Preliminary Study On The Mechanism Of Dendrobium Officinale Glucomannan Against Colon Cancer Through Autophagy And Mitochondrial Dysfunction

Dendrobium officinale is a rare and valuable kind of medicinal plant of Orchidaceae.Its biological components have good active functions,such as hypoglycemic,anti-tumor and so on.Therefore,it has great medicinal value and market potential.At present,it has been cultivated artificially.In this study,Dendrobium officinale polysaccharide(DOP,2,3-O-acetyl-glucomannan)was used as the research object and the effect of DOP on the proliferation of colon cancer cells was detected by cell experiments.RNA-seq and bioinformatics were used to predict the possible anti-tumor mechanism.Further experiments showed that DOP treatment resulted in increased autophagy,decreased mitochondrial membrane potential(MMP),accumulation of ROS and imbalance of mitochondrial energy metabolism in colon cancer cell line CT26.Meanwhile,western blot was adopted to detect the expression of key proteins in the signaling pathway of autophagy induced by DOP,and the interaction among DOP-induced autophagy,mitochondrial dysfunction and ROS was further explored in combination with an antioxidant.The main conclusions are as follows:1.Detecting the inhibitory effect of DOP on the viability of colon cancer cells.The influence of DOP on cell viability was detected by CCK-8,our results showed that DOP significantly inhibited the cell viability of colon cancer cells CT26 and HCT-116 in a dose and time-dependent manner,but showed no cytotoxicity to normal intestinal cells IEC-6 in the concentration range of 800?g/mL.2.Detecting the effect of DOP on the gene expression of colon cancer cell line CT26.The transcriptome information of CT26 cells treated with DOP(800 ?g/mL and 0)was obtained by RNA-seq and bioinformatics analysis.A total of 2553 differentially expressed genes between DOP treated group and the control group,of which 1228 genes were up-regulated and the others were down regulated.Go enrichment analysis showed that autophagy,oxidative stress,AMPK pathway and cell death were significantly up-regulated,while mTOR signaling,respiratory system,lysosome and other pathways were significantly down regulated in DOP treated group.At the same time,GSEA enrichment analysis results also demonstrated that DOP treatment group enriched selective autophagy,oxidative stress,programmed cell death and activation of downstream AMPK pathways.3.Exploring the effect of DOP on autophagy of CT26 cells.Based on m RFP-EGFP-LC3 double fluorescence reporter gene and western blot analysis,we found that DOP significantly induced the formation of autophagosomes,the conversion of LC3 II and the down-regulation of p62 expression.These data demonstrated that DOP treatment up-regulated autophagy in CT26 cells.4.Detecting the effect of DOP on ROS production and mitochondrial function of CT26 cells.The mitochondrial membrane potential(MMP)and content of ROS in CT26 cells treated with DOP were analyzed by Mito Tracker staining and DHE staining,respectively.The results of analysis showed that DOP induced ROS accumulation and MMP down-regulation.Furthermore,the oxygen consumption rate(OCR)was measured by the hippocampal extracellular flux analyzer(XFp,Agilent),and then the Basal Respiration,Non-Mitochondrial Oxygen Consumption,ATP Production,Proton Leak,Spare Respiratory Capacity and Maximal Respiration were detected.Our results showed that DOP inhibited OCR and various mitochondrial pressure indexes,leading to mitochondrial dysfunction.5.Exploring the relationship between molecular mechanism of autophagy induced by DOP and mitochondrial dysfunction.Combined with RNA-Seq analysis and western blot assay,the expression level of the upstream protein of autophagy signaling pathway was detected,and the results showed that DOP activated the AMPK/mTOR autophagy signaling pathway in CT26 cells.In addition,DOP inhibits the synthesis of ATP in mitochondria,which is the key inducement of AMPK activation.In conclusion,DOP could trigger AMPK/mTOR autophagy signaling pathway in CT26 cells by inhibiting mitochondrial energy synthesis.6.Exploring the relationship between DOP-induced ROS and autophagy.Antioxidant NAC was adopted to pretreat CT26 cells,the influences of DOP on cell activity,autophagy,ROS content and mitochondrial function of CT26 cells were detected.The data showed that the elimination of ROS by NAC inhibited DOP-induced AMPK/mTOR autophagy signaling and reversed cancer cell death and mitochondrial dysfunction.These results suggested that autophagy activated by DOP is regulated by ROS,which was realized through ATP-AMPK-mTOR signaling axis.