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Akt and MMPs signalling pathway
Biomedicine & Pharmacotherapy 125 (2020) 109893
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Green tea and black tea inhibit proliferation and migration of HepG2 cells via the PI3K/Akt and MMPs signalling pathway
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Lingli Suna, Yuanlong Zhangb, Wenji Zhanga, Xingfei Laia, Qiuhua Lia, Lingyun Zhangb,*, Shili Suna,* a Tea Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of Tea Plant Resources Innovation & Utilization, Guangzhou, 510640, PR China b College of Horticulture, South China Agricultural University, Guangzhou, 510642, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Tea HepG2 Tumour proliferation Tumour migration PI3K/Akt pathway
Background and aims: Black tea and green tea were produced via different processing techniques from the same tea leave variety. Then, biochemical components of the two water extracts were analysed to study cell apoptosis, migration and invasion of HepG2 cells induced by black tea and green tea. Method: The monomer components of the black tea and green tea extracts were analysed by colorimetry and HPLC, with MTT assay and colony formation assays used to assess cell proliferation and viability. The effects of black tea and green tea on apoptosis of HepG2 cells were verified by flow cytometry, with wound healing and Transwell experiments used to detect cell invasion and metastasis. The expression of PI3K/Akt signalling and apoptosis-related proteins as well as epithelial-mesenchymal transition (EMT) regulatory factor in HepG2 cells were determined by western blotting after black tea and green tea treatment. Results and conclusions: Black tea and green tea extracts demonstrated different degrees of inhibition of cell migration and invasion, with green tea inducing more HepG2 cell apoptosis. In addition, green tea and black tea extracts inhibited the growth of HepG2 cells and induced apoptosis via PI3K/Akt, and inhibited cell migration and invasion through the MMPs signalling pathway. This study revealed the effects of fermented (black tea) and non-fermented tea (green tea) on liver cancer cells, providing a basis for the investigation of tea extracts for their anti-tumour potential.
1. Introduction
doxorubicin, as well as some targeted therapeutic drugs such as sorafenib, but the prognosis is often poor and is associated with strong toxicity and side effects [4]. Therefore, in recent years, the development of drugs with anti-tumour effects using natural products as raw materials has become a potential trend. Tea is derived from Camellia sinensis and is the second most popular non-alcoholic beverage in the world [5]. A variety of studies have shown that tea has a variety of health effects, such as, alleviate the metabolic syndrome, anti-tumour ability and enhance immunity [6,7]. The most important teas are black tea and green tea, which due to the different processing techniques, possess very diverse taste quality characteristics and biological active ingredients [8]. Studies have shown that black tea and green tea have the potential to prevent liver
Liver cancer, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), is a common malignant tumour in digestive system, which is one of the leading causes of death in cancer patients [1]. Liver cancer often has no obvious clinical signs in the early stage of the disease, and is likely to progress to the middle and late stages when there are obvious symptoms [2]. As liver cancer patients often have hepatitis B virus (HBV) infection, liver function is frequently severely impaired in advanced liver cancer [3]. The treatment of liver cancer is currently based on surgery, chemical treatment is considered if surgery is not possible. Chemotherapeutic drugs conventionally used for the treatment of liver cancer include cisplatin and
Abbreviations: CG, Catechin gallate; EGCG, Epigallocatechin gallate; C, Catechin; EGC, Epigallocatechin; EC, Epicatechin; GC, Gallocatechin; GCG, Gallocatechin gallate; ECG, Epicatechin gallate; GA, Gallic acid; TP, Tea polyphenol; AA, Amino acid; TFs, Theaflavins; TRs, Thearubigins; TBs, Theabrownings; MTT, 3-45Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B; Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma 2; MMP-2/9, matrix metalloproteinases ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Zhang), [email protected] (S. Sun). https://doi.org/10.1016/j.biopha.2020.109893 Received 24 October 2019; Received in revised form 26 December 2019; Accepted 29 December 2019 0753-3322/ © 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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cancer. Black tea extract protected the liver from carbon tetrachlorideinduced lipid peroxidation, delaying the progression of liver inflammation and liver cancer in mice [9]. Epigallocatechin gallate (EGCG) and theaflavin (TF) in green tea and black tea can down-regulate Wnt/β-catenin and Hh/Gli1 signalling pathways to delay the progression of liver cancer [10]. However, the difference in therapeutic effects between black tea and green tea, and the mechanism of treatment for liver cancer have not been reported. Phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signal transduction pathway has a role in regulating cell proliferation and apoptosis, and is closely related to the occurrence and development of liver cancer [11]. Activation of the PI3K/Akt signalling pathway in liver cancer causes a series of responses, including promotion of cell growth, proliferation, epithelial-mesenchymal transition (EMT) and angiogenesis [12]. During the progression of malignant tumours, EMT appears to change from a highly differentiated epithelial cell phenotype to a fibroblast-like or mesenchymal phenotype, which causes tumour invasion and metastasis [13]. The MMP protein family, including MMP-2, MMP-3 and MMP-9, mediate the EMT process [14]. Black tea and green tea extracts, as potential therapeutic agents for liver cancer, have not previously been shown to affect PI3K/Akt/MMPs-mediated invasion and metastasis in liver cancer. In this study, we processed the fresh leaves of the same variety of tea trees into black tea and green tea via different processing techniques. Using HepG2 cells as a cell model, we studied the efficacy and molecular mechanism of black tea and green tea extracts to inhibit tumour cell growth and cell migration, revealing the novel anti-tumour potential of black tea and green tea in liver cancer.
Table 1 The contents of bioactive components in green tea and black tea. Component
Black tea
Green tea
Tea polyphenols (%) Amino acid (%) Soluble sugar (%) Flavonols (mg/g) TFs (%) TRs (%) TBs (%) Caffeine (mg/g) GA (mg/g) GC (mg/g) EGC (mg/g) C (mg/g) EC (mg/g) EGCG (mg/g) GCG (mg/g) ECG (mg/g) CG (mg/g) Total catechins (mg/g)
12.19 ± 0.25 2.70 ± 0.01 9.68 ± 1.06 27.26 ± 0.10* 0.11 ± 0.00** 1.78 ± 0.20** 5.96 ± 0.18** 95.63 ± 0.10* 13.63 ± 0.14* 7.27 ± 0.60 31.66 ± 0.63 14.98 ± 0.22 7.32 ± 0.24 5.77 ± 0.39 2.24 ± 0.12 5.39 ± 0.03 1.57 ± 0.43 76.20 ± 1.61
21.74 ± 0.80** 2.72 ± 0.01 9.11 ± 0.91 20.19 ± 0.02 / / / 83.09 ± 0.12 6.17 ± 0.08 8.72 ± 0.25* 44.51 ± 0.96** 26.52 ± 0.69** 15.01 ± 0.68** 77.84 ± 0.33** 2.48 ± 0.11 33.08 ± 0.75** 1.03 ± 0.10 209.20 ± 3.26**
Values represent means ± SD (n = 3). * are significantly different at p < 0.05. ** are significantly different at p < 0.01.
for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). HepG2 cells were cultured in DMEM media, supplemented with 10 % FBS, and grown in an incubator (37 °C, 5 % CO2, and saturated humidity). The cells used in the experiments were all recovered within two months from storage in liquid nitrogen.
2. Materials and methods
2.4. Cell viability assay
2.1. Chemicals and reagents
The effects of black tea and green tea on the cell viability of HepG2 cells was examined by MTT assay. In brief, 1 × 104 HepG2 cells were seeded per well in 96-well plates containing 100 μL of complete medium. After 24 h, the cells were adherent, then a specific concentration of black tea and green tea extract was added to the cells for 24 or 48 h. MTT solution (20 μL of 5 mg / mL in phosphate buffered saline, Sigma Aldrich, USA) was added to each well and incubated at 37 °C for 4 h in the dark. The medium was removed, 150 μL DMSO was added and shaken in the dark for 10 min, before the absorbance of the supernatant was measured at 570 nm using a microplate reader (BioRad, USA). The IC50 values were calculated by GraphPad Prism 7.0 Software and each treatment group and control group contained four independent replicates.
