Research On The Effect And Mechanism That Emodin Inhibits Invasion And Induces Apoptosis Of Hepatocellular Carcinoma

Background: Hepatocellular carcinoma(HCC)is the most common type of liver cancer with a high incidence and cancer-related death rate.Unfortunately,most patients are not eligible for curable resection when they were diagnosed as HCC since the symptoms of HCC were concealed.On the other hand,other treatments such as transarterial chemoembolization(TACE)or chemotherapy were far from satisfied.Therefore,exploring effective medicine from traditional Chinese medicine(TCM)has become a hot point among medical scientists.Emodin is an active ingredient derived from root and rhizome of Rheum palmatum L,which has been proved that it may inhibit several kinds of tumors,such as lung cancer,prostatic cancer,brain glioma and breast cancer.The functions of anti-tumor may be related to its antiproliferation and apoptosis-inducing effects,as well as anti-angiogenesis,which may work through multiple signaling pathways.Therefore,the anti-tumor function of emodin was characterized by broad spectrum and multi-target activity.As reported,emodin can both inhibit the proliferation and induce the apoptosis of HCC,but there still lacks potent information of the specific mechanism.Our research group has long been engaged in the study about the anti-liver cancer effects of TCM and its mechanism,so we has the good technical foundation and accumulated experience in this field.In our preliminary experiments we found that emodin in low-dose may suppress the metastasis and invasion while higher doses may obviously induce HCC cells apoptosis.Therefore,in this study we mainly focus on the effects of HCC cells proliferation,apoptosis and metastasis with different concentrations of emodin and the molecular mechanisms underlying these processes.We hope our result may provide some basic support for the clinical application of emodin.Objective: 1.Observe the effect of emodin on migration and invasion of HCC cell line MHCC-97 H in vitro and the molecular mechanism behind them.2.Observe the effect of emodin on the proliferation and apoptosis of HCC cell line SMMC-7721 in vitro and the molecular mechanisms.We also try to verify these effects with a nude mice model.Methods: 1.The influence of emodin with different concentrations on the proliferation of HCC cell lines such as MHCC-97 H and SMMC-7721 was measured with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)and Cell Counting Kit-8(CCK-8).2.Cell apoptosis after stimulation with different dose of emodin was detected by using the Annexin V-FITC apoptosis detection kit.3.Appropriate emodin concentration was selected from the above experimental results.We use Transwell chamber and Matrige invasion chamber to measure the effects of emodin on the migration and invasion of MHCC-97 H cells,which have a high invasion and migration characteristics.4.To explore the specific molecular mechanisms and the related signaling pathways of cells apoptosis and metastasis,Western Blot was used to detect the change of MAPK,AKT signaling pathways and their related protein molecules such as p-ERK1/2,ERK1/2,p-JNK,JNK,p-P38,P38,p-AKT and AKT.We also detected matrix metalloproteinases-2(MMP-2)and MMP-9 since they are closely related to the cell invasion and migration.Apoptosis-related molecules such as cleaved caspase-3,9 and pro-caspase-3,9 were also explore in our study.5.In order to establish a liver cancer model in nude mice,SMMC-7721 cells were inoculated subcutaneously into the right flank of the mice.After treating with emodin of different concentrations,we observe the suppression of tumor growth.The tumor samples were sectioned and either stained with hematoxylin and eosin(H&E)to reveal tumor tissue necrosis or immunohistochemistry(IHC)stained using antibodies against Ki67(1:400)or PCNA(1:500)to detect the tumor cells proliferation.We also detected the apoptosis of the tumor sections by using TUNEL assay.Finally,we analyzed the levels of ALT,AST,AKP,GGT,Cr and BUN to determine the safety of emodin.Results: 1.As MTT assay shown,emodin may evidently inhibit the proliferation of MHCC-97 H cells in a concentration-and time-dependent manner compared with the control.However,when the treatment concentration was less than 50 ?mol/L and the treatment time was shorter than 24 h,the viability of the cells changed very little.2.Flow cytometric analysis shows that emodin may induce apoptosis in MHCC-97 H cells but the change was mild when the emodin concentration was less than 50 ?mol/L.3.As Transwell chamber assay shown,emodin treatment can significantly inhibit the migration and invasion of MHCC-97 H cells in a dose-dependent manner when the emodin concentration was less than 50 ?mol/L.4.After treated with 50 ?mol/L of emodin,the MMP-2,MMP-9,p-ERK1/2 and p-AKT expression levels were significantly decreased in a time-dependent manner whereas the tendency of p-p38 expression levels were inverse.However,the ERK1/2,p38 and AKT expression levels were remained essentially unchanged.Therefore,we inferred that emodin may inhibit MHCC-97 H cells migration and invasion through the inhibition of MMP-2,MMP-9 expression,which may be regulated by the MAPK and AKT signaling pathways.5.Using the CCK-8 and Flow cytometric assays,we found that emodin may also suppress the proliferation and induce the apoptosis of SMMC-7721 cells in a concentration-and time-dependent manner.6.100 ?mol/L emodin may inhibited the expression of p-AKT and promote the expression of p-ERK and p-P38 significantly,but the protein lever of p-JNK was suppressed mildly.Meanwhile,the total AKT,ERK,P38 and JNK remained unchanged.On the other hand,cleaved caspase-3 and cleaved caspase-9 were clearly increased in a time-dependent manner while pro caspase-3,9 were mildly decreased.7.In vivo study,we found that emodin suppressed the tumor growth in a dose-dependent manner,while little influence on the body weight of mice was observe.H&E staining showed that emodin may induced cell death and caused symptoms of necrosis in the tumor masses.In IHC analysis,Ki-67 and PCNA were showed in a significant reduction while TUNEL staining indicated that the apoptotic index was significantly increased.8.By detected the levels of serum ALT,AST,AKP,GGT,Cr and BUN,we found that emodin did not damage the liver and kidney function in mice,which indicated that emodin may be a safe medicine for HCC patients.Conclusion: 1.Emodin can inhibit MHCC-97 H cells' migration and invasion at low doses and its molecular mechanism may relate to the expression of MMP-2,MMP-9 and MAPK,AKT signaling pathways.2.Emodin could effectively induce apoptosis of SMMC-7721 cells at high doses,which may be work through stimulating MAPK and inhibiting AKT signaling pathways.This result was confirmed in vivo studies and the drug observed no significant side effects.