Abstract: Background and Objective: Stellera chamaejasme L., was found to have anti-tumor effects. This study aimed to investigate the anti-proliferative effects of the ethanol extract of S. chamaejasme L. (EES), on human Hepatocellular Carcinoma (HC) cell line and explore the mechanism of EES inhibit HC cell proliferation. Methodology: In this study 3-(4,5)-dimethylthiazol (-z-y1)-3,5-di-phenyltetrazoliumbromide (MTT) assay was used to assess the viability of tumor cells submitted to EES. Application of luciferase report gene and Western Blotting (WB) were employed to investigate transforming growth factor β (TGF-β), Smad4 and p-Akt expression. Results: The EES significantly decreased hepatoma cell (HepG2, BEL-7402, SMMC-7721) proliferation in a dose-dependent manner with an IC50 37.75, 28.60 and 29.22 μg mL–1 in vitro (p<0.05). The EES could strongly inhibit TGF-β and down-regulated its downstream factors, such as TβR-I, Smad4, p-Akt in protein level. Conclusion: The EES decreased hepatoma cell proliferation via down-regulation Smad4-mediated TGF-β signaling pathway.
INTRODUCTION
The ethanol extract of S. chamaejasme L. (EES), top-quality materials of Stellera chamaejasme was earliest recorded in the oldest herbal medicine book. In this ancient book, it was recorded be pungent, bitter, natured and toxic and could be through lung, liver and spleen meridian, had the infection of removing stasis, alleviating water retention and eliminating phlegm, mainly treated the heavy food stayed on the stomach and complex combination between cold heat water and gas1-3. It was mainly used to treat malignant tumor in liver, lung, stomach, intestines and breast. Liu and Zhu4 showed that this traditional material medica had on inhibiting tumor cell proliferation, inducing apoptosis, regulating cell cycle and anti-tumor metastasis.
Liu et al.5 indicated that S. chamaejasme L., exert anti-tumor activity through a lot of screening experiment in vivo and in vitro using this midica extract. Liu et al.5 ensured the strong inhibiting infection of extractive EES to high expression transforming growth factor β ( TGF-β) according to the result of luciferase reporter gene screening. The TGF-β is important immunosuppressive factor and plays the important role in tumor development and progression. The TGF-β often was considered to cause tumor and plays the key role in malignant evolution process of metastasis1,6,7. The TGF-β in malignant cells from epithelium was often over-express and the anti-proliferative effect of Smads which is on the signaling pathways downstream becomes weaken even lost. The over-expressed TGF-β activates p-Akt to play the role of resistance to apoptosis and further to promote tumor cell proliferation, increase the risk of metastatic potential. Moreover, TGF-β makes tumor cells escape from immune surveillance and promote tumor growth and metastasis through inducing matrix environment around the tumor change, inducing between epithelial cells and transition mesenchyme and promoting angiogenesis and depressing host immune system8-10. At present, it becomes hot spot to prevent tumor using TGF-β inhibitor and a large number of studies have proved that this therapeutic schedule is feasible and superior. This study explored how EES inhibit human hepatocellular carcinoma cell line BEL-7402 by inhibiting TGF-β signaling pathway.
MATERIALS AND METHODS
Cells: HepG2, BEL-7402 and SMMC-7721 were presented by New Drug Screening Center, China Pharmaceutical University.The HepG2 cell lines transfecting TGF-β gene was established by pharmacology of the Yale University School of Medicine. All cells were originated from human hepatocellular carcinoma.
Reagents and instruments: Luciferase kits Cat. #4550 and report gene passive lysis buffer (Cat. # E153A) were used from Promega Biotech Co., Ltd, USA. Primary antibody anti-hSmad4 (lot No. UVH015011) was purchased from R and D Co., Ltd and the dilute concentration in Western blot was 1:1000. Primary antibody Akt and phospho-Akt ser473 was purchased from cell signaling Co., Ltd and the dilute concentration in Western blot were 1:1000. The β-actin mouse monoclonal antibody was purchased from BOSTER Biotech Co., Ltd, Wuhan, China. Secondary antibody HRP-labeled goat anti-mouse IgG (lot No. 82232, 77928) was the product of Beijing Golden Bridge Biotechnology Co., Ltd. (Beijing, China). The microplate reader TECAN Safire-2 was from Tecan Trading Co., Ltd. High Performance Liquid Chromatography Agilent-1100 was from Agilent Technologies Co., Ltd. (USA). Triple and quadrupole tandem mass spectrometer API4000 was from Applied Biosystem Co., Ltd. (USA). Mini-PROTEAN3 electrophoresis device and the gel imaging analyzer were from Bio-Rad Laboratories Inc.
