to changes in intestinal flora composition.Conclusions:1.Cafestol can inhibit the growth of ectopic carcinoma inoculated by colon cancer HCT116 cells in nude mice.2.Cafestol may activate the downstream autophagy related pathway AMPK/ULK1 by up-regulating the expression level of tumor suppressor gene LKB1,thereby inducing autophagy death of cells and ultimately inhibiting the proliferation activity of tumor tissue in nude mice.3.Cafestol can increase the content of beneficial bacteria in the intestinal tract of nude mice,such as Muribaculaceae,Bacteroides and Prevotellacece.And reduced harmful Lachnospiraceae,Helicobacter and Alistipes.It was also correlated with LC3B-II and Beclin-1 autophagy signature proteins and LKB1,AMPK and ULK1pathway proteins. |
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