Jianpi Bushen Recipe affects epithelial-mesenchymal transition in hepatocellular carcinoma through TGF-β and Twist

Hong-Jie Liu,You-Ying Lai,Zhi-Fen Han,Jian-Jie Chen,Xin-Wen Ma*

1Department of Oncology,Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China.

2Department of Hepatology,Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China.

Abstract Backgroud:To explore the effects of Jianpi Bushen Recipe on the epithelial-mesenchymal transition (EMT) of nude mice with transplanted human liver cancer SK-HEP-1 cells.Methods:To establish models with transplanted liver cancer cells,nude mice were divided randomly into three groups:blank group,traditional Chinese medicine(TCM) group and control group.They were given normal saline,Jianpi Bushen Recipe and solafeni through administration by gavage for four weeks.The expression of TGF-β and twist mRNA were detected by real-time PCR.The expression of E-cadherin was detected by Western blot.Results:Compared with the blank group,the expression of transforming growth factor-β (TGF-β) mRNA and twist mRNA in the TCM group and the control group decreased significantly (P <0.05).There was no significant difference between the two groups (P >0.05).The expression of E-cadherin protein in TCM group and control group increased significantly (P <0.01).There was no significant difference between the two groups (P >0.05).Conclusion:Jianpi Bushen Recipe can inhibit the EMT of liver cancer SK-HEP-1 cells in vivo by down-regulating the expression of TGF-β/twist signaling and indirectly up-regulating E-cadherin expression in transplanted liver cancer tissue,indicating the potential of Jianpi Bushen Recipe to inhibit invasion and metastasis of liver cancer.

Keywords:Jianpi Bushen Recipe,Liver cancer,Epithelial-mesenchymal transition,TGF-β,Twist,E-cadherin

Background

China shows the highest incidence of primary liver cancer in the world [1].Liver cancer develops rapidly and shows a high mortality rate and short survival duration,which are mainly attributable to its high invasiveness and metastatic potential.Studies have shown that epithelial-mesenchymal transition (EMT)is a critical step in the invasion and metastasis of liver cancer [2].Further research on regulatory mechanisms of EMT to block the local spread and distant metastasis of the primary tumor is imperative for the prevention and comprehensive treatment of liver cancer.According to traditional Chinese medicine (TCM),tumor recurrence and metastasis result from insufficient positive qi and presence of uneliminated toxins.Spleen and kidney deficiency syndromes are often concomitant with liver cancer [3].Accordingly,clinical treatments method including invigorating the spleen and nourishing the kidneys and detoxifying and dissipating mass can ameliorate symptoms,delay recurrence and metastasis,improve patients' quality of life,and prolong overall survival [4,5].The present study used a nude mouse xenograft model of hepatocellular carcinoma to investigate the effects of Jianpi Bushen Recipe on EMT of liver cancer and to explore the potential anti-tumor mechanisms.

Materials and methods

Experimental materials

Animals and cells.The animals were 30 male BALB/c nude mice (age,5 weeks;mean body weight,20 ± 2 g).The mice were purchased from Shanghai SIPPR-BK Laboratory Animal Research Center[animal production permit number,SCXK (Shanghai)2008-0016].The animals were maintained in a specific pathogen-free (SPF) environment at the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine and were used in accordance with the Animal Ethics and Welfare Committee of Shanghai University of Traditional Chinese Medicine (No.PZSHUTCM121021001).The human hepatocellular adenocarcinoma cell line SK-HEP-1 was provided by the Cell Bank of the Chinese Academy of Sciences.

Drugs and reagents.The Jianpi Bushen Recipe comprisedAstragalus membranaceus(Huangqi),Atractylodes macrocephala(Baishu),Radix Rehmanniae (Dihuang),Fructus Psoraleae (Buguzhi),Romanet Grapevine (Ye Pu Tao Teng),and Appendiculate Cremastra Pseudobulb (Shan Ci Gu).All Chinese herbal medicines were quality-checked through various assessments and were provided by the Traditional Chinese Medicine Pharmacy of Shuguang Hospital.For subsequent use,the herbal medicines were prepared as decoctions and alcoholic extracts.Sorafenib tablets are the product of Bayer Schering Pharma in Germany (CFDA Approval Certificate#H20090846);the tablets were powered for subsequent use.

Equipment.A biohazard laminar flow bench(TSAOHSIN Enterprise Co.,Ltd.);CO2 cell culture incubator (Heraeus Group,Germany);BH2 microscope (Olympus);Chemidox XRS System for chemiluminescence detection (Bio-Rad Laboratories);ELX800 Microplate reader (BioTek Instruments,USA);Precellys 24 tissue homogenizer (Bertin Instruments,France);and Eco Real-Time fluorescence quantitative PCR system (Illumina,USA) were used.