Ethanol extract of S. chamaejasme L. (EES) preparation
Extraction separation: The primary plant of Stellera chamaejasme L., was identified by specialist of Institute of Chinese Materia medica, China Academy of Chinese Medicine Sciences. They were extracted and isolated by researcher Liu An from Institute of Chinese Materia medica, China Academy of Chinese Medicine Sciences. The method of extraction was as followings: Raw medicinal material Stellera chamaejasme L. was extracted using absolute ethyl alcohol. The concentrated solution volatilizing till no alcohol taste was separated by efficiency using water, 30% alcohol, 60% alcohol and 100% alcohol. At last, the sample was obtained after 60% alcohol elution was dried.
Quality control
Chromatographic conditions: The ZORBAX SB-Cl8 analyzed chromatogram column and 30 column temperature were selected. The mobile phase was linear gradient 0.05% v/v formic acid (A) combined with acetonitrile (B): 0.01-2.5 min, 12-20% B (v/v); 2.50-12 min, 20-28% B; 12-18 min, 28-36% B; 20-25 min, 50-55% B; 27-30 min, 80-80% B; 30-32 min, 80-12% B; 32-40 min, 12-12% B. Flow rate was 0.3 mL min–1 and DAD detection wavelength was 190-400 nm.
| Fig. 1: | HPLC-MS fingerprint of efficient composition Zp111 in Stellera chamaejasme L. |
Mass spectrometer conditions: Parameters of ion spray was optimized to negative ion mode, 350°C. Declustering potential (DP) was -125 V and Collision Energy (CE) was -35. Nitrogen with ultra-high purity was used to be ion source gas (GS1 and GS2), curtain gas (CUR) and collision gas (CAD). They flow rate were respectively 55, 50, 35 and high. Enhanced Mass Spectrum (EMS) scanning range was from 115-1000. The analyzed result of HPLC-MS fingerprint is shown in Fig. 1.
Study drug preparation: After accurate weighing and being dissolved by DMSO, PBS was used to be made up into 10×200 μg mL–1 mother liquor, in which the final concentration of DMSO was 0.1%. It was necessary to make this mother liquor dilute to require concentration before it was added into cells and it was advised to use it right after it was ready.
MTT assay was used to assess the inhibitory effect of cells submitted to EES: Human hepatoma cells in exponential growth phase was dispersed with trypsin and seeded at 2×104 mL–1 in a 96-well plate according to Fennen et al.8 and 180 μL per well in a humidified 5% CO2 atmosphere at 37°C overnight before treatment. After 24 h, cells were treated with study sample 200, 100, 50, 25, 12.5 and 6.25 μg mL–1 and continued to be cultured 48 h. At the same time, zero well (only medium, MTT and DMSO) and control well (cells, the same drug solute, medium, MTT and DMSO) were set. Subsequently, 20 μL MTT solution was added and cells were incubated at 37°C for 4 h. The supernatant was removed and the insoluble formazan product was dissolved in 150 μL DMSO and shaking out 5 min in flat shaker. Absorbance value (A value) was measured with a microplate reader at a wavelength of 570 nm. After that, inhibition rate was calculated by the formula "(A value of negative control group-A value of treatment group)/(A value of negative control group-A value of zero control group)5". At last, the half maximal inhibitory concentration (IC50, μg mL–1) value was calculated trough linear regression method by Origin 7.5 software.
Fluorescent reporter gene method was used to select target gene to tumor cells from EES: HepG2 cells, which were stable line transferred target gene (2 ng mL–1 TGF-β) were cultured in 96-well plates with RPMI and 10% FBS in a humidified 5% CO2 atmosphere at 37°C. After 48 h, cells were merged and divided into groups to serve as 0 0.375, 1.25 and 3.75 μg mL–1. Continue incubation of 1 h and then added target gene stimulants (100 ng mL–1 TPA and 25 ng mL–1 TNF-α) to the cells that were treated with drugs. Continue incubation of 4 h was taken and added 20 μL 5×reporter gene passive pyrolysis liquid, then culture plate was kept at -70 overnight. In the next day, all spalling cells were scraped and 30 μL spalling liquids were moved to 96-wells ELISA plate. At last, fluorescence intensity was detected after adding 30 μL luciferase and under 430 nm excitation wavelength, 550 nm emission wavelength by multimode reader of TECAN company.
