Morusin Inhibits Cell Proliferation And Tumor Growth By Down-regulating C-Myc In Human Gastric Cancer

Despite the fact that the incidence of gastric cancer has been reduced over past decades,it remains one of the most common cancers in the world.Gastric cancer is currently the third leading cause of cancer-related deaths in developing countries,and it accounts for nearly one hundred thousand new cases each year.Although tremendous advances have been made in therapies to treat gastric cancer,including surgery,radiation and chemotherapy,overall clinical outcomes in affected patients remain poor,with a 5-year survival rate lower than 30%.Therefore,identifying efficient crude drugs and developing of chemo-preventive agents that can serve as alternative strategic options are crucial for improving outcomes in gastric cancer.Flavonoids,including flavone,flavanone,isoflavone,isoflavanone,dihydrochalcone,have been shown in previous studies to have some anti-tumor activity.For example,baicalein is derived from a flavonoid that possesses diverse biological properties and has been reported to induce apoptosis in bladder cancer and inhibit proliferation in non-small cell lung cancer both in vitro and in vivo.Chalcone,a flavonoid precursor,suppresses gastric cancer cell growth by inactivating NF-κB and induces apoptosis in human colon cancer and hepatocellular carcinoma cells by up-regulating DR5 and DR4.Morusin,a prenylated flavonoid that was isolated from the root bark of Morus australis(Moraceae),has been reported to exhibit anti-tumor biological activity in some types of human cancers.Notably,in human colorectal cancer and cervical cancer,morusin induced apoptosis and suppressd the activity of NF-κB and induced cell death by inactivating STAT3 signaling in prostate cancer cells.We hypothesized that morusin might inhibit cell proliferation and tumor growth in human gastric cancer.MTT assays,western blot assays and cell cycle assays demonstrated that morusin could inhibit cell proliferation and tumor growth in human gastric cancer.The main results are as follows: 1.Morusin inhibits gastric cancer cells proliferation through arresting cell cycleGastric cancer cell lines(MKN45 and SGC7901)were treated with different concentrations morusin for 72 h,the results of proliferation rate assays indicated that morusin efficiently inhibited on gastric cancer cell proliferation,MTT assays and BrdU assays demonstrate that gastric cancer cell proliferation was significantly inhibited by morusin.To gain insight into the mechanisms underlying the ability of morusin to inhibit gastric cancer cells,we performed cell cycle assays in cells treated with morusin,the major result of cell cycle assays was that morusin arrest gastric cancer cells at G1 phase.To determine the molecular mechanism underlying morusin-mediated cell cycle arrest,we performed western blot assays and found that the expression levels of CDK2,CDK4,Cyclin D1 and Cyclin E1 were decreased in morusin-treated cells.These results suggest that morusin induced cell cycle arrest at G1 phase and inhibited gastric cancer cell proliferation by inhibiting the expression CDKs and Cyclins.2.Morusin suppresses tumor growth in vitro and in vivoTo investigate the effect of morusin on tumor growth in gastric cancer cells in vitro,the result of soft agar assays indicated that fewer and smaller colonies were produced in the morusin-treated group than in the DMSO-treated group,morusin inhibits tumor growth in vitro.In vivo,we performed xenograft tumor assays and found that tumor growth was significantly inhibited by treatment with morusin.IHC staining demonstrated that the present of Ki67-positive cells was decreased in the morusin-treated group.Additionally,we detected the levels of cell cycle-related proteins in xenograft tumors and found that the expression levels of CDK2,CDK4 and CyclinE1 were lower in the morusin-treated group.These data show that the morusin-mediated inhibition of tumor growth resulted from an increase cell cycle arrest in vitro and in vivo.3.Morusin inhibits cell cycle related proteins expression through decreasing c-MycexpressionThe result of western blot assays indicated that when gastric cancer cells were treated with morusin,c-Myc was gradually down-regulated in a concentration-or time dependent manner,and the xenograft tumor data supported similar conclusion.Next,we sought to determine whether overexpressing of c-Myc would rescue the morusin-induced inhibition of cell growth,we successfully induced the overexpression of c-Myc in MKN45 and SGC7901 gastric cancer cells.Cell growth was markedly higher in c-Myc overexpressing cells that were treated with morusin,indicating that c-Myc rescued the morusin-induced inhibition of cell growth.When c-Myc was overexpressed,the expression levels of CDK2,CDK4,CyclinD1 and CyclinE1 were increased to varying degrees.Because c-Myc regulates gene expression by binding to “E-box” motifs in target gene promoters,to confirm whether c-Myc up-regulates the expression of CDKs and Cyclins by binding to their promoters,chromatin immunoprecipitation(ChIP)assays were performed using gastric cancer MKN45 and SGC7901 cells.The results of ChIP assays indicated that c-Myc significantly bind to the CDK4,CyclinD1 and CyclinE1 gene promoter regions that contained E-boxes.Unfortunately,despite the fact that the CDK2 gene promoter contains an E-box,we did not observe that c-Myc binds directly to the CDK2 gene promoter.In general,our data indicate that morusin inhibits cell cycle related proteins expression through decreasing c-Myc expression.4.Morusin arrests cell cycle and inhibits tumor growth by down-regulating c-Myc expressionBased on our previously results,we hypothesized that morusin might arrest the cell cycle by down-regulating c-Myc expression in gastric cancer cells.To confirm this hypotheses,cell cycle assays were performed in c-Myc overexpressing and control cells that were treated with morusin.Our quantification of the resulting flow cytometry data showed that overexpression c-Myc decreased the number of cells in G1 phase even in cells that were treated with morusin.Western blot assays indicated that,the expression levels of CDKs and Cyclins were higher in morusin-treated c-Myc overexpressing cells than in morusin-treated controls.The data of cell cycle assays demonstrate that c-Myc overexpression rescued morusin-induced cell cycle arrest in gastric cancer cells.Based we found morusin inhibits c-Myc expression and because c-Myc is an oncogene that has been shown to play an important role in tumor genesis and tumor growth,we next examined tumor growth ability in vitro in morusin-treated c-Myc-overexpressing cells,more clones were produced in the c-Myc-overexpressing group than in the controls.Xenograft tumors result indicated that c-Myc overexpression rescued morusin-induced tumor growth inhibition in gastric cancer cells.These data suggest that morusin arrests cell cycle and inhibits tumor growth by down-regulating c-Myc expression.In summary,the results of cell proliferation rate assays,MTT assays and BrdU staining indicated that morusin significantly inhibits cell proliferation.We found that morusin arrests cell cycle and inhibits cell proliferation by down-regulating cell cycle-related proteins expression,at the same time,morusin could inhibit tumor growth in vitro and in vivo.In addition,we demonstrate that morusin arrests cell cycle and inhibits tumor growth by down-regulating c-Myc expression.Because c-Myc is overexpressed in many gastric cancer patients,and this always relate with their poor survival.This study found that morusin significantly inhibit c-Myc expression and indicated that morusin might be a therapeutic option or alternative strategic option for gastric cancer patients.