Black tea and green tea samples were obtained from the Tea Research Institute, Guangdong Academy of Agricultural Sciences in China. Antibodies specific for a variety of proteins as well as all secondary antibodies were purchased from Cell Signalling Technology (Cell Signalling Technology, USA). LY294002 was purchased from Sigma Aldrich. DMEM media, foetal bovine serum (FBS), and trypsin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 2.2. Preparation and biochemical analysis of black tea and green tea extracts Two different kinds of tea were made from the same species of tea tree via different processing techniques. Briefly, in the manufacture of green tea, the tea leaves were heated to inactivate the enzymes, rolled and dried, whereas for black tea production, the tea leaves were withered, crushed, and allowed to under undergo enzyme-mediated oxidation. The black tea and green tea were powdered, then placed in boiling distilled water for 30 min (tea/water, 1:20 w:v) and continuously extracted three times. The three extracted hot tea solutions were combined and filtered immediately, and concentrated at 60 °C, before freeze drying. The free amino acids, total soluble sugars and polyphenol content of the tea solutions were determined by the ninhydrin method, the anthrone-sulphuric acid colorimetric method and the Folin-phenol method, respectively. The content of caffeine and catechins in the extract was quantified by high performance liquid chromatography (HPLC).
2.5. Colony formation assay HepG2 cells were treated with black tea and green tea extract for 48 h in the logarithmic growth phase and the culture broth was discarded. The treated cells were trypsinised to prepare a single cell suspension and re-inoculated into a six-well plate (1500 cells/well). The cells were cultured for 10 days at 37 °C in 5 % CO2 until large colonies visible to the naked eye appeared. The cells were washed with PBS, fixed (methanol : glacial acetic acid : ddH2O = 1:1:8) for 10 min, then stained with 0.1 % crystal violet for 30 min. The stained cell colonies were photographed and counted. 2.6. Cell migration assay A wound healing assay was performed to detect cell migration. HepG2 cells were seeded in six-well culture plates and after the cells were fully attached, a wound was made by artificially using a sterilised tip to draw a line of equal width on the underside of the dish. The suspended cell debris was washed away with PBS, and FBS-free basal medium was added. The 0 h initial wound photographs were taken
2.3. Cell line and cell culture The human hepatocellular carcinoma cell line HepG2 was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes 2
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Fig. 1. Black tea and green tea inhibit the growth and proliferation of HepG2 cells. (A). Different concentrations (0, 0.2 mg/ml, 0.8 mg/ml) of black tea and green tea extract-treated HepG2 cells were photographed by microscopy. (B). HepG2 cells were subjected to colony formation experiments after treatment with black tea and green tea extracts. (C). MTT assay was used to detect cell viability of HepG2 cells treated with different concentrations of black tea and green tea extract for 24 h or 48 h. (D). IC50 values of black tea and green tea extracts in HepG2 cells. IC50, concentrations that induced 50 % cell growth inhibition. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
Fig. 2. Black tea and green tea promoted the proportion of apoptotic HepG2 cells. Apoptosis was detected by flow cytometry in HepG2 cells treated with various concentrations of black tea (A) and green tea (B) extracts. (C). The proportion of apoptotic HepG2 cells (FITC-annexin V positive cells).
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Fig. 3. Black tea and green tea slow the migration of HepG2 cells. (A). HepG2 cells were treated with black tea and green tea extracts (0.05 and 0.20 mg/ml) and cell migration ability was assessed by the wound healing assay. (B). The ratio of HepG2 cell migration distance was counted. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
Fig. 4. Black tea and green tea inhibited the invasion of HepG2 cells. (A). HepG2 cells were treated with black tea and green tea extracts (0.2 and 0.8 mg/ml) and the cell invasion ability was examined by Transwell assay. (B). The proportion of the invading HepG2 cells was counted. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
Alamos, NM).
under an Olympus inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan) and marked on the 6-well plate lid at the corresponding position. A specific dose of black tea or green tea extract was then added to the treated cells for 24 or 48 h, and photographs of the treated cell wound were obtained. Four fields of view at a fixed position were taken at each time. Finally, Image Tool software (Bechtel Nevada Inc., Los Alamos, NM) to calculate the migration distance of the treated and control cells.
2.8. Flow cytometric analysis Flow cytometry analysis was performed using a BD FACS Accuri C6 plus flow cytometer (BD Biosciences, San Jose, CA, USA) to determine the proportion of apoptotic HepG2 cells. HepG2 cells were digested and resuspended in PBS after treatment with the tea solution, then stained with PI and annexin V solutions, respectively, according to the manufacturer's instructions in the Apoptosis Assay Kit (Beyotime, China). The proportion of apoptotic cells was analysed by flow cytometry using FlowJo (Becton, Dickinson and Company, US) software.
2.7. Transwell cell invasion assay First, a Matrigel-containing Transwell chamber (Corning Glass Works, Corning, NY) was placed in a 24-well plate and incubated at 37 °C. The treated HepG2 cells were digested and resuspended in FBS-free basal medium. A 0.5 ml cell suspension containing 5 × 104 cells was added to the Transwell upper chamber, and 0.5 ml of complete medium was added to the lower chamber and incubated at 37 °C. After 24 h, the medium in the upper and lower chambers was discarded, and the cells on the inside of upper chamber were removed with a cotton swab. The cells on the outside were fixed with cold ethanol for 30 min, then stained with 0.1 % crystal violet for 10 min, the excess stained background washed with running water and allow to dry. Four fields of view were imaged under an inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan) for each treatment group and control group. Invasive cells were counted by Image Tool software (Bechtel Nevada Inc., Los
2.9. Western blot assay The HepG2 cells treated with the tea solution were lysed with RIPA buffer, and the protein content was quantified by the BCA method. A 50 μg protein sample was placed in loading buffer and boiled for 5 min, then electrophoresed by SDS-PAGE and transferred to a PVDF membrane (Millipore, MA, USA). The PVDF membrane was then blocked with 5 % skimmed milk for 2 h at room temperature and incubated with the primary antibody overnight at 4 °C. The primary antibodies used in this study included anti-PI3K, anti-p-PI3K, anti-Akt, anti-p-Akt, antiBcl-2, anti-Bax and anti-β-actin 1:1000; Cell Signalling Technology, USA. The membrane was then washed three times with TBST and 4
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Fig. 5. Black tea and green tea time-dependently reduced PI3K/Akt signalling pathway activity in HepG2 cells. (A). HepG2 cells were treated with 0.2 mg/ml black tea and green tea extracts, and protein expression at various time points in the cells was detected by western blotting. The absolute amounts of p-PI3K (B) and p-Akt (C) were simultaneously quantified. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
incubated with HRP-labelled anti-rabbit or anti-mouse secondary antibody 1:10,000; Cell Signalling Technology, USA for 1 h at room temperature. Specific protein bands were detected using Chemiluminescence ECL Detection Kit Thermo Scientific, USA and Chemi Doc System Bio-Rad, USA. Protein bands were quantified by Image J software (Bethesda, MD, USA). When inhibitors were employed, cells were pretreated for 8 h with inhibitor (LY294002, 20 μM) before the addition of black tea and green tea extract.
significantly higher than in green tea extract, with the polyphenol content of green tea being almost twice that of black tea. Furthermore, the catechins content was almost three-fold higher in green tea, with black tea containing a specific amount of theaflavins (TFs), thearubigins (TRs) and theabrownines (TBs). More importantly, the content of EGCG, which is recognised as the most important active substance in tea, in the green tea extract was 13 times higher than that in black tea extract.
2.10. Statistical analysis
3.2. The antineoplastic effect of black tea and green tea
All data are expressed as mean ± SD of at least three independent experiments. Two-way ANOVA analysis of variance was used for comparison between multiple groups. GraphPad Prism 7.0 statistical software (GraphPad Software Inc., San Diego, CA, USA) was used to analyse the data. P < 0.05 and P < 0.01 were considered to be statistically significant.
