Epigallocatechin-3-gallate inhibits cell growth, induces apoptosis and causes S phase arrest in hepatocellular carcinoma by suppressing the AKT pathway

  • Authors:
    • Xiaoyun Shen
    • Yong Zhang
    • Yan Feng
    • Litu Zhang
    • Jilin Li
    • Yu-An Xie
    • Xiaoling Luo
  • View Affiliations

  • Published online on: January 8, 2014     https://doi.org/10.3892/ijo.2014.2251
  • Pages: 791-796
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Abstract

Epigallocatechin-3-gallate (EGCG) has been shown to inhibit the growth and induce apoptosis of certain cancer cells. The aim of this study was to determine the role of EGCG in hepatocellular carcinoma (HCC) and the underlying mechanism(s) thereof. MTT assay was used to determine the cell growth inhibition by EGCG. Apoptosis induced by EGCG was investigated by both AO/EB staining and flow cytometry. The cell cycle distribution was analyzed by flow cytometry. The mRNA levels of the AKT pathway were analyzed by quantitative PCR. The expression of AKT and its phosphorylation at Ser473 were detected by western blotting. The IC50 of EGCG at 48 h for HepG2, SMMC7721 and SK-hep1 cells were 74.7, 59.6 and 61.3 µg/ml, respectively. Significantly higher proportion of SMMC7721 cells entered the S phase upon treatment with EGCG for 48 h compared with control cells. EGCG decreased the mRNA levels of PI3K, AKT and NF-κB. The protein levels of AKT decreased and its phosphorylation at Ser473 was downregulated with EGCG treatment. EGCG inhibited growth by affecting the cell cycle and induced apoptosis in different HCC cells by downregulating PI3K/AKT activity. The results suggest the potential of EGCG as an anticancer agent in the prevention or treatment of HCC.

Introduction

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, characterized by high mortality rate and poor prognosis (1). Lack of effective treatment options and late diagnosis are major reasons for the high mortality rate in HCC (2). A new therapeutic strategy to address these challenges cannot be overstated.

Green tea, compared with other teas, has a higher concentration of catechin. Epigallocatechin-3-gallate (EGCG) is a water-soluble catechin, which suppresses the multiplication of cancer cells and induces apoptosis (35). EGCG suppressed adhesion and invasion of hepatocarcinoma cells through antioxidant activity (6). EGCG inhibited the growth and metastasis of pancreatic cancer (7,8) and colorectal cancer (9), reduced the incidence of esophageal cancer (10) and improved the prognosis of breast cancer (11). It also inhibited the growth of HepG2 cells (12).

The EGCG anticancer effect is mediated by the PI3K/AKT signaling pathway. EGCG inhibited PI3K/AKT/mTOR signaling and promoted the apoptosis of B lymphocytes (5). It decreased the transcription of FoxO1 via the PI3K/AKT and MEK/ERK pathways in 3T3-L1 differentiation (13). EGCG inhibited PI3K/AKT activation, which enhanced apoptosis of T24 human bladder cancer cells (14).

The role of EGCG in proliferation or apoptosis via AKT pathway in HCC has yet to be reported. In this study, we reported on the ability of EGCG to inhibit cell growth in HepG2, SMMC7721 and SK-hep1 cell lines, in vitro. We also discovered that EGCG induced apoptosis and S phase arrest by affecting the PI3K/AKT pathway in SMMC7721 cells, in vitro.

Materials and methods

Cell lines and cell culture

All human hepatocellular carcinoma cell lines were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Science, China. HepG2, SMMC7721 and SK-hep1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Hyclone, USA) at 37°C in a humidified incubator containing 5% CO2. Cells were subcultured every two days when the density of cells reached 80%.

MTT assay

The cell growth inhibition effect of EGCG (purity ≥95%, Sigma-Aldrich, USA) was investigated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In brief, HepG2, SMMC7721 and SK-hep1 cells were seeded in 96-well culture plates at a density of 4×103 cells per well overnight. After culture in serum-free DMEM for 2 h, cells were treated with 0, 40, 80 and 120 μg/ml EGCG in DMEM with 10% FBS for 24, 48 or 72 h. To evaluate the cell viability, 20 μl MTT (5 μg/ml in culture medium, Sigma Chemical Co., USA) was added to each well and incubated at 37°C for 4 h. After removing the supernatants, 150 μl DMSO was added to each well. The optical density (OD) was measured at 492 nm using a microplate reader (Thermo Multiskan MK3, USA). The growth inhibition rate was calculated as follows: Growth inhibition rate (%) = [1 - (OD of treatment cells/OD of control cells)] × 100%.