Use of Western blot to detect Smad4 and p-Akt expression of BEL-7402 cells in vitro before and after taking EES: Human hepatoma cells BEL-7402 in exponential growth phase were dispersed and seeded at 4.0×105 per plate in a 6-well plate and overnight before treatment. The next day, they were treated to three different concentration gradient study drugs: 50, 25 and 12.5 μg mL–1. Among these, control group was not added drugs and in usual culture. After 48 h, protein was extracted according to total protein kits. The quantitative determination was used to detect the protein expression levels and standard curve was draw. The samples were boiled for 5 min at 95°C and then loaded sample, electrophoresis, transferred membrane and blocked per Western blot steps. The β-actin mice antibody and primary antibodies Smad4, Akt, p-Akt were incubated overnight. After that, goat-anti-mouse second antibody was added in suitable concentration. Incubation 1 h in room temperature and gel imaging analyzer was used to analysis in 30 sec, 60 sec, 10 min and 20 min, at the same time images were collected.
Statistical analysis: The was used to analysis and calculate the band Integrated Optical Density (IOD).
RESULTS
IC50 of EES in human hepatoma cells in vitro: The EES exert significantly inhibition effect to HepG2, BEL-7402 and SMMC-7721 and the IC50 value was 37.75, 28.60 and 29.22 μg mL–1 respectively.
Screen result of HepG2 cells gene expression mediated by EES: The result showed that EES had up-regulated effect at a moderate intensity level to NFκB and AP1 factors of HepG2 cells. However, it had not showed up-regulated effect to high concentration AP1 reporter genes and had weak up-regulated effect to high concentration NFκB. It had significant down-regulated effect to high concentration TGF-β (Table 1, p<0.05).
Dose-effect relationship between TGF-β genes of HepG2 from human hepatoma cells in vitro and EES: The fluorescence intensity of HepG2 was weak before stimulating by exogenous TGF-β, it showed the level of TGF-β that cells HepG2 expressed, was low and EES had lesser effect on TGF-β expression. Meanwhile, this fluorescence intensity result was not changed even when the EES concentration increased. When 2 ng mL–1 exogenous TGF-β was put in to stimulate TGF-β signal pathway, fluorescence intensity increased to be 466,000 after 4 h reaction. While different concentration EES were put into cells and then put in exogenous TGF-β, the fluorescence intensity decreased significantly (p<0.05). The over half down-regulation expression of TGF-β was showed when EES concentration was 0.375 μg mL–1 (Table 2, Fig. 2).
Effect of EES on expression of protein Smad4 and protein p-Akt from cells BEL-7402 in vitro: The EES can inhibit expression of protein p-Akt and Smad4 significantly, which were signaling pathways downstream of TGF-β from HepG2 cells in vitro. The expression of protein p-Akt decreased obviously after putting into EES. It decreased by nearly half compared with control group when concentration of EES was 12.5 μg mL–1 and the biggest decrease in p-Akt expression was 25 μg mL–1 EES which was about one-seventh of control group (p<0.05). The decrease of Smad4 after being effected by EES was in an obvious dose-dependent relationship.
| Fig. 2: | Concentration effect curve between EES and HepG2 cells reporter genes |
| 1: After stimulating by TGF-β, 2: Before stimulating by TGF-β | |
| Table 1: | Changes of HepG2 cells reporter genes TGF-β, NFκB and AP1 in vitro before and after taking EES |
Low and high concentration represented for fluorescence intensity before and after adding stimulant when reporter genes detected. *p<0.05, -: Negative reaction, +, ++ and +++: Weakness, moderate degree and strong degree of positive reaction, ↓: Down-regulated, ↑: Up-regulated |
|
| Table 2: | Inhibiting effect of EES on TGF-β from HepG2 cells in vitro |
| EES: Ethanol extract of S. chamaejasme L. | |
| Fig. 3: | Effect on expression of protein Smad4 and protein p-Akt from cells BEL-7402 in vitro before and after adding EES |
| Fig. 4: | Comparison of Smad4 and p-Akt protein content from BEL-7402 cells between before and after adding EES |
When EES was on 12.5 μg mL–1, the decrease of Smad4 was not significant, while EES was on 25 μg mL–1, it was down to one-third of control group and on 50 μg mL–1 its expression was quite low (p<0.05). So the expression of p-Akt and Smad4 all showed a downward trend after being effected by EES. The p-Akt expression showed the obvious changes in inhibiting when EES was on a lower concentration. The relative expressions of p-Akt and Smad4 were all significantly low when EES was 50 μg mL–1 and among this p-Akt expression was lower than Smad4 expression. This EES concentration might be the key effective concentration that effected on regulation of cells survival and death via TGF-β signal pathway (Fig. 3, 4).