To determine whether black tea and green tea could inhibit HepG2 cell proliferation, the morphology and proliferation of HepG2 cells was determined. As shown in Fig. 1A, shrunken cells and plasma membrane blebs were present after treatment with black tea and green tea. Furthermore, the colony formation assay demonstrated that both black tea and green tea enhanced the inhibition of cell proliferation (P < 0.05), which increased with increasing concentration (Fig. 1B). The cytotoxic effects of the tea extracts on human HepG2 cells were evaluated by the MTT assay. After 24 and 48 h of treatment, the proliferation of HepG2 cells was inhibited at the dose of more than 0.25 mg/ml, but the concentrations below 0.25 mg/ml extract could affect the survival of cancer cells. Black tea and green tea extracts appeared to different efficacies of growth inhibition of HepG2 cell, with estimated IC50 values of 1.11 mg/ml and 0.88 mg/ml at 24 h of 0.57 mg/ml and 0.52 mg/ml at 48 h treatment, respectively. More importantly, the inhibitory effect
3. Results 3.1. The composition of black tea and green tea extracts As shown in Table 1 (the HPLC chromatogram in Fig. S1), the content of amino acid and soluble sugar in the extracts of black tea and green tea were not significantly different. The amount of flavanols, caffeine and γ-aminobutyric acid (GA) in the black tea extract were 5
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Fig. 6. Black tea and green tea decreased PI3K/Akt signalling pathway activity in a concentration-dependent manner. (A) HepG2 cells were treated with various concentrations of black tea and green tea extracts for 24 h, and protein expression was detected by western blotting. The absolute amounts of p-PI3K (B) and p-Akt (C) were simultaneously quantified. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
the invasion of HepG2 cells (Fig. 4A). Compared with the control group, the ability of the treated cells in the black and green tea group to penetrate the Matrigel was significantly decreased (P < 0.05), with the number of migrated cells in the black tea group significantly higher than that in the green tea group at the concentration of 0.8 mg / ml (P < 0.05, Fig. 4B). These results indicate that both black tea and green tea could inhibit the migration and invasion of HepG2 cells, and green tea was more effective than the black tea group.
of green tea was stronger than that of black tea, but the difference was not significant (Fig. 1C, D). 3.3. Black tea and green tea promotes HepG2 cell apoptosis Flow cytometric analysis showed that treatment with black tea or green tea for 24 h induced apoptosis of HepG2 cells in a concentrationdependent manner. As shown in Fig. 2, after treatment of HepG2 cells with 1.0 mg/ml of black tea or green tea for 24 h, the proportion of apoptotic cells increased to 38.5 % and 64.0 %, indicating that high concentrations of black and green tea induce apoptosis in HepG2 cells.
3.5. Black tea and green tea enhance PI3K/Akt signalling inhibition The PI3K/Akt signalling pathway regulates tumour cell proliferation and death [16,17], so the western blotting assay was used to assess the effect of black and green tea on the expression of pro-survival proteins associated with the PI3K/Akt pathway in HepG2 cells. Intriguingly, treatment with black and green tea markedly suppressed the levels of phosho-PI3K (p-PI3K) and p-Akt proteins in a time-dependent (Fig. 5A) and concentration-dependent manner (Fig. 6A), whereas it had almost no effect on total PI3K and Akt protein expression. In addition, quantitative analysis of protein levels was undertaken (Fig. 5B, C and Fig. 6B, C), showing that like effects on cell growth inhibition, the regulatory effect of green tea on the PI3K/Akt signalling pathway in HepG2 cells was more significant than that of black tea. These results indicate that the black tea and green tea may partially inactivate PI3K/ Akt signalling, thereby affecting HepG2 cell proliferation.
3.4. Inhibition of cell migration and invasion Tumour metastasis is characterised by invasion and migration of tumour cells [15], so we verified whether black tea and green tea could inhibit tumour metastasis by blocking the invasion and migration of tumour cells. Scratch adhesion assays showed that black tea and green tea treatment inhibited HepG2 cell migration in a time and concentration-dependent manner (Fig. 3A). Quantitatively, the tumour cell migration distance of the tea treatment group was significantly shorter than that of the control group (P < 0.05), with the migration distance of HepG2 cells treated with green tea for 48 h significantly higher than that of the black tea-treated cells (P < 0.05, Fig. 3B). Transwell cell migration assays demonstrated that black tea and green tea inhibited 6
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Fig. 7. The influence of black tea and green tea on the PI3K/Akt pathway in liver cancer cells. (A) Cells were treated LY294002 (20 μM), or black tea and green tea (0.2 mg/mL), or their combination. After 48 h, cell viability was determined by MTT analysis. Cells were treated with black tea or green tea alone or in combination with LY294002. After 48 h, the levels of p-PI3K, PI3K, p-Akt, and Akt proteins were analysed by western blotting and β-actin served as the loading control. (B) Representative blot of expression. (C and D) Quantitative analysis of the proteins in (B) was undertaken. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01.
cytoplasm to promote apoptosis [19]. Next, we verified whether black tea and green tea induced apoptosis in HepG2 cells is dependent on the Bcl-2/Bax signalling pathway. Western blotting data showed that the expression of Bcl-2 in the HepG2 cells decreased gradually with treatment time (Fig. 8A, C) and concentration (Fig. 8B, D) of black and green tea. However, the expression of Bax was not affected by the treatment with black tea and green tea. It is generally believed that there is a balance between Bcl-2 and Bax, which is related to the occurrence of apoptosis [20]. We compared the Bax / Bcl-2 ratios of the black tea group and the green tea group, finding that the green tea treatment group had a higher Bax / Bcl-2 ratio. Thus, treatment with green tea decreased the Bcl-2 / Bax ratio in HepG2 cells, thereby activating a mitochondria-dependent apoptotic pathway.
To confirm the involvement of the PI3K/Akt pathway in the inhibition of tumour cell growth by black tea and green tea, HepG2 cells were pretreated with the PI3K-specific inhibitor, LY294002 (20 μM), for 8 h, followed by co-treatment with black tea or green tea. After 48 h, cell viability was analysed using MTT assay. As shown in Fig. 7A, treatment with LY294002 significantly inhibited the growth of cells, whereas combined treatment with LY294002, followed by black tea and green tea had no significant effect on cell viability. In addition, treatment with LY294002 reduced the phosphorylation levels of PI3K and Akt. These results indicate that treatment with black tea and green tea may partially inactivate PI3K/Akt signalling, thereby affecting HepG2 cell proliferation. 3.6. Green tea decreases the Bcl-2 / Bax ratio to regulate apoptosis of HepG2 cells
3.7. Effects on migration and invasion related protein activities in HepG2 cells
Mitochondrial pathway-dependent apoptosis is mainly regulated by Bcl-2 family proteins, including the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax [18]. Bcl-2 blocks the transfer of cytochrome c from mitochondria to the cytoplasm and inhibits caspase activation, thereby inhibiting apoptosis, while Bax permeabilises the mitochondrial outer membrane and induces cytochrome c release into the
Cancer cell invasion and migration result in the diffuse and invasive growth of cancer cells, which makes these tumours difficult to eradicate using conventional therapeutic methods. MMP-2/9, important members of the MMP family, can promote cell migration through disruption of the extracellular matrix [21]. As markers of cell invasion, N-cadherin 7
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Fig. 8. Black tea and green tea induce Bax/Bcl-2-dependent apoptosis in HepG2 cells. HepG2 cells were treated with time (A) and concentration gradients (B) of black and green tea extracts, and Bax and Bcl-2 protein expression was detected by western blotting. At the same time, the ratio between Bax/Bcl-2 was quantified (C and D). The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
then rolled into a rope. Black tea needs to be fermented and dried during its production, which causes the polyphenols in cells to be oxidised by the action of intracellular oxidase [23]. Polyphenols are considered to be the important functional substances in tea and it is generally believed that the content and composition of polyphenols in the aqueous extracts of green tea and black tea may be the main factor leading to their difference in biological activity of aqueous extracts [24]. Our data revealed that tea polyphenols, especially a variety of catechin monomer, such as EGCG and other substances, were high in green tea, which may cause green tea to down-regulate Bcl-2 to promote apoptosis in HepG2 cells. The occurrence of liver cancer is generally a malignant tumour of HCC or ICC, which is characterised by high malignancy and rapid progress [25]. However, due to the lack of effective drugs, the treatment of liver cancer is still difficult. This study showed that green tea and black tea extracts had a significant inhibitory effect on liver cancer cells in vitro. Green tea and black tea extracts significantly promoted apoptosis of liver cancer cells and inhibited cell invasion and metastasis. The higher the concentration of the extract, the more obvious the inhibition of cell proliferation was dose-dependent. In addition, green
and vimentin were detected in the present study (Fig. 9 and Fig. 10). Our results demonstrated that treatment with either black tea or green tea led to significant downregulation of MMP-2/9, N-cadherin, and vimentin in a time-dependent (Fig. 9A-E) and concentration-dependent manner (Fig. 10A-E). Similar to the above results, the protein expression regulation effect in green tea treatment groups were stronger than that in black tea treatment groups. Taken together, these results support the conclusion that combined treatment with black tea and green tea potentiates the suppression of tumour cell migration and invasion through modulation of MMPs signalling.
