Research On The Anti-Tumor Effects And Mechanism Of Licoricidin In Gastric Cancer

Background:The development for novel anti-gastric cancer therapeutic agents or molecules with high efficiency and low toxicity is an urgent task as the treatment strategy for gastric cancer is still facing many challenges.Natural drugs and their derivatives,which are widely available and structurally diverse,often inspire and inform the discovery of new drugs and have been an excellent source for the development of new drugs.Many of the anti-tumor drugs currently being developed on their basis have already benefited the majority of clinical patients.Licoricidin is one of the main active components of Glycyrrhiza glabra and has an extensive pharmacological action.In recent years,its markable anti-tumor effects have attracted much attention from scholars,however,whether Licoricidin has anti-gastric cancer activity has not been reported yet.This work is intended to evaluate the anti-gastric cancer effect of Licoricidin using in vitro and in vivo experiments,and to elucidate its mechanism of action by means of proteomics techniques and clinical data analysis,so that we could prepare the foundation for the future development of Licoricidin as a candidate molecule for anti-gastric cancer drugs.Methods:Human gastric cancer cell lines MGC-803 cells and HGC-27 cells were treated with different concentrations of licoricidin.In addition,5-fluorouracil（5-FU）positive control group was set up.The half maximal inhibitory concentration（IC50）of licoricidin on gastric cancer cells was determined by CCK-8 method,and the inhibition rates at different time points were calculated based on the OD values.The cell proliferation was examined by the cell colony formation assay and the Ed U assay.The cell cycle was detected by flow cytometry after PI staining.We performed Annexin V-FITC/PI dual staining of gastric cells via flow cytometry,and Giemsa staining was used to evaluate the morphological changes in apoptosis.Both transwell invasion/migration assays and wound healing assay were used to discover the effect of licoricidin on the motility ability of MGC-803 cells.The expression levels of cell cycle-related proteins（cyclinD1 and CDK4）,apoptosis-related proteins（Bcl-2,Bax,Cyt-C,Caspase-3）and metastasis-related proteins（MMP-2,MMP-9）were detected by Western Blot assay.The anti-cancer activities of licoricidin in vivo were determined based on the xenografted tumor model of MGC-803 gastric cancer cells.Different concentrations of licoricidin were injected intraperitoneally into nude mice once every other day（7 times in total）.Body weight of the nude mice and the growth of their transplanted tumors were recorded every 3 days,and growth curves were plotted.Then we performed the Western Blot assay and the immunohistochemical staining to detect the expression levels of Ki-67 and Bcl-2 and Bax.Hematoxylin-eosin staining（HE）was used to evaluate the toxic response of licoricidin in vivo.Quantitative proteomics research strategy of Tandem Mass Tag（TMT）technology was used to screen the differentially expressed proteins in MGC-803 cells after the treatment with licoricidin.Bioinformatics analysis was conducted to predict the effective anti-gastric cancer targets of licoricidin,and then molecular docking models,Western Blot assay and immunohistochemical staining were preformed to validate targets.We collected the clinical data and tissue specimens from patients with gastric cancer.The different expressions of target proteins between gastric cancer and adjacent tissues were compared by PCR assay and immunohistochemical staining,respectively.Finally,the correlations between target proteins and the clinical characteristics of patients suffered with gastric cancer were analyzed.Results:Licoricidin showed notable inhibitory effects on gastric cancer cell lines.The IC₅₀of HGC-27 cells and MGC-803 cells was 7.558μM and 10.41μM,respectively.Compared with the control group,licoricidin exerted a variety of anti-gastric cancer effects in a concentration-dependent manner:it significantly suppressed the proliferation of gastric cancer cells,inhibited the formation of clonal colonies,and reduced the number of Ed U-positive cells.It also arrested the cell cycle of MGC-803cells at the G₀/G₁ phase,and down-regulated the expression of cyclin D1 and CDK4 proteins.Moreover,licoricidin induced apoptosis of gastric cancer cells,and the typical morphological changes of apoptotic cells were also distinctly observed.Consistently,licoricidin decreased the expression of Bcl-2,and promoted the expression of Bax,Cyt-C and Caspase-3.Besides,licoricidin had the capacity to inhibit the migration and invasion of gastric cancer cells,impeded the healing of wounds,and down-regulated the proteins levels of MMP-2 and MMP-9.Licoricidin inhibited the growth of MGC-803 gastric cancer xenografts in a concentration-dependent manner.Namely,it inhibited the growth of transplanted tumors in nude mice,repressed Ki-67 protein to combat proliferation,and induced apoptosis by correcting the imbalance of Bcl-2 and Bax.No pathological changes were found in the vital organs of nude mice in each group.There were 19 differentially expressed proteins identified by TMT technology,including 2 up-regulated proteins（ADAT2 and PLA2G12A）and 17 down-regulated proteins（CRYL1,PRPSAP1,ACTR3B,DUSP11,LSM5,DNM1,CLIP2,EIF1,DOT1L,GTPBP3,DERL1,TBC1D23,NOS2,ICMT,DPM1,PRAF2,TRAPPC5）.The differential proteins were distributed in cytoplasm（39.13%）,nucleus（26.09%）,mitochondria（13.04%）,plasma membrane（13.04%）,extracellular（4.35%）and cytoskeleton（4.35%）.Isoprenyl carboxyl methyltransferase（ICMT）was one of the most significantly differentially expressed proteins（p<0.001）,whose fold change（FC）was 0.734805226881093.The differential proteins were significantly enriched in domains such as’ICMT family’,the molecular function of’protein C-terminal S-prenylcysteine carboxyl O-methyltransferase activity’changed significantly,the’terpenoid skeleton biosynthesis’signaling pathway was effectively down-regulated.Therefore,we hypothesized that ICMT may be the target protein for the anti-gastric cancer activity of licoricidin.Subsequently,licoricidin was found to bind to the tyrosine（TYR）residue（TYR-131）of ICMT protein by forming a hydrogen bond in a molecular docking model with a docking fraction of-9.165.Furtherly,the expression level of ICMT in gastric cancer cells and transplanted tumor tissues was significantly reduced with increasing concentrations of licoricidin exposure,and its downstream signaling molecules（Ras-GTP,p-Raf and p-ERK）were also effectively suppressed（p<0.05）.Both TIMER and GEPIA databases showed that the expression level of ICMT was significantly higher in gastric cancer tissues compared to normal tissues（p<0.05）.The relative expression of ICMT m RNA in gastric cancer tissues of 60 patients was significantly higher than that in normal tissues adjacent to cancer（p<0.05）.Patients with high ICMT expression often had the following characteristics:pancreatic cancer（63.16%）,mixed type of Lauren’s staging（80%）,poorly differentiated（57.45%）,T3-T4 stages（63.64%）,and mutated p53 common（68.42%）.All these correlations were considered to statistically significant（p<0.05）.Conclusion:In the present study,we firstly demonstrated that licoricidin has a beneficial anti-gastric cancer activity,the mechanism of which may be associated to the down-regulation of ICMT/Ras signaling pathway.It is expected to become as a promising candidate molecule for new anti-gastric cancer drugs in the future.