| Objective: Pancreatic cancer is a lethal malignancy with occult onset,difficult early diagnosis,rapid progress,poor therapeutic response,short median survival time,and poor prognosis.Only 15-20% of the patients are eligible for surgical resection because of late diagnosis,but the strong tendency to metastasize and high recurrence rate limit the effect of surgery.Gemcitabine and other chemotherapy agents can effectively improve the prognosis of patients with pancreatic cancer,the emergence of drug resistance has lowered the efficacy of these treatments.Meanwhile,the toxicity,negative side effects,and associated high costs of chemotherapy remain a challenge.Therefore,it is necessary to develop new anti-pancreatic cancer treatment methods.In recent years,natural products have become a research hotspot due to their novel structure,high anti-tumor activity,multi-target and low toxicity.Previous studies have shown that natural products can regulate the proliferation,autophagy,apoptosis,invasion and metastasis,drug resistance,and the activity of pancreatic stem cells through a variety of pathways.Scoparone is a natural product of phenylpropanoids,which has a wide range of pharmacological properties and has attracted considerable attention due to its anti-tumor activity,and its anti-tumor activity has been confirmed in leukemia,laryngeal cancer,and prostate cancer.However,to date,no study has demonstrated its effect on other tumors or determined its underlaying molecular mechanism.Bioinformatics has developed rapidly over the past decades,especially in tumor research.Through the application of computer and informatics technology,a large number of tumor data are collected,sorted out and analyzed,and databases with different functions are constructed to enrich the means of tumor research.The aim of this study was to explore the potential anti-tumor effect of scoparone in pancreatic cancer and its possible mechanism by combining bioinformatics and tumor basic research methods.Methods: 1.Pub Chem,Swiss Target Prediction,STITCH,Gene Cards,CTD,STRING,Web Gestalt,Cytoscape and other bioinformatics databases and analysis platforms were used to determine the possible signaling pathways and key genes mediated by scoparone in pancreatic cancer.2.Firstly,the cell assays in vitro were carried out to verify the effect of scoparone on pancreatic cancer.The IC50 values of scoparone in pancreatic cancer cell lines were obtained by CCK8,and the effect on the proliferation of pancreatic cancer cells was detected.Wound healing assay was used to detect the effect on the migration of pancreatic cancer cells,Transwell assay was used to detect the effect on the migration and invasion of pancreatic cancer cells,and flow cytometry was used to detect the effect on the cell apoptosis and cell cycle of pancreatic cancer cells;Western blot was used to detect expression difference of the metastasis related protein MMP9,apoptosis related protein Bcl-2,Bax,cleaved caspase-3 and PI3K/Akt signaling pathway related molecules;then the effect of scoparone on the proliferation of pancreatic cancer in vivo was detected by xenograft tumor model in nude mice and immunohistochemistry.The effects of scoparone on the biological behavior of pancreatic cancer cells under different conditions were thoroughly observed.3.Finally,the Akt activator SC79 was used to perform the rescue experiment.CCK8 assay was used to detect the effect on the proliferation of pancreatic cancer cells,wound healing assay was used to detect the effect on the migration of pancreatic cancer cells,Transwell assay was used to detect the effect on the migration and invasion of pancreatic cancer cells,and flow cytometry was used to detect the effect on the cell apoptosis and cell cycle of pancreatic cancer cells;The expression of metastasis related protein MMP9,apoptosis related protein Bcl-2,Bax,cleaved caspase-3 and PI3K/Akt signaling pathway related molecules were detected by western blot to further confirm the role of signaling pathway in the pancreatic cancer.Results: 1.One hundred and thirteen predicted scoparone targets and 4719 pancreatic cancerrelated targets were obtained through the databases.A total of 83 therapeutic pancreatic cancer targets of scoparone were obtained by overlapping disease and drug targets.2.The MAPK signaling pathway,the PI3K/Akt signaling pathway,signaling pathways in cancer,and the TNF signaling pathway may be involved in tumor related signaling pathways.Four pathways involved 34 targets,among which two targets,IKBKB and Akt1,participated in four pathways,whereas nine targets,including FLT4,EGFR,IGF1 R,MET,PDGFRb,ERBB2,MAPK8,MAPK10,and RPS6KA5,were involved in three pathways 3.The protein-protein interaction network was visualized by Cytoscape software.The top five targets were Akt1,SRC,HSP90AA1,MAPK8,EGFR,and PTGS2(EGFR and PTGS were both in the fifth place),which overlapped with targets related to the signaling pathways.Akt1 was the most likely candidate key gene to be influenced by scoparone in pancreatic cancer,followed by MAPK8 and EGFR.4.Akt1 and MAPK8 expression differed significantly between pancreatic cancer and normal pancreatic tissues,but the expression of EGFR was not significantly different.It can be inferred that scoparone may suppress pancreatic cancer by inhibiting the expression of Akt1,followed by that of MAPK8.5.The IC50 values of scoparone in Capan-2 and SW1990 cells were 225.2 ?M and 209.1 ?M,respectively.Compared with the control group,scoparone significantly inhibited the proliferation,migration and invasion of pancreatic cancer cells,induced cell cycle arrest and cell apoptosis in a dose-dependent manner.6.Compared with the control group,the expressions of MMP9 and Bcl-2 were downregulated,and the expressions of Bax and cleaved caspase-3 were up-regulated in 100,200 and 400 ?M scoparone groups.7.q RT-PCR assay confirmed that scoparone inhibited the expression of Akt1,but did not inhibit the expression of MAPK8.Scoparone was most likely to play an anti-tumor role through Akt1.8.Compared with the control group,scoparone treatment did not decrease the total expression of PI3 K and Akt,but significantly inhibited the expression of p-Akt.9.In vivo xenograft tumor model in nude mice,scoparone inhibited the growth of xenograft tumor,significantly reduced the volume and weight of xenograft tumor,and decreased the expression of proliferation related proteins Ki65 and PCNA.10.SC79 can reverse the inhibitory effect of scoparone on proliferation,migration and invasion of pancreatic cancer cells,and block the induction of cell cycle arrest and apoptosis.11.SC79 can reverse the inhibitory effect of scoparone on metastasis related protein MMP9 and anti-apoptotic protein Bcl-2,and the promoting effect of pro-apoptotic protein Bax and cleaved caspase-3.12.SC79 blocked the inhibitory effect of scoparone on p-Akt.Conclusion: 1.Through network bioinformatics analysis using public databases and platforms,the candidate key genes of scoparone on pancreatic cancer were obtained,and then Akt1 was identified as the key gene by q RT-PCR.2.Scoparone significantly inhibited the proliferation in vitro and in vivo,inhibit migration and invasion,and caused cell cycle arrest and induced apoptosis of pancreatic cancer in vitro;and scoparone down regulated the expression of metastasis related protein MMP9 and anti-apoptotic protein Bcl-2,and up regulated the expression of pro-apoptotic protein Bax and cleaved caspase-3.3.Scoparone may inhibit the proliferation,migration and invasion,and induce cell cycle arrest and apoptosis of pancreatic cancer cells through PI3K/Akt signaling pathway,.4.Akt pathway activator SC79 can block the inhibitory effect of scoparone on pancreatic cancer. |
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