Oridonin Inhibits The Colorectal Cancer By Reducing PKM2 Dimer Formation And Preventing Its Entry Into The Nucleus

Background:Colorectal cancer is one of the most common malignant tumours;PKM2 is the rate-limiting enzyme of glycolysis,and its dimeric form has low enzymatic activity but can translocate to the nucleus to promote tumour development;oridonin(ORI)is a herbal extract with anticancer activity,and its anticancer mechanism remains to be elucidated.Objective:To investigate the mechanism of anti colorectal cancer by oridonin.Methods:1.To observe the anti-cancer activity of ORI,the effect of different concentrations of ORI on the viability of colorectal cancer HCT116,HCT8 and RKO cells was first examined using the CCK8 assay,and the effect of ORI on the proliferation of colorectal cancer HCT116,HCT8 and RKO cells was examined using the EDU assay,and finally the effect of ORI on the apoptosis of HCT116 cells was analysed using flow cytometry.2.To find the specific mechanism of ORI against colorectal cancer cells,we used high-throughput sequencing to analyse the changes in the transcriptome of HCT116 cells by ORI,followed by glucose,lactate and ATP content assay kits to detect the effects of ORI on glucose uptake,lactate production and ATP production in HCT116 cells.3.To observe the effect of ORI on PKM2,qPCR was used to detect the expression of PKM2 mRNA and WB to detect the expression of PKM2 protein;DSS protein cross-linking technique was used to cross-link the total cellular protein and the level of PKM2 multimers was detected by WB technique;nuclear protein was extracted from HCT116 cells using nuclear protein extraction kit and nuclear changes of PKM2 were verified by cellular immunofluorescence assay.4.To investigate how ORI inhibits dimeric PKM2 and nuclear PKM2,we examined the activity of EGFR/ERK1/2 pathway that regulates nuclear translocation of PKM2 by WB assay,the binding ability of Importin-α5 to PKM2 by IP assay,and performed computerized molecular docking prediction to observe the potential interaction between ORI and PKM2;subsequently,we separately HCT116 cells were treated with the PKM2 inhibitor Cys and activator FDP,and the drug was added to the cells on this basis.The changes of dimeric PKM2 were observed by protein cross-linking assay,nuclear proteins were extracted and subjected to WB to detect the changes of nuclear PKM2,and total proteins were extracted for WB to detect the expression of c-Myc,p-STAT3 and GLUT1 downstream of PKM2.The effect of ORI on glucose uptake,lactate production and ATP production in HCT116 cells was again examined using glucose,lactate and ATP content assay kits.5.To further confirm whether Cys or FDP alters the effect of ORI on HCT116 cell viability,proliferation and apoptosis,we used CCK-8,EDU and flow cytometry to detect cell viability,proliferation and apoptosis.6.To verify whether ORI can inhibit tumour growth in vivo,we established an ectopic transplantation tumour model of HCT116 in nude mice,randomly grouped and subjected to ORI treatment experiments,and after the experiments,the tumours were removed for weighing and volume measurement,Immunohistochemistry was performed to detect the abundance of Ki67 in tumour tissues,tissue nuclear proteins were extracted and WB detected the changes of nuclear PKM2,total proteins were extracted and WB detected the expression of c-Myc,p-STAT3 and GLUT1 expression.Results:1.ORI significantly inhibited the proliferation of colorectal cancer cells and promoted apoptosis.ORI significantly inhibited the viability of three colon cancer cells,HCT116,HCT8 and RKO,with IC50 values of 26.84,17.71 and 14.59 μM at 24 h.The chromatin replication of the three cells was significantly inhibited by ORI after 24 h.The apoptosis level of HCT116 cells was significantly up-regulated by ORI.2.ORI inhibits the Warburg effect in colon cancer cellsSignificant down-regulation of the cAMP pathway and a pentose/glucuronide interconversion event in ORI-treated cells compared with controls;ORI inhibited cellular glucose uptake and lactate production,but increased intracellular ATP levels.3.ORI reduced the dimerization level of PKM2 and prevented the nuclear translocation of dimeric PKM2.ORI had no significant effect on the transcription and translation of PKM2,but ORI reduced the dimerization level of PKM2 and inhibited the nuclear translocation of PKM2,with a consequent decrease in the expression of p-STAT3,c-Myc and GLUT1 downstream of nuclear PKM2.4.ORI reduces the binding of importin-alpha5 to dimeric PKM2 and stabilises the formation of tetrameric PKM2ORI can interact with tetrameric PKM2 protein in various forms:forming hydrogen bonds with Arg400,van der Waals forces with Arg399,Ile404,Glu396,Arg422 and Arg447,and Pi-alkyl interactions with Phe395,Phe421 and Leu392,thus stabilising the spatial conformation;in addition,ORI reduced the binding ability of Importin-α5 to PKM2;the PKM2 dimer activator Cys reversed the effects of ORI,i.The PKM2 dimer activator Cys reversed the effects of ORI,i.e.increased levels of PKM2 dimers,enhanced nuclear translocation of nuclear PKM2,upregulated expression of p-STAT3 and c-Myc downstream of nuclear PKM2,increased cellular glucose uptake and lactate production,while the PKM2 tetramer activator FDP enhanced the effect of ORI.5.Cys or FDP reversed or enhanced the effects of ORI on HCT116 cell viability,proliferation and apoptosis.Cys significantly reversed the inhibitory effects of ORI on cell viability and proliferation and reduced the pro-apoptotic effects of ORI.However,FDP potentiated the inhibitory effects of ORI on cell viability and proliferation and enhanced the pro-apoptotic effects of ORI..6.ORI inhibits colorectal cancer growth in vivo by reducing PKM2 dimer formation and nuclear translocationCompared with the control group,the weight and volume of transplanted tumours in experimental nude mice were significantly reduced and Ki67 expression was significantly inhibited;nuclear PKM2 was also reduced in tumour cells and the expression of p-STAT3,c-Myc and GLUT1 downstream of nuclear PKM2 was also inhibited..Conclusions:ORI inhibited colorectal cancer cell proliferation and apoptosis in vitro and colorectal cancer growth in vivo.ORI promoted the conversion of PKM2 dimers to tetramers,reduced the level of glycolysis in CRC cells,decreased the nuclear translocation of PKM2 dimers and inhibited the activity of the PKM2/STAT3/c-Myc axis.The reduction in c-Myc expression further downregulated downstream GLUT1 expression,which then inhibited the Warburg effect,leading to apoptosis and inhibition of proliferation in tumour cells.