Establishment of the mouse xenograft model

SK-HEP-1 cells were cultured in RPMI-1640 medium(supplemented with 10% fetal bovine serum,100 IU/mL penicillin,and 100 μg/mL streptomycin) in an incubator at 37°C with saturated humidity and 5%CO2.Every 2-3 days,the adherent cells were passaged at 1:3 following digestion with 0.25% trypsin.Concentration of the tumor cell suspension was adjusted to 1 × 107cells/mL,and each mouse was inoculated with 0.1 mL of the suspension in the right axilla.On the third day after inoculation,the axillary tumor nodules were palpable subcutaneously.One week after the inoculation,when the tumor nodules reached the size of a rice grain,the inoculation was considered successful.

Experimental groups and interventions

The mice were divided into three groups according to body weight using the random number table,with 10 animals each in the blank (saline),TCM group(aqueous extract of Jianpi Bushen Recipe at a concentration of 0.68 g/mL),and control (sorafenib suspension at a concentration of 3.5 mg/mL) groups.After successful inoculation,the corresponding interventions were administered by gavage (0.6 mL)for four consecutive weeks.The weight of each mouse was recorded weekly.During the experiments,the animals were provided food and water ad libitum.At the end of the fourth week,the mice were sacrificed by cervical dislocation,and the tumors were removed under aseptic conditions.Skin at the surgical site was sterilized with iodine.Axillary skin at the tumor growth site was grasped with tweezers and cut with surgical scissors to expose the tumor.The tumor was bluntly peeled with surgical scissors.After blot drying with a filter paper,the tumors were placed on ice,transferred to centrifuge tubes,frozen in liquid nitrogen,and stored at -80°C in a freezer.

Test items and methods

Detection of Twist and transforming growth factor-β (TGF-β) mRNA by fluorescence real-time PCR.Total RNA was extracted from 30 mg tumor tissue using a commercial kit,following the manufacturer's instructions.Total RNA (2 μg) was used to constitute the reaction system (final volume,12µL) and reverse transcribed into cDNA.The PCR reaction system (final volume,20 µL) comprised 2 µL cDNA,10 µL SYBR® green,and 5 µM upstream and downstream primers (Table 1) (95°C 30 s).PCR amplification was performed with 40 cycles at 95°C for 30 s,followed by 60°C for 30 s.Relative expression levels of the target genes were analyzed using the ΔΔCT method.

Table 1 The primers of genes

Detection of E-cadherin protein by western blotting.

Tumor tissues masses (100 mg each) were cut into as many pieces as possible and placed in a homogenizer.The tissue was homogenized on ice in 400 µL of RIPA lysis buffer (containing 1% PMSF) and lysed for 30 min.The lysate was centrifuged at 12,000 rpm for 5 min at 4°C.The supernatant was used to determine the protein concentration.A total of 50 µg protein was mixed with the loading buffer and boiled for 5 min to denature the protein.The samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE),and the separated proteins were electrotransferred onto polyvinylidene fluoride(PVDF) membranes.The blotted membranes were blocked with 5% bovine serum albumin,treated with a primary antibody (1:1000,Cell Signaling Technology),and incubated at 4°C overnight while shaking.Next,the corresponding secondary antibody was added,and the membranes were incubated at room temperature for 60 min.The blotted membranes were then treated with an equal volume of a chemiluminescence agent and AB solution and placed into the Chemidox XRS System for chemiluminescence detection.E-cadherin protein levels were analyzed quantitatively.

Statistical analysis

Quantitative data are described as mean ± standard deviation.Analysis of variance was adopted.Differences between two groups were compared using the t-test.All data were analyzed using SPSS 19.0,and aP<0.05 was considered significant

Results

Effects on general conditions of the mice

Following the inoculation of SK-HEP-1 cells,the mice became slow to move and react.One week after inoculation,when subcutaneous axillary tumor nodules reached the size of a rice grain,administration of the interventions was initiated.Following this,activity levels of the mice increased gradually,their reactions improved,and their body weight elevated steadily.

Before treatment,body weights of the mice were comparable among the groups without significant differences (P>0.05).After inoculation of the tumor cells,the mice showed a trend of initial decrease of body weight for a short while,followed by gradual weight gain.At 3 and 5 weeks,body weight of mice in the TCM group had increased significantly compared with that of mice in the blank group (P<0.05).However,there was no significant difference in weights of mice between the TCM group and control group (P>0.05).(Table 2)

Table 2 The change of body weight（g）

Effect on TGF-β mRNA expression

Fluorescence real-time PCR revealed that TGF-β mRNA expression in the tumor tissue was significantly reduced in TCM and control groups compared with that in the blank group (P<0.05).In addition,there was no significant difference in TGF-β mRNA expression between the TCM and control groups (P>0.05).(Table 3)