Apoptosis assay

HepG2, SMMC7721 and SK-hep1 cells were seeded in 6-well plates at a density of 2×104 cells per well overnight. HepG2, SMMC7721 and SK-hep1 were then treated with EGCG at concentration of 74.7, 59.6 and 61.3 μg/ml respectively. Later, cells were stained with acridine orange (AO) and ethidium bromide (EB) using Normal/Apoptosis/Necrosis identification kit (KeyGen, China), following the manufacturer’s instructions. Apoptosis of HCC cells was observed under an IX51 inverted fluorescent microscope (Olympus, Japan).

Flow cytometry

SMMC7721 cells were treated with or without 59.6 μg/ml EGCG in 6-well culture plates for 48 h. Approximately 1×106 cells were collected, washed with 1X PBS and fixed with 70% ethanol at −20°C overnight. Cells were then centrifuged and washed with PBS three times, re-suspended in 100 μl RNase A, incubated at 37°C for 30 min, followed by staining with 400 μl propidium iodide (PI) staining solution (BD Sciences, USA) and incubated at 4°C in the dark. Finally, DNA contents in stained nuclei were analyzed with a flow cytometer (Beckman coulter Epics XL, USA).

Annexin V-FITC analysis

SMMC7721 cells were treated with or without 59.6 μg/ml EGCG for 48 h. Cells were then washed twice with PBS. Then they were stained with Annexin V and PI (BD Sciences) in binding buffer. Cells were then analyzed using flow cytometry (BD Caliber, USA).

RNA extraction and reverse transcriptase PCR

Total RNA was extracted from SMMC7721 cells treated with or without 59.6 μg/ml EGCG for 48 h, using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, USA) and quantified by NanoDrop2000 (Thermo Scientific, USA). The cDNA was generated from 0.5 μg total RNA using a ReverTra Ace® qPCR RT kit (Toyobo, Japan) following the manufacturer’s protocol and stored at −20°C.

Quantitative PCR

The mRNA levels of PI3K, AKT and NF-κB were determined by qPCR using GoTaq®qPCR Master Mix (Promega, USA) following the manufacturer’s instructions. The GAPDH mRNA was used as an internal control to normalize the amount of the above mRNA in each sample. The primers designed were as follows: GAPDH forward, 5′-AAG GTG AAG GTC GGA GTC AAC-3′, reverse: 5′-GGG GTC ATT GAT GGC AAC AAT A-3′; PI3K forward: 5′-TGG AAG CAG CAA CCG AAA C-3′, reverse: 5′-CAT TGA GGG AGT CGT TGT-3′; AKT forward: 5′-GGC AAG GTG ATC CTG GTG AA-3′, reverse: 5′-GGG ACA GGT GGA AGA ACA GC-3′; NF-κB forward: 5′-GTC ACT GCC CAG ACT TTA CT-3′, reverse: 5′-GCT TCT CCA CTG AAA ATC CT-3′. All assays were performed in triplicate and calculated on the basis of 2−ΔΔCt method.

Western blot analysis

SMMC7721 cells were pretreated with EGCG, LY294002 (Cell Signaling Technology, USA), IGF-1 (R&D Systems, USA) alone or with the combination of EGCG and LY294002 or IGF-1 for 48 h. Protein extracts were prepared by RIPA lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 1 mM Na3VO4] adding 0.5 μg/ml leupeptin, 1 mM PMSF and 10 mM NaF. Protein concentration was quantified by BCA kit (Beyotime, China) according to the manufacturer’s protocol. Proteins (40 μg) were electrophoresed on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Membranes were probed with antibodies to AKT, p-AKT (Ser473) and β-actin. All antibodies were purchased from Cell Signaling Technology. Immunoreactive bands were visualized using the DyLight®680 Conjugate-anti-rabbit IgG (Cell Signaling Technology). Bands were then scanned and analyzed by Odyssey two-color infrared imaging system (LI-COR, USA).