4. Discussion The black tea and green tea used in this study were derived from the same varieties of fresh tea leaves, which were processed separately by different processing techniques. For green tea production, the tea leaves are first distributed to allow the fresh leaves to lose water, then the tea leaves are inactivated by high temperature (280–300 °C), before being rolled into strips and dried to make green tea [22]. For black tea production, the tea leaves are placed to allow the fresh leaves to lose water, 8
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Fig. 9. Black tea and green tea time-dependently reduced MMP protein expression and inhibited EMT progression. (A). Black tea and green tea extracts were treated with HegG2 cells in a time gradient, and the expression of EMT-related proteins in the cells was detected by western blotting. (B–E). Quantitative analysis of expression of EMT-related proteins. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
further investigate the molecular mechanism of apoptosis induced by black and green tea in liver cancer cells. Studies have shown that PI3Kmediated Akt phosphorylation activation further directly phosphorylates caspase-3/9, which disrupts the activation of a series of apoptotic factors triggered by caspase-3/9, thus terminating the apoptotic pathway [29,30]. EGCG is the highest and most active polyphenolic compound in green and black tea. Previous studies revealed that EGCG promoted tumour cell apoptosis by altering the PI3K / Akt signalling pathway. In breast cancer, EGCG increased PTEN, caspase 3 and caspase 9 and decreased the expression of PI3K, Akt and hTERT. In this way, EGCG promoted breast cancer cell apoptosis by regulating the
tea had a stronger inhibitory effect on liver cancer cells than black tea, which may be due to high content of important active substances, such as EGCG, in green tea. Previous studies also reported that tea extracts inhibited the growth, proliferation and differentiation of liver cancer cells [26,27]. Green tea polyphenols inhibited the proliferation and growth of HepG2 cells, and polyphenol monomer EGCG induced apoptosis of HepG2 cells by increasing the activity of caspase-3 protein [28]. Therefore, our study revealed the potential of extracts from fermented tea (black tea) and non-fermented tea (green tea) in the treatment of liver cancer. Western blotting of PI3K / Akt signalling proteins was performed to
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Fig. 10. Black tea and green tea concentration-dependently reduced MMP protein expression and inhibited EMT progression. (A). HepG2 cells were treated with a concentration gradient of black tea and green tea extracts, and the expression of EMT-related proteins in the cells was detected by western blotting. (B–E). Quantitative analysis of expression of EMT-related proteins. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
growth of liver cancer cells, which makes these tumours difficult to eradicate using conventional therapeutic methods. Invasion and metastasis of liver cancer is a multi-stage process, of which MMP-2/9mediated destruction of extracellular matrix (ECM) is a key step [35]. It has been shown that EGCG down-regulated mRNA and protein expression of NF-κB and MMP-9 in a dose-dependent manner and inhibits bladder cancer migration [36]. In gastric cancer, green tea polyphenol extracts reduce the invasiveness of gastric cancer cells by reducing inflammatory factors-induced MMP-2/9 activation [37], but the tea extract does not affect all EMT-related proteins in tumour metastasis. For
PI3K / Akt pathway and telomerase activity [31]. In bladder cancer T24 and 5637 cells, EGCG up-regulated PTEN and reduced the expression of phosphorylated PI3K and Akt, which inhibited cell proliferation [32]. In addition, apigenin in tea reduced the phosphorylation of Akt, P70RSK, and S6 in choriocarcinoma cells, thereby promoting tumour cell apoptosis and inhibiting metastasis [33]. Theaflavins in black tea blocked the PI3K / Akt / Bad pathway to trigger apoptosis in medullary thyroid cancer [34]. Our data also confirmed that the tea extract components, including EGCG, promoted apoptosis in HepG2 cells. Cancer cell migration and invasion result in the diffuse and invasive 10
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example, in ovarian cancer, EGCG inhibited tumour migration by down-regulating MMP-2, not MMP-9, and exerts its therapeutic value [38]. In our study, high concentrations of green tea and black tea extracts significantly reduced the expression of MMP-2 and MMP-9 in HepG2 cells, with a weak effect on vimentin. Therefore, we revealed the mechanism by which tea extract regulated MMP-2/9 mediated invasion and metastasis, providing a new basis for liver cancer treatment.
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Ethnopharmacol. 130 (2010) 536–544. [27] G.C. Yen, J.W. Ju, C.H. Wu, Modulation of tea and tea polyphenols on Benzo(a) pyrene-induced DNA damage in chang liver cells, Free Radic. Res. Commun. 38 (2004) 193–200. [28] A.E.N. Rn, S.S. Azab, E. Eldemerdash, S. Shaarawy, M. Elmerzabani, A. Elsm, Sensitization of TRAIL-induced apoptosis in human hepatocellular carcinoma HepG2 cells by phytochemicals, Life Sci. 92 (2013) 555–561. [29] J. Yuan, Y. Deng, Y. Zhang, X. Gan, S. Gao, H. Hu, S. Hu, J. Hu, H. Liu, L. Li, J. Wang, Bmp4 inhibits goose granulosa cell apoptosis via PI3K/AKT/Caspase-9 signaling pathway, Anim. Reprod. Sci. 200 (2019) 86–95. [30] D. Liu, P. You, Y. Luo, M. Yang, Y. Liu, Galangin induces apoptosis in MCF-7 human breast Cancer cells through mitochondrial pathway and phosphatidylinositol 3Kinase/Akt inhibition, Pharmacology 102 (2018) 58–66. [31] M. Moradzadeh, A. Hosseini, S. Erfanian, H. Rezaei, Epigallocatechin-3-gallate promotes apoptosis in human breast cancer T47D cells through down-regulation of PI3K/AKT and Telomerase, Pharmacol. Rep. 69 (2017) 924–928. [32] K.W. Luo, W.Y. Lung, Chun-Xie, X.L. Luo, W.R. Huang, EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway, Oncotarget 9 (2018) 12261–12272. [33] W. Lim, S. Park, F.W. Bazer, G. Song, Apigenin reduces survival of choriocarcinoma cells by inducing apoptosis via the PI3K/AKT and ERK1/2 MAPK pathways, J. Cell. Physiol. 231 (2016) 2690–2699. [34] M. Mazumdar, A. Adhikary, S. Chakraborty, S. Mukherjee, A. Manna, S. Saha, S. Mohanty, A. Dutta, P. Bhattacharjee, P. Ray, S. Chattopadhyay, S. Banerjee, J. Chakraborty, A.K. Ray, G. Sa, T. Das, Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/ caspase-8 pathways, Apoptosis. 18 (2013) 589–604. [35] M. Kunte, K. 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5. Conclusion In conclusion, our results show that both black tea and green tea can inhibit the proliferation and migration of HepG2 cells, with non-fermented green tea being more effective than fermented black tea in cell apoptosis induction. Black tea and green tea inhibited the growth of HepG2 cells, induced apoptosis through PI3K/Akt, and inhibited cell migration and invasion through the MMP signalling pathway. These results indicate that black tea and green tea have significant effects against liver cancer and more distinctive mechanisms, therefore have the potential to be used in cancer therapy and as functional foods. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements The research was supported by the National Natural Science Foundation of China (81803236, 31800295, 81903319), the Guangdong Science and Technology Programme (2017A070702004, 2016B090918118, 2017A020224015, 2018KJYZ002), the Natural Science Foundation of Guangdong Province (2017A030310504), Science and Technology Board of Yingde (JHXM2018029), Fund of the Technology Innovation of Guizhou Province([2014]45), Key Agricultural Project of department of science & technology of Guizhou ([2019]2377), and Pecial fund for scientific innovation strategy-construction of high level Academy of Agriculture Science (R2019PYJX004, R2018YJ-YB3002, R2016YJ-YB3003, R2018PY-QF005, R2018QD-101). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2020.109893. References [1] W. Okajima, S. Komatsu, D. Ichikawa, M. Miyamae, T. Ohashi, T. Imamura, J. Kiuchi, K. Nishibeppu, T. Arita, H. Konishi, A. Shiozaki, R. Morimura, H. Ikoma, K. Okamoto, E. Otsuji, Liquid biopsy in patients with hepatocellular carcinoma: circulating tumor cells and cell-free nucleic acids, World J. Gastroenterol. 23 (2017) 5650–5668. [2] R.A. Nesbit, M.H. Tattersall, R.M. Fox, R.L. Woods, Presentation of unknown primary cancer with metastatic liver disease–management and natural history, Aust. N. Z. J. Med. 11 (2010) 16–24. [3] W. Yeo, B. Zee, S. Zhong, P.K.S. Chan, W.L. Wong, W.M. Ho, K.C. Lam, P.J. Johnson, Comprehensive analysis of risk factors associating with Hepatitis B virus (HBV) reactivation in cancer patients undergoing cytotoxic chemotherapy, Br. J. Cancer 90 (2004) 1306–1311. [4] W. Yeo, T.S. Mok, B. Zee, T.W.T. Leung, P.B.S. Lai, Y.L. Wan, J. Koh, F.K.F. Mo, S.C.H. Yu, A.T. Chan, A randomized phase III study of doxorubicin versus Cisplatin/ Interferon α-2b/Doxorubicin/Fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma, J. Natl. Cancer Inst. 97 (2005) 1532–1538. [5] L. Zeng, Y. Zhou, X. Fu, X. Mei, S. Cheng, J. Gui, F. Dong, J. Tang, S. Ma, Z. Yang, Does oolong tea (Camellia sinensis) made from a combination of leaf and stem smell more aromatic than leaf-only tea? Contribution of the stem to oolong tea aroma, Food Chem. 237 (2017) 488. [6] A. Basu, N.M. Betts, A. Mulugeta, C. Tong, E. Newman, T.J. Lyons, Green tea supplementation increases glutathione and plasma antioxidant capacity in adults with the metabolic syndrome, Nutr. Res. 33 (2013) 180–187. [7] N. Sheikhzadeh, K. Nofouzi, A. Delazar, A.K. Oushani, Immunomodulatory effects of decaffeinated green tea (Camellia sinensis) on the immune system of rainbow
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inhibited bladder cancer SW780 cell proliferation and migration both in vitro and in vivo via down-regulation of NF-κB and MMP-9, J. Nutr. Biochem. 41 (2017) 56–64. [37] R. Arcone, M. Palma, V. Pagliara, G. Graziani, M. Masullo, G. Nardone, Green tea polyphenols affect invasiveness of human gastric MKN-28 cells by inhibition of LPS
or TNF-α induced Matrix Metalloproteinase-9/2, Biochim Open. 3 (2016) 56–63. [38] F. Wang, Z. Chang, Q. Fan, L. Wang, Epigallocatechin‑3‑gallate inhibits the proliferation and migration of human ovarian carcinoma cells by modulating p38 kinase and matrix metalloproteinase‑2, Mol. Med. Rep. 9 (2014) 1085–1089.
