Role Of Scorpion In Epithelial-Mesenchymal Transition And Metastasis Of Hepatocellular Carcinoma

IntroductionHepatocellular carcinoma(HCC)is the third leading cause of cancer deaths in our country.The unfavorable clinical outcome and poor prognosis are due to high rates of recurrence and metastasis after treatments.Therefore,the key point of effective therapy for liver cancer is the prevention of HCC metastasis.Some scholars of traditional Chinese medicine suggested that endogenous wind-evil had played an important role in metastasis of malignant tumor.In this study we investigated the Scorpion water extraction,the major method of administration in clinics of Chinese Medicine,to determine its influence on HCC cells in vitro and in vivo,and to explore the potential associated mechanisms.Our study focused on the role of Scorpion in EMT and metastasis of HCC.Experiment 1 Role of Scorpion in Epithelial-Mesenchymal Transition and Metastasis of Hepatocellular Carcinoma in vivo[Aims]To investigate the role of Scorpion in EMT and metastasis of HCC in vivo.[Methods]The male nude mice were randomly divided into three groups:control,Scorpion and normal.Luciferase-expressing HCC(Hepa1-6-luc~+)cells were used to establish liver cancer metastatic models in nude mice via tail intravenous injection.And one week later,each test group was treated with different way:tumor bearing with PBS treated(control),tumor bearing with Scorpion water extraction treated(Scorpion),non-tumor bearing with PBS treated(normal).Control group and Normal group received PBS 0.2ml/day by intragastric administration;the Scorpion group was treated with the same volume of Scorpion solution(containing Scorpion 0.091g/20g·d)by intragastric administration.The general condition of mice was observed every two or three days and bioluminescent imaging was conducted by using the IVIS Lumina II System(Caliper Life Sciences,Boston,MA)twice a week.The metastatic tumor burden was estimated by photons.All mice were sacrificed,and their lung and liver tissues were collected for histological analysis by hematoxylin and eosin(H&E)staining.Then we examined the expression change of epithelial marker E-cadherin and mesenchymal marker N-cadherin with immunohistochemical staining and Western Blot.[Results]There was no significant difference in body weights between the mice in each group and no mice died during the whole experimental stage.Bioluminescent imaging results showed that the mice which were treated with Scorpion decoction had fewer metastatic tumors(P<0.05).There was no significant difference in body weights between the mice in each group during the whole experimental stage.H&E staining showed that both the metastasis number and relative area of lung decreased significantly in Scorpion group than that of control group(P<0.05).The area of positive staining for E-cadherin of lung tissue samples in Scorpion group was mainly identified in the tumor cells membrane and adjacent benign tissues which was increased significantly than control group.And compared with the control,area of staining for N-cadherin decreased significantly in Scorpion-treated group.The Western Blot statistics showed that there was an increased expression of E-cadherin in the samples from Scorpion group(P<0.001);meanwhile the expression of N-cadherin was all declined with Scorpion-treated group(P<0.01).[Conclusions]Scorpion restrained the growth and metastases of Hepa1-6 cells in vivo and inhibited EMT process,which is characterized by increased epithelial marker E-cadherin expression and decreased mesenchymal markers N-cadherin,in the mice tumor metastasis models.Meanwhile Scorpion may have no toxic effect on normal cells.Experiment 2 The influence of Scorpion on HCC cells in vitro[Aims]To investigate the influence of Scorpion on HCC cells in vitro.[Methods]Forty male Sprague-Dawley(SD)rats were randomly divided into control group and Scorpion group.Twenty male SD rats in Scorpion group were given administration by intragastrical gavage with Scorpion solution at 0.315g/100g·d and twenty rats in control group were given same volume of PBS every day.All rats were maintained with normal diet and blood samples were obtained from heart 1 h after the last administration at the third day.HCC(Hepa1-6)cells were divided into control group and Scorpion group and were respectively cultured by the control serum and serum containing Scorpion.We use EdU assay to evaluate the cellular proliferative activity.The TUNEL staining was used to detect the apoptotic Hepa1-6 cells.The influence of Scorpion-medicated serum on hepatoma carcinoma cell migration and invasion was investigated by wound healing assay and Transwell analysis.[Results]The positive rate of Hepa1-6 EdU staining in Scorpion group was lower than that in control group(P<0.01),and the Positive rate of Hepa1-6TUNEL staining in Scorpion group was higher than that in control group(P<0.001).Compared with the control group,the migration and invasion abilities of the hepatoma carcinoma cells in Scorpion-medicated serum group were also inhibited(P<0.001,P<0.05).[Conclusions]Scorpion inhibited the proliferation of HCC cells and induced apoptosis of the cells,and decreased migration and invasion capacity of Hepa1-6 cells in vitro.Experiment 3 The influence of Scorpion on EMT in vitro and in vivo[Aims]To explore the potential mechanism of the influence of Scorpion on EMT and metastasis of HCC in vitro and in vivo.[Methods]qRT-PCR and Western Blot were used to detect the expression level of E-cadherin,N-cadherin,and Snail which were EMT-related factors in established EMT-induced Hepa1-6 cells which had already been treated with TGF-b1(10ng/mL)in Scorpion-medicated serum or control serum,respectively,for 72 h.Then tested the expression change of Snail in lung tissues from experiment 1 with immunohistochemical staining and Western Blot.[Results]Compared with the blank group,E-cadherin was down-regulated,and N-cadherin was up-regulated in the control group(P<0.01,P<0.001).Compared with the control group,the expression of E-cadherin was increased,and the expression of N-cadherin was decreased in the Scorpion group(P<0.05).And we found that the expression of EMT transcription factor Snail was changedremarkablyafterScorpion-medicatedserumtreatment.The immunohistochemical staining indicated that compared with control group,there was a significant decrease in the staining area of the Snail in Scorpion group.We investigated the protein expression of Snail in lung samples from different groups and the statistics showed that there was an increased expression of Snail in control group(P<0.01);meanwhile a declined expression of Snail in Scorpion group(P<0.01).These data point to as a sign that Scorpion could inhibit EMT process of HCC and then inhibit metastasis of HCC in the mice tumor metastasis models.[Conclusions]Scorpion may inhibit EMT process to reduce metastasis of HCC by suppressing the expression of Snail,the EMT transcription factor.Scorpion could inhibit Hepa1-6 cells’invasion and metastasis in part by reversing EMT both in vitro and in vivo,and providing a possible potential approach for preventing HCC metastasis.