Statistical analysis

Differences between the two groups were analyzed with the Student’s t-test unless indicated otherwise. Results were considered statistically significant at a P<0.05.

Results

EGCG inhibits the growth of human hepatocellular carcinoma cells in vitro

To investigate the effect of EGCG on hepatocellular carcinoma cells, HepG2, SMMC7721 and SK-hep1 cells were treated with EGCG at different concentrations and time-points. The inhibition rate was calculated, based on the cell viability, determined by MTT assay. As shown in Fig. 1, the inhibition rate increased with the increase in concentration of EGCG across all three cell lines (Fig. 1). The cell growth inhibition rates at 48 h were significantly higher compared with rates at 24 h, at each concentration in all cell lines. However, the difference between rates at 48 and 72 h following treatment was not significant. As a result, we chose 48-h pretreatment in the following experiments. The half maximal (50%) inhibitory concentrations (IC50) of EGCG at 48 h for HepG2, SMMC7721 and SK-hep1 were 74.7, 59.6 and 61.3 μg/ml, respectively (Fig. 1).

EGCG induces apoptosis

We next measured apoptosis in the presence of EGCG, as apoptosis was closely related to cell growth. Each cell line was treated with EGCG at IC50 concentration for 48 h. The cell numbers of EGCG-treated group were significantly less than the control group (Fig. 2). Untreated cells (control) showed normal structure without prominent apoptosis and necrosis. The AO/EB staining indicated late apoptosis in 48-h incubated cells (Fig. 2), represented by orange staining. Flow cytometry also confirmed induction of apoptosis by EGCG in SMMC7721 cells. In EGCG-treated SMMC7721 cells, increased early and late apoptosis were observed (Fig. 3A). The total percentage of apoptosis (sum up of early stage and late stage) was 25.1±4.2% in EGCG-treated cells, compared with 9.0±2.7% in the non-treated control cells (Fig. 3B).

EGCG treatment arrests SMMC7721 cells at S phase

To further investigate the underlying mechanism, we analyzed distribution of the cell cycle by flow cytometry (Fig. 4). After 48-h treatment, the percentage of EGCG-treated SMMC7721 cells in S phase was significantly higher compared with non-treated cells (49.7±1.2 vs. 22.1±1.5%, Fig. 4). This result suggested that EGCG arrested SMMC7721 cells at S phase.

EGCG decreases the transcription of PI3K and AKT

We further investigated the role of AKT pathway mediating EGCG function in SMMC7721 cells. The mRNA level of AKT was quantified by qPCR (Fig. 5). The results showed that relative mRNA levels of PI3K and AKT decreased ∼31 and 29% respectively, after treatment with EGCG compared with control. NF-κB was the major downstream effector of AKT. The transcription of NF-κB was 43% lower in the SMMC7721 cells treated with EGCG than control cells (P<0.05).

EGCG downregulates the expression of AKT and its phosphorylation

EGCG downregulated the activity of AKT. We also verified the phosphorylation of AKT at Ser473 in SMMC7721 cells treated with EGCG using western blotting. As expected, the expression and phosphorylation of AKT were both reduced following treatment (Fig. 6).

Discussion

HCC is characterized by high mortality rate and poor prognosis (1). HepG2, SMMC7721 and SK-hep1 are classic cell lines for studying HCC. Studies have shown that EGCG induced tumor apoptosis, and prevented tumor invasion and metastasis (6,8,1517). We therefore, used these cell lines to investigate the effect of EGCG on inhibition of cell growth and the underlying mechanism. We observed that EGCG halted cell growth and induced apoptosis of SMMC7721 cells, which was also confirmed in other HCC cell lines (Figs. 13). Further, we observed that the IC50 of these three HCC cell lines at 48 h ranged from 60 to 75 μg/ml (Fig. 1), which was in accordance with previous studies (1719).