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Green tea and black tea inhibit proliferation and migration of HepG2 cells via the PI3K/Akt and MMPs signalling pathway
T
Lingli Suna, Yuanlong Zhangb, Wenji Zhanga, Xingfei Laia, Qiuhua Lia, Lingyun Zhangb,*, Shili Suna,* a Tea Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of Tea Plant Resources Innovation & Utilization, Guangzhou, 510640, PR China b College of Horticulture, South China Agricultural University, Guangzhou, 510642, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Tea HepG2 Tumour proliferation Tumour migration PI3K/Akt pathway
Background and aims: Black tea and green tea were produced via different processing techniques from the same tea leave variety. Then, biochemical components of the two water extracts were analysed to study cell apoptosis, migration and invasion of HepG2 cells induced by black tea and green tea. Method: The monomer components of the black tea and green tea extracts were analysed by colorimetry and HPLC, with MTT assay and colony formation assays used to assess cell proliferation and viability. The effects of black tea and green tea on apoptosis of HepG2 cells were verified by flow cytometry, with wound healing and Transwell experiments used to detect cell invasion and metastasis. The expression of PI3K/Akt signalling and apoptosis-related proteins as well as epithelial-mesenchymal transition (EMT) regulatory factor in HepG2 cells were determined by western blotting after black tea and green tea treatment. Results and conclusions: Black tea and green tea extracts demonstrated different degrees of inhibition of cell migration and invasion, with green tea inducing more HepG2 cell apoptosis. In addition, green tea and black tea extracts inhibited the growth of HepG2 cells and induced apoptosis via PI3K/Akt, and inhibited cell migration and invasion through the MMPs signalling pathway. This study revealed the effects of fermented (black tea) and non-fermented tea (green tea) on liver cancer cells, providing a basis for the investigation of tea extracts for their anti-tumour potential.
1. Introduction
doxorubicin, as well as some targeted therapeutic drugs such as sorafenib, but the prognosis is often poor and is associated with strong toxicity and side effects [4]. Therefore, in recent years, the development of drugs with anti-tumour effects using natural products as raw materials has become a potential trend. Tea is derived from Camellia sinensis and is the second most popular non-alcoholic beverage in the world [5]. A variety of studies have shown that tea has a variety of health effects, such as, alleviate the metabolic syndrome, anti-tumour ability and enhance immunity [6,7]. The most important teas are black tea and green tea, which due to the different processing techniques, possess very diverse taste quality characteristics and biological active ingredients [8]. Studies have shown that black tea and green tea have the potential to prevent liver
Liver cancer, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), is a common malignant tumour in digestive system, which is one of the leading causes of death in cancer patients [1]. Liver cancer often has no obvious clinical signs in the early stage of the disease, and is likely to progress to the middle and late stages when there are obvious symptoms [2]. As liver cancer patients often have hepatitis B virus (HBV) infection, liver function is frequently severely impaired in advanced liver cancer [3]. The treatment of liver cancer is currently based on surgery, chemical treatment is considered if surgery is not possible. Chemotherapeutic drugs conventionally used for the treatment of liver cancer include cisplatin and
Abbreviations: CG, Catechin gallate; EGCG, Epigallocatechin gallate; C, Catechin; EGC, Epigallocatechin; EC, Epicatechin; GC, Gallocatechin; GCG, Gallocatechin gallate; ECG, Epicatechin gallate; GA, Gallic acid; TP, Tea polyphenol; AA, Amino acid; TFs, Theaflavins; TRs, Thearubigins; TBs, Theabrownings; MTT, 3-45Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B; Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma 2; MMP-2/9, matrix metalloproteinases ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Zhang), [email protected] (S. Sun). https://doi.org/10.1016/j.biopha.2020.109893 Received 24 October 2019; Received in revised form 26 December 2019; Accepted 29 December 2019 0753-3322/ © 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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cancer. Black tea extract protected the liver from carbon tetrachlorideinduced lipid peroxidation, delaying the progression of liver inflammation and liver cancer in mice [9]. Epigallocatechin gallate (EGCG) and theaflavin (TF) in green tea and black tea can down-regulate Wnt/β-catenin and Hh/Gli1 signalling pathways to delay the progression of liver cancer [10]. However, the difference in therapeutic effects between black tea and green tea, and the mechanism of treatment for liver cancer have not been reported. Phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signal transduction pathway has a role in regulating cell proliferation and apoptosis, and is closely related to the occurrence and development of liver cancer [11]. Activation of the PI3K/Akt signalling pathway in liver cancer causes a series of responses, including promotion of cell growth, proliferation, epithelial-mesenchymal transition (EMT) and angiogenesis [12]. During the progression of malignant tumours, EMT appears to change from a highly differentiated epithelial cell phenotype to a fibroblast-like or mesenchymal phenotype, which causes tumour invasion and metastasis [13]. The MMP protein family, including MMP-2, MMP-3 and MMP-9, mediate the EMT process [14]. Black tea and green tea extracts, as potential therapeutic agents for liver cancer, have not previously been shown to affect PI3K/Akt/MMPs-mediated invasion and metastasis in liver cancer. In this study, we processed the fresh leaves of the same variety of tea trees into black tea and green tea via different processing techniques. Using HepG2 cells as a cell model, we studied the efficacy and molecular mechanism of black tea and green tea extracts to inhibit tumour cell growth and cell migration, revealing the novel anti-tumour potential of black tea and green tea in liver cancer.
Table 1 The contents of bioactive components in green tea and black tea. Component
Black tea
Green tea
Tea polyphenols (%) Amino acid (%) Soluble sugar (%) Flavonols (mg/g) TFs (%) TRs (%) TBs (%) Caffeine (mg/g) GA (mg/g) GC (mg/g) EGC (mg/g) C (mg/g) EC (mg/g) EGCG (mg/g) GCG (mg/g) ECG (mg/g) CG (mg/g) Total catechins (mg/g)
12.19 ± 0.25 2.70 ± 0.01 9.68 ± 1.06 27.26 ± 0.10* 0.11 ± 0.00** 1.78 ± 0.20** 5.96 ± 0.18** 95.63 ± 0.10* 13.63 ± 0.14* 7.27 ± 0.60 31.66 ± 0.63 14.98 ± 0.22 7.32 ± 0.24 5.77 ± 0.39 2.24 ± 0.12 5.39 ± 0.03 1.57 ± 0.43 76.20 ± 1.61
21.74 ± 0.80** 2.72 ± 0.01 9.11 ± 0.91 20.19 ± 0.02 / / / 83.09 ± 0.12 6.17 ± 0.08 8.72 ± 0.25* 44.51 ± 0.96** 26.52 ± 0.69** 15.01 ± 0.68** 77.84 ± 0.33** 2.48 ± 0.11 33.08 ± 0.75** 1.03 ± 0.10 209.20 ± 3.26**
Values represent means ± SD (n = 3). * are significantly different at p < 0.05. ** are significantly different at p < 0.01.
for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). HepG2 cells were cultured in DMEM media, supplemented with 10 % FBS, and grown in an incubator (37 °C, 5 % CO2, and saturated humidity). The cells used in the experiments were all recovered within two months from storage in liquid nitrogen.
