| Liver cancer is a common malignant tumor.In January 2019,the National Cancer Center released the latest cancer statistics,primary liver cancer ranks fourth in the incidence spectrum of malignant tumors in China,the mortality rate is the second in the country.Comparisons with new cases and deaths of malignant tumors worldwide,liver cancer and other gastrointestinal cancers account for half of the world's total in China.With the development of modern medical science and technology,great progress has been made in the treatment of liver cancer.But the clinical symptoms of early liver cancer were not obvious,70% ~ 80% of the patients were in a progressive stage.At present,surgery,transcatheter arterial chemoembolization,radiotherapy and combined therapy are recommended by Chinese experts.Therefore if it can inhibit the metastasis and invasion of liver cancer cells,it will significantly improve the treatment effect and prognosis of patients.All-trans retinoic acid(ATRA)has been used in the treatment of acute promyelocytic leukemia by Chinese scientists in the 1980 s,and has achieved good results.Its anti-cancer effect was praised as one of the three major discoveries of international anti-cancer drugs in the 1990 s.At that time,it attracted much attention from experts in China and western country.When used in the treatment of liver cancer,it was found that the concentration was 10 times higher than of leukemia to produce curative effect.Such a high drug concentration is not suitable for clinical application,because retinoic acid syndrome will accompany it.Vitamin A analogues of concern are synthetic retinoic acid N-(4-hydroxyphenyl)vitamin formamide(fenretinide)for chemical prevention of breast cancer.It is a derivative of ATRA,which is manually added with an amide group on the basis of ATRA.It is a drug with less side effects.Early studies have shown that it can prevent and treat tumors and promote apoptosis of tumors.Recent studies have shown that it can prevent angiogenesis and inhibit metastasis of tumors.This study focused on the effect of fenretinide on the proliferation and migration of hepatocellular carcinoma cells.Tumor metastasis is closely related to its increased mobility.Increased expression and activation of myosin light chain kinase(MLCK)is an important factor in cell movement.Does fenretinide inhibit the migration of cancer cells by reducing cell motility? It's not clear yet.Fenretinide inhibits migration in hepatocellular carcinoma cells and its related mechanisms are rarely reported until now.The aim of this study was to investigate the effects of fenretinide on the biological behavior of liver cancer Hep G2 and SMMC-7721 cells.Whether it can reduce the migration of liver cancer cells and whether this effect is related to the expression and activity of MLCK or not.Its validity and related mechanism were further demonstrated by in vivo animal experiments.It lays a theoretical foundation for fenretinide to be better used in the clinical treatment of liver cancer.Aim To investigate the effect of fenretinide on Hep G2,SMMC-7721 cell proliferation,migration and colony formation in vitro,the effect of fenretinide on tumorigenesis of hepatocellular carcinoma cell line SMMC-7721 in vivo,and study on the preliminary mechanisims.Methods MTS cell viability assay,plate colony assay were used to detect cell proliferation,plate colony assay were used to detect cell cloning ability,wound healing assay were performed to detect cell migration,Western blot analysis was applied to investigate the inner molecular mechanisms,and detection of the expression of MLCK,p38-MAPK signaling pathway protein and migration-related protein,etc.Fenretinide,ATRA,SB203580 and ML-7 were added into cells respectively and in combination,cell migration was observed by wound healing assay,Western blot analysis was applied to see the related molecular mechanisms.Using the subcutaneous transplantation model of SMMC-7721 cells in nude mice,the volume of tumors and weight of mice were monitored regularly after treatment,and the inhibitory effect on tumorigenesis was observed.The effects of HE staining on the pathological morphology of transplanted tumors,Masson staining on the fibers distribution of transplanted tumors,immunohistochemistry and western blotting on the MLCK and phosphorylation of p38-MAPK,MLC were observed.Results Fenretinide inhibited HepG2,SMMC-7721 proliferation.In the concentration range of 1 ~ 25 u M,the growth inhibition rate of Hep G2 cells was from 1.32% to95.13%,SMMC-7721 cells was from 0.27% to 78.36%.The dose-effect relationship of fenretinide was obvious.The inhibition time-effect relationship was also obvious.The inhibition rate of Hep G2 cells treated with fenretinide 15 u M was close to 50% at 48 h and 90% at 72 h,SMMC-7721 cells was close to 30% at 48 h and 85% at 72 h.After treated with fenretinide,the migration and colony formation of the cells were inhibited significantly in the two cell lines.The inhibitory effect of ATRA on cell proliferation,plate cloning and migration was weaker than that of fenretinide at the same concentration.The protein expression of MLCK and MLC phosphorylation were inhibited by fenretinide in the Hep G2 cells,and the MLC phosphorylation was also reduce in the SMMC-7721 cells.At the same time,the expression of migration-related proteins E-cadherin and F-actin was changed.In the signaling pathway,fenretinide can activate the p38-MAPK pathway.Added SB203580 reversed the inhibitory effect of drugs on migration,and increased phosphorylation of MLC.The expression of migration-related proteins E-cadherin and F-actin was reversed by SB203580 compared with fenretinide treatment.To further observe whether fenretinide can reduce cell migration by reducing the activity of MLCK,using MLCK inhibitor ML-7,the migration ability of cells was significantly inhibited,and the migration ability of cells was decreased more significantly when ML-7 combined with fenretinide.Protein level detection showed that the phosphorylation of MLC decreased,phosphorylation of p38 increased,migration-related protein E-cadherin expression increased,and F-actin expression decreased,which was similar to the effect of fenretinide alone.Fenretinide significantly inhibited SMMC-7721 cell tumorigenesis in nude mice xenograft model in vivo.The inhibiting ability increased with the prolongation of drug use,and the weight of mice did not decrease significantly during drug using.Microscopically,the nuclei were arranged regularly,necrosis was rare,the distribution of fibrous tissue increased,the phosphorylation of MLC decreased,and the phosphorylation of p38-MAPK increased.Conclusions Fenretinide could inhibit Hep G2,SMMC-7721 proliferation,migration and colony formation.Fenretinide activates p38-MAPK signaling pathway,alters the expression of migration-related proteins E-cadherin and F-actin,down-regulates the expression and activity of MLCK,reduces cell mobility and inhibits cell migration.Fenretinide can effectively inhibit the transplantation of liver cancer,improve the morphological disorder and increase the distribution of fibers tissue.Its effect may be related to activate p38-MAPK pathway and reduce the phosphorylation of MLC. |
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