Effect on Twist mRNA expression

Fluorescence real-time PCR revealed that Twist mRNA expression in the tumor tissue was significantly decreased in TCM and control groups compared with that in the blank group (P<0.05).Besides,there was no significant difference in Twist mRNA expression between the TCM and control groups (P>0.05).(Table 3)

Table 3 Effect on TGF-β and Twist mRNA expression (2-ΔΔCT value)

Effect on E-cadherin expression

Western blotting analysis revealed that E-cadherin protein expression in the tumor tissue was significantly increased in the TCM and control groups compared with that in the blank group (P<0.01).Moreover,there was no significant difference in E-cadherin protein expression between the TCM and control groups (P>0.05).(Table 4)

Table 4 Effect on E-cadherin protein expression

Discussion

During EMT of hepatocellular carcinoma cells,polarity and E-cadherin expression are lost but vimentin and N-cadherin expressions are upregulated.The cells transform to a mesenchymal phenotype and acquire increased migration and invasion capabilities[6].Studies have found that multiple signaling pathways,such as the TGF-β [7],Notch [8],and HIF-1α [9]pathways are involved in the induction of EMT of tumor cells.TGF-β belongs to the structural regulatory protein superfamily.Increased activated TGF-β levels have been observed in cancer cells [10].Studies have shown that TGF-β induces the expression of EMT-related genes,resulting in corneal epithelial dysfunction [11].In animal models of pancreatic cancer,downregulation of TGF-β type III receptor suppressed EMT progression and inhibited pancreatic cancer invasion and metastasis [12].Twist is a transcription factor belonging to the basic helix-loop-helix protein family and is a key regulator of EMT.Twist silencing has been shown to enhance E-cadherin mRNA expression and block cell migration and invasion in lung cancer [13].

In general,the pathogenesis of hepatocellular carcinoma in TCM is considered to be due to essential empty and out solid.Deficiencies are commonly noted in the spleen and kidneys,and excess are commonly in qi stagnation,blood stasis,coagulated phlegm,and damp-heat.Retrospective studies have suggested that the syndrome patterns of liver cancer include liver stagnation,spleen deficiency,damp-stasis accumulation,Yin deficiency of the liver and kidney,qi stagnation,blood stasis,and damp-heat stagnation[3].According to Jinkui Yaolue,“seeing a liver disease,know that it will spread to the spleen,and thus [you]should first reinforce the spleen.”All medical doctors of the past Chinese dynasties have underscored spleen reinforcement in liver diseases.The treatment strategies for spleen and kidney deficiency syndromes in liver cancer include invigorating the spleen and nourishing the kidneys as well as detoxifying and dissipating the tumors.When the water (kidney) and soil (spleen) are treated together,the wood (liver) can be healed.The Jianpi Bushen Recipe used in this study comprisedAstragalus membranaceus,Atractylodes macrocephala,Radix Rehmanniae,Fructus Psoraleae,Romanet Grapevine,and Appendiculate Cremastra Pseudobulb.Astragalus membranaceussupplements qi and strengthens immunity,serving as the monarch drug.Atractylodes macrocephalainvigorates the spleen and removes dampness;Radix Rehmanniae clears heat and nourishes Yin;and Fructus Psoraleae warms the kidney and assists Yang;together,these three herbs serve as the ministers.Romanet Grapevine clears heat and removes toxins,and Appendiculate Cremastra Pseudobulb disperses phlegm and dissipates the tumors;together,these two serve as the assistant and the guide,respectively.Thus,these ingredients act synergistically to achieve the effect of invigorating the spleen and nourishing the kidney as well as detoxifying and dissipating the tumors.Clinically,the use of Jianpi Bushen Recipe to treat spleen and kidney deficiencies and damp-stasis accumulation in patients with liver cancer has achieved good treatment efficacy in terms of improving symptoms and prolonging disease-free survival.Studies have found thatAstragalusmembranaceusinhibits tumor metastasis by increasing E-cadherin protein expression and suppressing MMP-9 expression [14].Appendiculate Cremastra Pseudobulb has been shown to inhibit EMT of cells and suppress the invasion and metastasis of breast cancer by influencing VEGF-A-related angiogenesis [15].

Our research group previously studied and found that Jianpi Bushen recipe suppresses the invasion and metastasis of colorectal cancer [16,17],inhibits the proliferation and migration of liver cancer cells [18],and induces the apoptosis and inhibits EMT of liver cancer cells [19].Resultsof the present study indicated that Jianpi Bushen Recipe could increase the body weight of nude mice and inhibit EMT of SK-HEP-1 cells.The mechanism may be through decreasing TGF-β expression in the tumor xenograft tissue of liver cancer,leading to Twist downregulation and indirect E-cadherin upregulation,thereby inhibiting tumor invasion and metastasis.Possible involvement of other signaling pathways in the EMT inhibition of liver cancer cells by Jianpi Bushen Recipe needs further research.