2. Materials and methods
2.4. Cell viability assay
2.1. Chemicals and reagents
The effects of black tea and green tea on the cell viability of HepG2 cells was examined by MTT assay. In brief, 1 × 104 HepG2 cells were seeded per well in 96-well plates containing 100 μL of complete medium. After 24 h, the cells were adherent, then a specific concentration of black tea and green tea extract was added to the cells for 24 or 48 h. MTT solution (20 μL of 5 mg / mL in phosphate buffered saline, Sigma Aldrich, USA) was added to each well and incubated at 37 °C for 4 h in the dark. The medium was removed, 150 μL DMSO was added and shaken in the dark for 10 min, before the absorbance of the supernatant was measured at 570 nm using a microplate reader (BioRad, USA). The IC50 values were calculated by GraphPad Prism 7.0 Software and each treatment group and control group contained four independent replicates.
Black tea and green tea samples were obtained from the Tea Research Institute, Guangdong Academy of Agricultural Sciences in China. Antibodies specific for a variety of proteins as well as all secondary antibodies were purchased from Cell Signalling Technology (Cell Signalling Technology, USA). LY294002 was purchased from Sigma Aldrich. DMEM media, foetal bovine serum (FBS), and trypsin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 2.2. Preparation and biochemical analysis of black tea and green tea extracts Two different kinds of tea were made from the same species of tea tree via different processing techniques. Briefly, in the manufacture of green tea, the tea leaves were heated to inactivate the enzymes, rolled and dried, whereas for black tea production, the tea leaves were withered, crushed, and allowed to under undergo enzyme-mediated oxidation. The black tea and green tea were powdered, then placed in boiling distilled water for 30 min (tea/water, 1:20 w:v) and continuously extracted three times. The three extracted hot tea solutions were combined and filtered immediately, and concentrated at 60 °C, before freeze drying. The free amino acids, total soluble sugars and polyphenol content of the tea solutions were determined by the ninhydrin method, the anthrone-sulphuric acid colorimetric method and the Folin-phenol method, respectively. The content of caffeine and catechins in the extract was quantified by high performance liquid chromatography (HPLC).
2.5. Colony formation assay HepG2 cells were treated with black tea and green tea extract for 48 h in the logarithmic growth phase and the culture broth was discarded. The treated cells were trypsinised to prepare a single cell suspension and re-inoculated into a six-well plate (1500 cells/well). The cells were cultured for 10 days at 37 °C in 5 % CO2 until large colonies visible to the naked eye appeared. The cells were washed with PBS, fixed (methanol : glacial acetic acid : ddH2O = 1:1:8) for 10 min, then stained with 0.1 % crystal violet for 30 min. The stained cell colonies were photographed and counted. 2.6. Cell migration assay A wound healing assay was performed to detect cell migration. HepG2 cells were seeded in six-well culture plates and after the cells were fully attached, a wound was made by artificially using a sterilised tip to draw a line of equal width on the underside of the dish. The suspended cell debris was washed away with PBS, and FBS-free basal medium was added. The 0 h initial wound photographs were taken
2.3. Cell line and cell culture The human hepatocellular carcinoma cell line HepG2 was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes 2
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Fig. 1. Black tea and green tea inhibit the growth and proliferation of HepG2 cells. (A). Different concentrations (0, 0.2 mg/ml, 0.8 mg/ml) of black tea and green tea extract-treated HepG2 cells were photographed by microscopy. (B). HepG2 cells were subjected to colony formation experiments after treatment with black tea and green tea extracts. (C). MTT assay was used to detect cell viability of HepG2 cells treated with different concentrations of black tea and green tea extract for 24 h or 48 h. (D). IC50 values of black tea and green tea extracts in HepG2 cells. IC50, concentrations that induced 50 % cell growth inhibition. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
Fig. 2. Black tea and green tea promoted the proportion of apoptotic HepG2 cells. Apoptosis was detected by flow cytometry in HepG2 cells treated with various concentrations of black tea (A) and green tea (B) extracts. (C). The proportion of apoptotic HepG2 cells (FITC-annexin V positive cells).
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Fig. 3. Black tea and green tea slow the migration of HepG2 cells. (A). HepG2 cells were treated with black tea and green tea extracts (0.05 and 0.20 mg/ml) and cell migration ability was assessed by the wound healing assay. (B). The ratio of HepG2 cell migration distance was counted. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
Fig. 4. Black tea and green tea inhibited the invasion of HepG2 cells. (A). HepG2 cells were treated with black tea and green tea extracts (0.2 and 0.8 mg/ml) and the cell invasion ability was examined by Transwell assay. (B). The proportion of the invading HepG2 cells was counted. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
Alamos, NM).
under an Olympus inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan) and marked on the 6-well plate lid at the corresponding position. A specific dose of black tea or green tea extract was then added to the treated cells for 24 or 48 h, and photographs of the treated cell wound were obtained. Four fields of view at a fixed position were taken at each time. Finally, Image Tool software (Bechtel Nevada Inc., Los Alamos, NM) to calculate the migration distance of the treated and control cells.
2.8. Flow cytometric analysis Flow cytometry analysis was performed using a BD FACS Accuri C6 plus flow cytometer (BD Biosciences, San Jose, CA, USA) to determine the proportion of apoptotic HepG2 cells. HepG2 cells were digested and resuspended in PBS after treatment with the tea solution, then stained with PI and annexin V solutions, respectively, according to the manufacturer's instructions in the Apoptosis Assay Kit (Beyotime, China). The proportion of apoptotic cells was analysed by flow cytometry using FlowJo (Becton, Dickinson and Company, US) software.
2.7. Transwell cell invasion assay First, a Matrigel-containing Transwell chamber (Corning Glass Works, Corning, NY) was placed in a 24-well plate and incubated at 37 °C. The treated HepG2 cells were digested and resuspended in FBS-free basal medium. A 0.5 ml cell suspension containing 5 × 104 cells was added to the Transwell upper chamber, and 0.5 ml of complete medium was added to the lower chamber and incubated at 37 °C. After 24 h, the medium in the upper and lower chambers was discarded, and the cells on the inside of upper chamber were removed with a cotton swab. The cells on the outside were fixed with cold ethanol for 30 min, then stained with 0.1 % crystal violet for 10 min, the excess stained background washed with running water and allow to dry. Four fields of view were imaged under an inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan) for each treatment group and control group. Invasive cells were counted by Image Tool software (Bechtel Nevada Inc., Los
2.9. Western blot assay The HepG2 cells treated with the tea solution were lysed with RIPA buffer, and the protein content was quantified by the BCA method. A 50 μg protein sample was placed in loading buffer and boiled for 5 min, then electrophoresed by SDS-PAGE and transferred to a PVDF membrane (Millipore, MA, USA). The PVDF membrane was then blocked with 5 % skimmed milk for 2 h at room temperature and incubated with the primary antibody overnight at 4 °C. The primary antibodies used in this study included anti-PI3K, anti-p-PI3K, anti-Akt, anti-p-Akt, antiBcl-2, anti-Bax and anti-β-actin 1:1000; Cell Signalling Technology, USA. The membrane was then washed three times with TBST and 4
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Fig. 5. Black tea and green tea time-dependently reduced PI3K/Akt signalling pathway activity in HepG2 cells. (A). HepG2 cells were treated with 0.2 mg/ml black tea and green tea extracts, and protein expression at various time points in the cells was detected by western blotting. The absolute amounts of p-PI3K (B) and p-Akt (C) were simultaneously quantified. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
incubated with HRP-labelled anti-rabbit or anti-mouse secondary antibody 1:10,000; Cell Signalling Technology, USA for 1 h at room temperature. Specific protein bands were detected using Chemiluminescence ECL Detection Kit Thermo Scientific, USA and Chemi Doc System Bio-Rad, USA. Protein bands were quantified by Image J software (Bethesda, MD, USA). When inhibitors were employed, cells were pretreated for 8 h with inhibitor (LY294002, 20 μM) before the addition of black tea and green tea extract.
significantly higher than in green tea extract, with the polyphenol content of green tea being almost twice that of black tea. Furthermore, the catechins content was almost three-fold higher in green tea, with black tea containing a specific amount of theaflavins (TFs), thearubigins (TRs) and theabrownines (TBs). More importantly, the content of EGCG, which is recognised as the most important active substance in tea, in the green tea extract was 13 times higher than that in black tea extract.
2.10. Statistical analysis
3.2. The antineoplastic effect of black tea and green tea
All data are expressed as mean ± SD of at least three independent experiments. Two-way ANOVA analysis of variance was used for comparison between multiple groups. GraphPad Prism 7.0 statistical software (GraphPad Software Inc., San Diego, CA, USA) was used to analyse the data. P < 0.05 and P < 0.01 were considered to be statistically significant.