DISCUSSION
Fluorescence reporter gene showed that EES has obvious inhibition effect on TGF-β from stimulated HepG2. The EES strongly decreased hepatoma cell proliferation via down-regulation Smad4-mediated TGF-β signaling pathway. The EES may have potential in hepatocarcinoma therapy as a kind of TGF-β inhibitors. The immunity inhibition factor TGF-β played the important role in promoting the tumor development of tumor, especially in leading to tumor invasion and metastasis and further to malignant tumor. The TGF-β from epithelium originated malignant tumor cells often over express and every branch of its downstream signal pathway had mutations will lead to that cells have the strength of self-selective growth. This would induce the development of tumor, after that it could promote the proliferation invasion and metastasis of tumor in tumor progressive stage11-13. The TGF-β signal pathway could inhibit cells proliferation and this function may occur with obstacles because form factor receptor and Smads have variation, down-regulation and inactivation. When malignant tumor cells and endogenous TGF-β over expressed and meanwhile the function of was lost, the other anti-apoptosis pathway of Smads anchorage-independent signal pathway was activated and further to negatively regulate the tumor proliferation14,15.
The TGF-β signaling networks were complex. Smads anchorage-independent signal pathway and Smads dependent signal pathway had relationship of interaction and combination in downstream. They all played critical roles in regulating cellular homeostasis. During the process of tumor formation, the lower expression or functional inactivation of Smad4 could make tumor cells escape from growth inhibition of TGF-β and growth out of control was found. However, these cells still had response sensibility to TGF-β and they did not depend on Smad4 to regulate the expression of endogenous gene16-18.
| Fig. 5: | Interaction of downstream factors from TGF-β signal pathway |
Previous studies19,20 found that TGF-β signal pathway and PI3K-Akt signal pathway had multilevel interaction effect from extracellular ligand to cell membrane receptors, from cytoplasm to cell nucleus (Fig. 5). Among so many tumors, researchers found that TGF-β/Smads signal pathway was blocked and PI3K-Akt signal pathway was activated abnormally. This indicated that TGF-β/Smads and PI3K-Akt signal pathway played crucial role in occurrence and progress of tumor. Many researches had shown that PI3K-Akt could mediate the process of growth inhibition of TGF-β from tumor cells and promote the proliferation and metastasis of tumor cells. Thereby, TGF-β/Smads and PI3K-Akt participated jointly mechanism of regulation in regulation of intercellular environment, balance of organ development, maintenance of tissue steady state and tumorigenesis process.
Fluorescence reporter gene showed efficient composition of S. chamaejasme L., EES has obvious inhibition effect on TGF-β from stimulated HepG2. Approximately 0.375 μg mL–1 EES could down-regulate over half expression of reporter gene TGF-β. The EES could inhibit phosphorylation of Akt and inhibit activation of Akt downstream factors and intranuclear anti-apoptotic gene via down-regulating TGF-β. These blocked the proliferation inhibition effect which was mediated by downstream factors Smad4 of TGF-β signaling pathways. The TGF-β signaling networks were complicated and it had positive and negative regulation pathway to cell proliferation at the same time. The TGF-β signaling pathway that Smads depended from normal cells had effect on inducing apoptosis and leading cell cycle arrest. While in tumor progression period, TGF-β expression abnormally increased was often found in tumor cells. Smads especially Smad2 and Smad4, which were the classical downstream factors of TGF-β, often lead to block of apoptotic pathways because of variation or down-regulation and further to activate normally anti-apoptosis signaling pathways PI3K-Akt so as to show inhibition effect on tumor cell growth.
CONCLUSION
It is concluded that EES strongly decreased the expression levels of p-Akt and Smad4 which were involved in TGF-β signaling pathway, EES could inhibit hepatoma cell proliferation and so it may have potential in hepatocarcinoma therapy as a kind of TGF-β inhibitors.
SIGNIFICANCE STATEMENTS
Fluorescence reporter gene showed that EES has obvious inhibition effect on TGF-β from stimulated HepG2. The EES could strongly decrease the proliferation of hepatoma cells and down-regulate p-Akt expression. It was thought that EES could be TGF-β inhibitor and had potential in hepatocarcinoma therapy.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial assistance of The National Natural Science Foundation of China (Grant No. 81001564), Beijing Nova Program (No. Z141107001814061), Beijing Municipality Health Technology High level Talent (2014-3-046), Research Project of Beijing Integrative Medicine Institute of Cancer and Construction Project of Beijing Integrative Medicine Treatment Center of Cancer. The authors would like to thank the scientific editor at American Journal Experts for providing professional English language for editing of this paper.