| Esophageal cancer seriously affects the body and mental health of Chinese residents.Esophageal cancer ranked sixth in the incidence of malignant tumors released in 2015,with more than 90% of these cancers being squamous cell carcinoma.With the development of the times,its incidence has shown an upward trend.The incidence varies widely around the regions of the world,especially in East Asia,and the rural areas are higher than the city.At present,the pathogenesis of esophageal cancer is related to many factors,including smoking,drinking,diet,genetic factors,and some chronic esophageal diseases.Invasion and metastasis are the important pathophysiological characteristics of esophageal cancer.Most patients with esophageal cancer are already in the middle or advanced stage.They lose the opportunity of surgery and can only choose chemoradiotherapy,targeted or immunotherapy.Although our country has made great progress in esophageal cancer,the final effect is not ideal.The 5-year survival rate is still no more than 20%.Therefore,the mechanisms of esophageal cancer and the development of more effective natural drugs are of great significance to improve the prognosis of patients with esophageal cancer.Elemene is a kind of olefin monomer extracted from the Chinese herbal medicine Wenyujin and β-elemene is its main component.As an auxiliary drug for chemotherapy,it has a wide range of anti-tumor effects.In esophageal cancer,β-elemene inhibits the growth of tumor,but the specific mechanism is entirely unclear.Therefore,the effects of β-elemene on the proliferation,invasion,and migration of esophageal cancer cells were first investigated.Then,we used network pharmacology and bioinformatics methods to find the hub gene and enrichment pathway of β-elemene in the treatment of ESCC,and to understand the binding ability of drugs and gene targets through molecular docking;later analyzed the effect of β-elemene on the malignant biological behavior of ESCC in the cell and tissue level,and explored its possible anti-tumor mechanism from PI3K/Akt/NF-κB/MMP-9 pathway;finally,by establishing nude mice subcutaneously transplanted tumor models,we observed the therapeutic effects of β-elemene on tumors,to provide clinical treatment for ESCC.The paper contains four parts as below:Part one Effect of β-elemene on proliferation and invasion of esophageal squamous cell carcinomaObjective: To observe the proliferation,invasion,and migration ability of different concentrations,different time period of β-elemene on ESCC,and to explore the effect of β-elemene on the biological behavior of ESCC.Methods:1.ESCC cell lines TE-1 and KYSE-150 were cultured and treated with different concentrations of β-elemene(0,5,10,20,30,40,50μg/ml).Select two minimum inhibitory concentrations that can inhibit cell activity for follow-up experiments.2.CCK-8 was used to detect the cell activity of ESCC cells with the two minimum concentrations of β-elemene for 24 h,48h,and 72 h.3.After implantation of squamous cell carcinoma cells into the Transwell cell,the change of tumor cell invasion capacity was detected with two minimum inhibitory concentrations of β-elemene treatment for 24 hours(concentration of β-elemene concentration as control group).4.Cell scratch assay was used to detect the migration of tumor cells treated with two minimum inhibitory concentrations of β-elemene for 24h(concentration of β-elemene concentration as control group).Results:1.With the increase of β-elemene concentration,the inhibitory ofβ-elemene on tumor cells increased gradually,which was concentration-dependent(P<0.001),and the two lowest concentrations ofβ-elemene(10μg/ml and 20μg/ml)inhibit the activity of ESCC were determined.2.At the lowest inhibition concentration,CCK-8 results showed that the activity of tumor cells decreased gradually with the time prolonging,and the inhibition of β-elemene on cell proliferation increased in a time-dependent manner(P<0.05).3.Transwell test demonstrated that the invasion ability of esophageal squamous cell carcinoma cells was decreased after 24 hours of β-elemene treatment(P<0.001).4.Cell scratch test showed that after 24 hours of β-elemene treatment,compared with the control group,the migration ability of esophageal squamous cell carcinoma cells was reduced(P<0.001).Summary:1.β-elemene inhibites ESCC cells in a concentration-and timedependent manner.2.β-elemene can inhibit the invasion and migration of esophageal squamous carcinoma cells.Part two Mechanism of β-elemene against esophageal squamous cell carcino ma based on bioinformatics and networkpharma cologyObjective: To study the possible mechanism of β-elemene in the treatment of ESCC based on network pharmacology and bioinformatics technology.Methods:1.Related genes of β-elemene are downloaded from the BATMAN-TCM database;Gene expression microarray of ESCC are downloaded from GEO database and Gene Cards database;Venny plot was plotted to make the intersection to obtain the common differentially expressed targets ofβ-elemene in the treatment of ESCC.2.