To determine whether black tea and green tea could inhibit HepG2 cell proliferation, the morphology and proliferation of HepG2 cells was determined. As shown in Fig. 1A, shrunken cells and plasma membrane blebs were present after treatment with black tea and green tea. Furthermore, the colony formation assay demonstrated that both black tea and green tea enhanced the inhibition of cell proliferation (P < 0.05), which increased with increasing concentration (Fig. 1B). The cytotoxic effects of the tea extracts on human HepG2 cells were evaluated by the MTT assay. After 24 and 48 h of treatment, the proliferation of HepG2 cells was inhibited at the dose of more than 0.25 mg/ml, but the concentrations below 0.25 mg/ml extract could affect the survival of cancer cells. Black tea and green tea extracts appeared to different efficacies of growth inhibition of HepG2 cell, with estimated IC50 values of 1.11 mg/ml and 0.88 mg/ml at 24 h of 0.57 mg/ml and 0.52 mg/ml at 48 h treatment, respectively. More importantly, the inhibitory effect
3. Results 3.1. The composition of black tea and green tea extracts As shown in Table 1 (the HPLC chromatogram in Fig. S1), the content of amino acid and soluble sugar in the extracts of black tea and green tea were not significantly different. The amount of flavanols, caffeine and γ-aminobutyric acid (GA) in the black tea extract were 5
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Fig. 6. Black tea and green tea decreased PI3K/Akt signalling pathway activity in a concentration-dependent manner. (A) HepG2 cells were treated with various concentrations of black tea and green tea extracts for 24 h, and protein expression was detected by western blotting. The absolute amounts of p-PI3K (B) and p-Akt (C) were simultaneously quantified. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
the invasion of HepG2 cells (Fig. 4A). Compared with the control group, the ability of the treated cells in the black and green tea group to penetrate the Matrigel was significantly decreased (P < 0.05), with the number of migrated cells in the black tea group significantly higher than that in the green tea group at the concentration of 0.8 mg / ml (P < 0.05, Fig. 4B). These results indicate that both black tea and green tea could inhibit the migration and invasion of HepG2 cells, and green tea was more effective than the black tea group.
of green tea was stronger than that of black tea, but the difference was not significant (Fig. 1C, D). 3.3. Black tea and green tea promotes HepG2 cell apoptosis Flow cytometric analysis showed that treatment with black tea or green tea for 24 h induced apoptosis of HepG2 cells in a concentrationdependent manner. As shown in Fig. 2, after treatment of HepG2 cells with 1.0 mg/ml of black tea or green tea for 24 h, the proportion of apoptotic cells increased to 38.5 % and 64.0 %, indicating that high concentrations of black and green tea induce apoptosis in HepG2 cells.
3.5. Black tea and green tea enhance PI3K/Akt signalling inhibition The PI3K/Akt signalling pathway regulates tumour cell proliferation and death [16,17], so the western blotting assay was used to assess the effect of black and green tea on the expression of pro-survival proteins associated with the PI3K/Akt pathway in HepG2 cells. Intriguingly, treatment with black and green tea markedly suppressed the levels of phosho-PI3K (p-PI3K) and p-Akt proteins in a time-dependent (Fig. 5A) and concentration-dependent manner (Fig. 6A), whereas it had almost no effect on total PI3K and Akt protein expression. In addition, quantitative analysis of protein levels was undertaken (Fig. 5B, C and Fig. 6B, C), showing that like effects on cell growth inhibition, the regulatory effect of green tea on the PI3K/Akt signalling pathway in HepG2 cells was more significant than that of black tea. These results indicate that the black tea and green tea may partially inactivate PI3K/ Akt signalling, thereby affecting HepG2 cell proliferation.
3.4. Inhibition of cell migration and invasion Tumour metastasis is characterised by invasion and migration of tumour cells [15], so we verified whether black tea and green tea could inhibit tumour metastasis by blocking the invasion and migration of tumour cells. Scratch adhesion assays showed that black tea and green tea treatment inhibited HepG2 cell migration in a time and concentration-dependent manner (Fig. 3A). Quantitatively, the tumour cell migration distance of the tea treatment group was significantly shorter than that of the control group (P < 0.05), with the migration distance of HepG2 cells treated with green tea for 48 h significantly higher than that of the black tea-treated cells (P < 0.05, Fig. 3B). Transwell cell migration assays demonstrated that black tea and green tea inhibited 6
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Fig. 7. The influence of black tea and green tea on the PI3K/Akt pathway in liver cancer cells. (A) Cells were treated LY294002 (20 μM), or black tea and green tea (0.2 mg/mL), or their combination. After 48 h, cell viability was determined by MTT analysis. Cells were treated with black tea or green tea alone or in combination with LY294002. After 48 h, the levels of p-PI3K, PI3K, p-Akt, and Akt proteins were analysed by western blotting and β-actin served as the loading control. (B) Representative blot of expression. (C and D) Quantitative analysis of the proteins in (B) was undertaken. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01.
cytoplasm to promote apoptosis [19]. Next, we verified whether black tea and green tea induced apoptosis in HepG2 cells is dependent on the Bcl-2/Bax signalling pathway. Western blotting data showed that the expression of Bcl-2 in the HepG2 cells decreased gradually with treatment time (Fig. 8A, C) and concentration (Fig. 8B, D) of black and green tea. However, the expression of Bax was not affected by the treatment with black tea and green tea. It is generally believed that there is a balance between Bcl-2 and Bax, which is related to the occurrence of apoptosis [20]. We compared the Bax / Bcl-2 ratios of the black tea group and the green tea group, finding that the green tea treatment group had a higher Bax / Bcl-2 ratio. Thus, treatment with green tea decreased the Bcl-2 / Bax ratio in HepG2 cells, thereby activating a mitochondria-dependent apoptotic pathway.
To confirm the involvement of the PI3K/Akt pathway in the inhibition of tumour cell growth by black tea and green tea, HepG2 cells were pretreated with the PI3K-specific inhibitor, LY294002 (20 μM), for 8 h, followed by co-treatment with black tea or green tea. After 48 h, cell viability was analysed using MTT assay. As shown in Fig. 7A, treatment with LY294002 significantly inhibited the growth of cells, whereas combined treatment with LY294002, followed by black tea and green tea had no significant effect on cell viability. In addition, treatment with LY294002 reduced the phosphorylation levels of PI3K and Akt. These results indicate that treatment with black tea and green tea may partially inactivate PI3K/Akt signalling, thereby affecting HepG2 cell proliferation. 3.6. Green tea decreases the Bcl-2 / Bax ratio to regulate apoptosis of HepG2 cells
3.7. Effects on migration and invasion related protein activities in HepG2 cells
Mitochondrial pathway-dependent apoptosis is mainly regulated by Bcl-2 family proteins, including the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax [18]. Bcl-2 blocks the transfer of cytochrome c from mitochondria to the cytoplasm and inhibits caspase activation, thereby inhibiting apoptosis, while Bax permeabilises the mitochondrial outer membrane and induces cytochrome c release into the
Cancer cell invasion and migration result in the diffuse and invasive growth of cancer cells, which makes these tumours difficult to eradicate using conventional therapeutic methods. MMP-2/9, important members of the MMP family, can promote cell migration through disruption of the extracellular matrix [21]. As markers of cell invasion, N-cadherin 7
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Fig. 8. Black tea and green tea induce Bax/Bcl-2-dependent apoptosis in HepG2 cells. HepG2 cells were treated with time (A) and concentration gradients (B) of black and green tea extracts, and Bax and Bcl-2 protein expression was detected by western blotting. At the same time, the ratio between Bax/Bcl-2 was quantified (C and D). The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
then rolled into a rope. Black tea needs to be fermented and dried during its production, which causes the polyphenols in cells to be oxidised by the action of intracellular oxidase [23]. Polyphenols are considered to be the important functional substances in tea and it is generally believed that the content and composition of polyphenols in the aqueous extracts of green tea and black tea may be the main factor leading to their difference in biological activity of aqueous extracts [24]. Our data revealed that tea polyphenols, especially a variety of catechin monomer, such as EGCG and other substances, were high in green tea, which may cause green tea to down-regulate Bcl-2 to promote apoptosis in HepG2 cells. The occurrence of liver cancer is generally a malignant tumour of HCC or ICC, which is characterised by high malignancy and rapid progress [25]. However, due to the lack of effective drugs, the treatment of liver cancer is still difficult. This study showed that green tea and black tea extracts had a significant inhibitory effect on liver cancer cells in vitro. Green tea and black tea extracts significantly promoted apoptosis of liver cancer cells and inhibited cell invasion and metastasis. The higher the concentration of the extract, the more obvious the inhibition of cell proliferation was dose-dependent. In addition, green
and vimentin were detected in the present study (Fig. 9 and Fig. 10). Our results demonstrated that treatment with either black tea or green tea led to significant downregulation of MMP-2/9, N-cadherin, and vimentin in a time-dependent (Fig. 9A-E) and concentration-dependent manner (Fig. 10A-E). Similar to the above results, the protein expression regulation effect in green tea treatment groups were stronger than that in black tea treatment groups. Taken together, these results support the conclusion that combined treatment with black tea and green tea potentiates the suppression of tumour cell migration and invasion through modulation of MMPs signalling.
