Studies On The Effect Of Ponicidin On Epithelial Mesenchymal Transformation And Metastasis Of Colorectal Cancer Cells And Its Mechanism

Colorectal cancer is one of the malignant tumors in the world,with its incidence ranking the fourth among all malignan t tumors,and the mortality ranking the second among all malignant tumors,posing a serious threat to human health.The metastasis of colorectal cancer is one of the important lethal factors in many patients with colorectal cancer.With the acceleration of the pace of life and the change of dietary structure,the incidence of colorectal cancer gradually appeared the trend of universalization and rejuvenation.At present,the treatment of colorectal cancer and its metastasis is mainly surgical resection combined with radiotherapy and chemotherapy,and has achieved a certain clinical effect.However,the condition of many patients with colorectal cancer does not meet the conditions of surgical resection.In addition,the side effects of radiotherapy and chemotherapy are very harmful to the patients.Therefore,how to expand the treatment of colorectal cancer-related diseases and alleviate the pain caused by chemotherapy and radiotherapy are urgent problems to be solved in the medical community.Traditional Chinese medicine is an important part of world medicine.After thousands of years of experience in drug use,traditional Chinese medicine has formed its unique medical theoretical system.Using traditional Chinese medicine theories and methods to treat tumors and its metastasis has achieved great success.Many traditional Chinese medical prescriptions,single herb and active monomers have shown strong anti_tumor and anti-tumor metastasis effects.Therefore,it is particularly important to explore the effective material basis of traditional Chinese medicine against tumor and its metastasis.Rabdosia rube.scens is a perennial herb,a subshrub plant or a small shrub plant,which is a commonly used herb in the clinical practice of traditional Chinese medicine.The nature of the Rabdosia rubescens is slightly cold and the flavor of it are bitter and sweet.Rabdosia rubescens belongs to the lung,stomach,liver meridians and has the efficacy of clearing away heat and detoxification,promoting blood circulation and relieving pain,and has good antibacterial,anti-tumor,antipyretic and noise-reducing effects.At present,it has been proved that Rabdosia rube.scens has a good therapeutic and palliative effect on esophageal cancer,breast cancer,liver cancer,cardiac cancer and colorectal cancer.As one of the main active components of Rabdosia rubescens,Ponicidin(PON)has obvious anti-tumor effect.However,whether PON has the effect of anti-tumor epithelial mesenchymal transformation(EMT)and metastasis has not been clearly reported in the literature.Therefore,through literature retrieval mining and pre-experiment,our research group put forward the hypothesis that PON may have the effect of anti-tumor EMT and metastasis,and the mechanism may be related to AKT/GSK-3?/Snail and AKT/GSK-3?/?-catenin signaling pathway.Based on the cell related studies in vitro,our research group first investigated the effects of PON on TNF-?-induced EMT and metastasis of colorectal cancer cells.First of all,through the experiment of the effect of drugs on EMT.it was preliminarily concluded that PON could inhibit EMT of HCT116 cells from the changes of cell phenotype.Then it was confirmed that PON had anti-tumor metastasis effect on HCT116 cells by cell scratch assay,Transwell migration and invasion assays.The possible molecular mechanisms of PON against EMT and metastasis of HCT116 cells were explored by qPCR,immunofluorescence and Western Blot assays,respectively and it was proved that PON had significant effects on EMT-related biomarkers of HCT116 cells,such as E-cadherin,N-cadherin and Vimentin,and could inhibit AKT/GSK-3?/Snail and AKT/GSK-3?/?-catenin pathway to a certain extent.On the basis of cell research in vitro,the liver metastasis model of colorectal cancer in nude mice of HCT116 cells was established through the whole animal experiment in vivo,and the effects of PON on EMT and metastasis of HCT116 cells in vivo were further studied.The results showed that PON could alleviate and inhibit the EMT and metastasis of colorectal cancer cells in nude mice,and could effectively reduce the volume of splenic tumors and the number of liver metastases and alleviate the weight loss of nude mice.Then,the pathological evaluation of liver tissue of nude mice was carried out by HE staining,and the molecular mechanism of PON against EMT and metastasis of colorectal cancer cells in vivo was studied by IHC assay.The results showed that PON could alleviate the infiltration of tumor cells in the liver of nude mice,and its anti-EMT and anti-metastasis mechanism of colorectal cancer cells may be related to promoting the expression of E-cadherin,inhibiting the expression of N-cadherin,p-AKT,p-GSK3?,?-catenin and transcription factor Snail.By using baculovirus expression vector system,the subtypes of AKT1 and AKT2 proteins of the AKT were expressed in the oocyte Sf9 of Spodoptera exigua,and the corresponding protein products were obtained.The affinity of PON to AKT1 and AKT2 was tested by biolayer interferometry technique.The results showed that PON may have affinity with AKT1 and AKT2 proteins to a certain extent.Finally,virtual molecular docking technology was used to dock PON with AKT1 and AKT2 with Schrodinger Suites software.The results showed that PON may have corresponding binding sites in the ATP active pockets of AKT1 and AKT2.The results showed that PON may have corresponding binding sites in the ATP active pockets of AKT1 and AKT2,and finally formed a non-covalent bond force with the corresponding amino acid residues,thus forming affinity with AKT1 and AKT2 proteins.To sum up,our group confirmed that PON m ay inhibit EMT and metastasis of colorectal cancer HCT116 cells in vitro and in vivo,and its related molecular mechanism may be related to the influence of EMT-related biomarkers and the inhibition of the activation of AKT signal pathway.In addition,the subtypes AKT1 and AKT2 of AKT protein were expressed by baculovirus expression vector system,and the possible affinity between AKT1 and AKT2 protein and PON was explored by biolayer interferometry.Furthermore,the possible binding patterns and binding sites of PON to AKT 1 and AKT2 proteins were predicted by molecular docking technique.These studies have revealed that PON,an active molecule of traditional Chinese medicine,may have the potential to become an anti-tumor metastasis drug.