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed on DEGs by R language,as to obtain enriched biological processes(BP),molecular functions(MF),cell composition(CC),and enriched pathways.3.We created a protein interaction network(PPI)for common differentially expressed targets based on the STRING website;the Cyto Hubba plugin in the Cytoscape was used to screen the 5 Hub genes with the highest links.Then Autodock and Py Mol software were applied to perform molecular docking of the top five Hub genes.Results:1.Through the BATMAN-TCM database,16359 genes related toβ-elemene were obtained.GSE17351 was selected as the research object for gene expression chip data in the GEO database,and the screening criteria were:P< 0.05,|log FC| ≥ 2.552 differentially related genes,including 194up-regulated genes and 358 down-regulated genes in ESCC were got.We entered "esophageal squamous cell carcinoma" in the Gene Cards database,and the screening criterion was relevance score≥10.1638 genes related to ESCC were obtained.Finally,71 common differentially expressed targets forβ? elemene against ESCC were mined by the Venny intersection.2.Using R language,we performed enrichment analysis on 71 common differentially expressed targets.GO enrichment analysis indicated that biological processes(BP)were enriched in epidermal development,extracellular matrix decomposition and collagen catabolism,etc;Molecular functions(MF)were enriched in endopeptidase activity,metallopeptidase activity,etc;Cell compositions(CC)were concentrated in extracellular matrix containing collagen,outside plasma membrane,etc.KEGG was concentrated in Phosphatidylinositol triple kinase-protein kinase B(PI3K-Akt)signaling pathway,focal adhesion,extracellular matrix-receptor interaction,transcription disorder in cancer,and so on.3.71 common differentially expressed targets were imported into the STRING database to build a PPI interaction network.Subsequently,they were introduced into Cytoscape for visual analysis.Through Cyto Hubba operation,according to the degree value,the top 5 Hub genes with the highest degree of linkage were obtained,mainly including MMP-9,CHEK1,COL1A1,SPP1,and MMP-3.4.The top 5 Hub genes with the highest degree value were selected for molecular docking by Autodock software,the results showed that the binding energy of β-elemene and MMP-9 was the highest.Py Mol software was Applied for visualization.Summary:1.Both β-elemene and ESCC have a large number of related targets.β-elemene exerts its anti-tumor effect through multi-pathway and multi-target mechanism.2.PI3K/AKT/NF-Κ B/MMP-9 pathway may be one of the antitumor pathways of β-elemene.Part three β-elemene inhibits invasion and migration of ESCC via PI3K/Akt/NF-κB/MMP-9 pathwayObjective: To investigate the possible mechanism of β-elemene inhibiting the biological behavior of ESCC by the PI3K/Akt/NF-κB/MMP-9pathway.Methods:1.A total of 82 patients with esophageal squamous cell carcinoma admitted to Cangzhou Central Hospital were selected from January 2019 to December 2019.The expression of Akt,NF-κB,and MMP-9 in esophageal carcinoma and normal tissues was detected by immunohistochemical staining.The correlation between the three indicators and clinicopathological features was analyzed.2.The cells TE-1 and KYSE-150 were both divided into control group,10μg/ml β-elemene group,10μg/ml β-elemene+SC79 group and 10μg/mlβ-elemene+PDTC group.3.CCK-8 array was used to detect the cell activity at 24 h,48h,and 72 h of treatment respectively.4.The effects of SC79 and PDTC on the migration and invasion of tumor cells after 48 hours were examined by cell scratch test and Transwell cell assay,respectively.5.Western blot was applied to detect the cell cycle and apoptosis-related protein,Akt,p-Akt,p-IκB,NF-κB p65,and MMP-9 after 48 hours.The effects of SC79 and PDTC on cell cycle and apoptosis level were examined by Flow cytometry.6.The expression levels of Akt1 m RNA,NF-κB m RNA,and MMP-9m RNA were detected by RT-PCR after 72 h,respectively.Results:1.Akt and MMP-9 were mainly expressed as brown-yellow granules in the cytoplasm,NF-κB was mainly expressed as brown-yellow granules in the nucleus.In ESCC,the positive rates of Akt,NF-κB,and MMP-9 were 82.93%,74.39%,and 84.15%.The positive expression rates in normal tissues were9.76%,7.32%,and 18.29%,respectively(P<0.01).The three indicators were not associated with gender and age(60 years)(P>0.05),but with TNM stage,pathological differentiation,and lymph node metastasis(P<0.05).2.CCK-8 experiment results showed that the proliferation activity of cells was further inhibited after adding PDTC(P<0.05).Nevertheless,the cell activity was gradually enhanced with the addition of SC79 in a time-dependent manner(P<0.05).3.Cell scratch experiment and Transwell assay showed that β-elemene combined with SC79 enhanced cell migration and invasion ability compared with β-elemene(P<0.05),while combined with PDTC reduced cell migration and invasion ability compared with β-elemene(P<0.01).4.Western blot analysis showed that p-Akt,p-IκB,NF-κB p65,MMP-9,and cell cycle-related proteins were decreased after 48 h treatment withβ-elemene(P<0.001).Apoptosis-related proteins were increased(P<0.05),but Akt had no significant change(P>0.05).Combined