4. Discussion The black tea and green tea used in this study were derived from the same varieties of fresh tea leaves, which were processed separately by different processing techniques. For green tea production, the tea leaves are first distributed to allow the fresh leaves to lose water, then the tea leaves are inactivated by high temperature (280–300 °C), before being rolled into strips and dried to make green tea [22]. For black tea production, the tea leaves are placed to allow the fresh leaves to lose water, 8
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Fig. 9. Black tea and green tea time-dependently reduced MMP protein expression and inhibited EMT progression. (A). Black tea and green tea extracts were treated with HegG2 cells in a time gradient, and the expression of EMT-related proteins in the cells was detected by western blotting. (B–E). Quantitative analysis of expression of EMT-related proteins. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
further investigate the molecular mechanism of apoptosis induced by black and green tea in liver cancer cells. Studies have shown that PI3Kmediated Akt phosphorylation activation further directly phosphorylates caspase-3/9, which disrupts the activation of a series of apoptotic factors triggered by caspase-3/9, thus terminating the apoptotic pathway [29,30]. EGCG is the highest and most active polyphenolic compound in green and black tea. Previous studies revealed that EGCG promoted tumour cell apoptosis by altering the PI3K / Akt signalling pathway. In breast cancer, EGCG increased PTEN, caspase 3 and caspase 9 and decreased the expression of PI3K, Akt and hTERT. In this way, EGCG promoted breast cancer cell apoptosis by regulating the
tea had a stronger inhibitory effect on liver cancer cells than black tea, which may be due to high content of important active substances, such as EGCG, in green tea. Previous studies also reported that tea extracts inhibited the growth, proliferation and differentiation of liver cancer cells [26,27]. Green tea polyphenols inhibited the proliferation and growth of HepG2 cells, and polyphenol monomer EGCG induced apoptosis of HepG2 cells by increasing the activity of caspase-3 protein [28]. Therefore, our study revealed the potential of extracts from fermented tea (black tea) and non-fermented tea (green tea) in the treatment of liver cancer. Western blotting of PI3K / Akt signalling proteins was performed to
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Fig. 10. Black tea and green tea concentration-dependently reduced MMP protein expression and inhibited EMT progression. (A). HepG2 cells were treated with a concentration gradient of black tea and green tea extracts, and the expression of EMT-related proteins in the cells was detected by western blotting. (B–E). Quantitative analysis of expression of EMT-related proteins. The level of significance versus the control group is indicated by *p < 0.05, ** p < 0.01 and the level of significance comparing the black tea group with the green tea group is indicated by # p < 0.05, ## p < 0.01.
growth of liver cancer cells, which makes these tumours difficult to eradicate using conventional therapeutic methods. Invasion and metastasis of liver cancer is a multi-stage process, of which MMP-2/9mediated destruction of extracellular matrix (ECM) is a key step [35]. It has been shown that EGCG down-regulated mRNA and protein expression of NF-κB and MMP-9 in a dose-dependent manner and inhibits bladder cancer migration [36]. In gastric cancer, green tea polyphenol extracts reduce the invasiveness of gastric cancer cells by reducing inflammatory factors-induced MMP-2/9 activation [37], but the tea extract does not affect all EMT-related proteins in tumour metastasis. For
PI3K / Akt pathway and telomerase activity [31]. In bladder cancer T24 and 5637 cells, EGCG up-regulated PTEN and reduced the expression of phosphorylated PI3K and Akt, which inhibited cell proliferation [32]. In addition, apigenin in tea reduced the phosphorylation of Akt, P70RSK, and S6 in choriocarcinoma cells, thereby promoting tumour cell apoptosis and inhibiting metastasis [33]. Theaflavins in black tea blocked the PI3K / Akt / Bad pathway to trigger apoptosis in medullary thyroid cancer [34]. Our data also confirmed that the tea extract components, including EGCG, promoted apoptosis in HepG2 cells. Cancer cell migration and invasion result in the diffuse and invasive 10
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example, in ovarian cancer, EGCG inhibited tumour migration by down-regulating MMP-2, not MMP-9, and exerts its therapeutic value [38]. In our study, high concentrations of green tea and black tea extracts significantly reduced the expression of MMP-2 and MMP-9 in HepG2 cells, with a weak effect on vimentin. Therefore, we revealed the mechanism by which tea extract regulated MMP-2/9 mediated invasion and metastasis, providing a new basis for liver cancer treatment.
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5. Conclusion In conclusion, our results show that both black tea and green tea can inhibit the proliferation and migration of HepG2 cells, with non-fermented green tea being more effective than fermented black tea in cell apoptosis induction. Black tea and green tea inhibited the growth of HepG2 cells, induced apoptosis through PI3K/Akt, and inhibited cell migration and invasion through the MMP signalling pathway. These results indicate that black tea and green tea have significant effects against liver cancer and more distinctive mechanisms, therefore have the potential to be used in cancer therapy and as functional foods. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements The research was supported by the National Natural Science Foundation of China (81803236, 31800295, 81903319), the Guangdong Science and Technology Programme (2017A070702004, 2016B090918118, 2017A020224015, 2018KJYZ002), the Natural Science Foundation of Guangdong Province (2017A030310504), Science and Technology Board of Yingde (JHXM2018029), Fund of the Technology Innovation of Guizhou Province([2014]45), Key Agricultural Project of department of science & technology of Guizhou ([2019]2377), and Pecial fund for scientific innovation strategy-construction of high level Academy of Agriculture Science (R2019PYJX004, R2018YJ-YB3002, R2016YJ-YB3003, R2018PY-QF005, R2018QD-101). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2020.109893. References [1] W. Okajima, S. Komatsu, D. Ichikawa, M. Miyamae, T. Ohashi, T. Imamura, J. Kiuchi, K. Nishibeppu, T. Arita, H. Konishi, A. Shiozaki, R. Morimura, H. Ikoma, K. Okamoto, E. Otsuji, Liquid biopsy in patients with hepatocellular carcinoma: circulating tumor cells and cell-free nucleic acids, World J. Gastroenterol. 23 (2017) 5650–5668. [2] R.A. Nesbit, M.H. Tattersall, R.M. Fox, R.L. Woods, Presentation of unknown primary cancer with metastatic liver disease–management and natural history, Aust. N. Z. J. Med. 11 (2010) 16–24. [3] W. Yeo, B. Zee, S. Zhong, P.K.S. Chan, W.L. Wong, W.M. Ho, K.C. Lam, P.J. Johnson, Comprehensive analysis of risk factors associating with Hepatitis B virus (HBV) reactivation in cancer patients undergoing cytotoxic chemotherapy, Br. J. Cancer 90 (2004) 1306–1311. [4] W. Yeo, T.S. Mok, B. Zee, T.W.T. Leung, P.B.S. Lai, Y.L. Wan, J. Koh, F.K.F. Mo, S.C.H. Yu, A.T. Chan, A randomized phase III study of doxorubicin versus Cisplatin/ Interferon α-2b/Doxorubicin/Fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma, J. Natl. Cancer Inst. 97 (2005) 1532–1538. [5] L. Zeng, Y. Zhou, X. Fu, X. Mei, S. Cheng, J. Gui, F. Dong, J. Tang, S. Ma, Z. Yang, Does oolong tea (Camellia sinensis) made from a combination of leaf and stem smell more aromatic than leaf-only tea? Contribution of the stem to oolong tea aroma, Food Chem. 237 (2017) 488. [6] A. Basu, N.M. Betts, A. Mulugeta, C. Tong, E. Newman, T.J. Lyons, Green tea supplementation increases glutathione and plasma antioxidant capacity in adults with the metabolic syndrome, Nutr. Res. 33 (2013) 180–187. [7] N. Sheikhzadeh, K. Nofouzi, A. Delazar, A.K. Oushani, Immunomodulatory effects of decaffeinated green tea (Camellia sinensis) on the immune system of rainbow
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