with PDTC can further reduce the levels of NF-κB p65 and MMP-9 protein,while combined with SC79 can partially reverse the inhibition and partially restore the levels of p-IκB,NF-κB p65,and MMP-9 protein(P<0.05).5.Flow cytometry showed that G0/G1 phase cells were increased significantly in β-elemene group or combined with PDTC(P<0.01),but S and G2/M phase cells were decreased(P<0.05).Nevertheless,after the SC79 combination,the apoptosis of tumor cells was decreased,G0/G1 stage cells were increased(P<0.05),S stage and G2/M stage cells were increased(P<0.05).The apoptosis rate was increased in β-elemene group or combined with PDTC(P<0.0001).The combination of SC79 could partially reverse apoptosis(P<0.001).6.RT-PCR showed that NF-κB m RNA and MMP-9 m RNA were increased after treatment of β-elemene combined with SC79 for 72h(P<0.001),However,after being treated with β-elemene and PDTC for 72 h,NF-κB m RNA and MMP-9 m RNA were decreased compared with β-elemene(P<0.001).Akt1 m RNA was not significantly changed(P>0.05).Summary:1.β-elemene can inhibit the proliferation,migration and invasion of ESCC,and promote cell apoptosis,NF-κB inhibitor can further inhibit the cell activity;while AKT activists can partly reverse the cell activity.2.β-elemene can reduce p-AKT,p-IκB,NF-κB p65,MMP-9 protein levels and cycle protein levels,increase the level of apoptosis;NF-κB inhibitors can further enhance this role,and AKT activator can partially reversed this effect.3.β-elemene can decrease the level of NF-κB m RNA and MMP-9m RNA,while NF-κB inhibitor can further decrease it;AKT activator could partially reverse the level of NF-ΚB m RNA and MMP-9 m RNA.Part four The effect of β-elemene on the tumorigenesis of esophageal cancer cells in vivoObjective: To detect the inhibitory effect of β-elemene on esophageal cancer cells in nude mice after tumorigenesis for providing an experimental basis for clinical treatment.Methods:1.Twenty-four nude mice were randomly divided into four groups with six mice in each group.They are: TE-1 cell control,TE-1 cell + β-elemene,KYSE-150 cell control,KYSE-150 cell + β-elemene.The ESCC cell lines TE-1 and KYSE-150 were cultured in vitro and into the right armpit subcutaneous of the corresponding group of nude mice respectively.It was established the subcutaneous transplantation model of the esophageal squamous cell in nude mice.From the seventh day,each mouse in theβ-elemene group was given the drug by gavage every day.After 17 days of continuous administration,the nude mice were killed and the subcutaneous tumor tissues were isolated.2.HE staining: to observe the morphological changes of tumor cells.3.TUNEL staining: to detect apoptosis in tumor tissue.4.Immunohistochemistry: to detect the expression of Ki-67 and MMP-9in tumor tissue.Western blot: to detect the expression of Akt,p-Akt(ser473),and PD-L1.Results:1.After subcutaneous injection of tumor cell lines,the tumor cells were uniform in size and clear in cytoplasm and nucleus after HE staining.However,after treatment with β-elemene,the cells were unevenly distributed with different sizes,and apoptotic cells and apoptotic bodies appeared.2.After TUNEL staining,apoptotic cells were discovered in the tumor tissue after β-elemene treatment,while no apoptotic cells existed in the control group.With the prolongation of time,apoptotic bodies increased gradually,and the apoptotic rate was higher than the control group(P<0.05).3.Immunohistochemistry showed that the Ki-67 and MMP-9 expression level increased,but both decreased after β-elemene intervention(P<0.0001).4.Western blot showed that,after the β-elemene intervention,the expression of p-Akt and PD-L1 decreased significantly(P<0.0001).However,no significant change was discovered in Akt protein expression(P>0.05).Summary:1.β-elemene can inhibit the proliferation and invasion of subcutaneous transplanted tumor.2.β-elemene can reduce the density of cells,promote cell apoptosis,and induce hyperplasia of fibrous connective tissue in the subcutaneous transplanted tumor.3.β-elemene can reduce the expression level of p-Akt and PD-L1 protein in the subcutaneous transplanted tumor.Conclusion:1.β-elemene plays an anti-tumor effect in ESCC through multiple targets and multiple pathways.It may inhibit ESCC through PI3K/Akt/NF-κB/MMP-9 signaling pathway.Pathway related proteins were associated with the TNM stage,pathological differentiation,and lymph node metastasis.This pathway may be one of the mechanisms of anti-tumor action for β-elemene.2.β-elemene could inhibit the proliferation of ESCC cells in a dose-and time-dependent manner.Simultaneously,β-elemene can accelerate the apoptosis of ESCC cells and delay the cell proliferation cycle.3.After treatment with β-elemene,the migration and invasion ability of ESCC cells are inhibited,and NF-κB inhibitors can further enhance the inhibition.However,the Akt activator can partially reverse the inhibitory ofβ-elemene on ESCC cells.4.β-elemene can significantly inhibit the effect of transplanted tumors in nude mice,which is related to the induction of tumor cell apoptosis. |
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