Progression through each phase of the cell cycle is regulated carefully to avoid proliferation under adverse conditions. Cells are arrested in G1, S and G2/M phases to prevent replication of damaged DNA or to prevent aberrant mitosis. Previous studies reported that EGCG blocked progression of the cell cycle at G1 phase in HCC lines (12), which is reportedly related to the activation of AMPK (12). However, we found that EGCG caused S phase but not G1 arrest in SMMC7721 cells (Fig. 4), which might be attributed to the unique features of HCC cell lines.

PI3K/AKT pathway was reported to play an important role in cancer proliferation and migration. Previous studies indicated the role of PI3K/AKT pathway in the development and progression of HCC cells, mainly reflected in the mechanism of liver cancer cell proliferation, differentiation and apoptosis (2022). The effect of EGCG on PI3K/AKT pathway in carcinogenesis, progression and metastasis in various type of tumors, have been widely studied in breast cancer (23), pancreatic cancer (8), endometrial cancer (24) and thyroid carcinoma (25). Here, we first detected the mRNA level of major components of AKT pathway, and found that EGCG downregulated the PI3K, AKT and NF-κB (Fig. 4). Phosphorylation at Ser473 and Thr308, AKT activates the downstream signaling molecules including the NF-κB and regulates cancer cell apoptosis (2628). Phosphorylation of AKT at Ser473 in SMMC7721 cells was reduced by EGCG (Fig. 5), which was consistent with a previous study of HepG2 cells, reporting that EGCG induced apoptosis and caused a decrease in the p-IGF-1R protein and its downstream signaling molecules including the p-ERK, p-Akt, p-Stat-3, and p-GSK-3β proteins (29).

EGCG affects both upstream and downstream targets of AKT. Previous reports showed that EGCG inhibited the thrombin-PAR1/PAR4-p42/p44 MAPKinase invasive signaling axis in hepatocellular carcinoma cells (30). HIF-1α/VEGF function was also a therapeutic target for EGCG in cancer chemoprevention and anticancer therapy (15,22). EGCG also delayed HCC cell growth through inhibition of Bcl-2 family (16,19) or induced apoptosis in HCC cells via downregulation of COX-2 and Bcl-2, and consequently activated caspase-9 and caspase-3 (31). The anti-metastatic effects of EGCG were associated with the inhibition of MMP-2 and MMP-9 activity (32,33).

In conclusion, our study provides evidence that EGCG inhibited HCC cell growth by affecting cell cycle and inducing apoptosis via downregulation of PI3K/AKT activity. The results suggested that EGCG is a potential anticancer agent in HCC therapy.

Acknowledgements

This study was supported by grants from the Youth Science Foundation of Guangxi Medical University (no. 02602211011) and Guangxi Natural Science Foundation (no. 2013GXNSFBA0191865).

References

1. 

Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar

2. 

Shek FH, Fatima S and Lee NP: Implications of the use of eukaryotic tanslation initiation factor 5A (eIF5A) for prognosis and treatment of hepatocellular carcinoma. Int J Hepatol. 2012:7609282012.PubMed/NCBI

3. 

Hagen RM, Chedea VS, Mintoff CP, Bowler E, Morse HR and Ladomery MR: Epigallocatechin-3-gallate promotes apoptosis and expression of the caspase 9a splice variant in PC3 prostate cancer cells. Int J Oncol. 43:194–200. 2013.PubMed/NCBI

4. 

Li JJ, Gu QH, Li M, Yang HP, Cao LM and Hu CP: Role of Ku70 and Bax in epigallocatechin-3-gallate-induced apoptosis of A549 cells in vivo. Oncol Lett. 5:101–106. 2013.PubMed/NCBI

5. 

Liu D, Li P, Song S, et al: Pro-apoptotic effect of epigallo-cate-chin-3-gallate on B lymphocytes through regulating BAFF/PI3K/Akt/mTOR signaling in rats with collagen-induced arthritis. Eur J Pharmacol. 690:214–225. 2012. View Article : Google Scholar : PubMed/NCBI

6. 

Zhang G, Miura Y and Yagasaki K: Suppression of adhesion and invasion of hepatoma cells in culture by tea compounds through antioxidative activity. Cancer Lett. 159:169–173. 2000. View Article : Google Scholar : PubMed/NCBI

7. 

Shankar S, Ganapathy S, Hingorani SR and Srivastava RK: EGCG inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer. Front Biosci. 13:440–452. 2008. View Article : Google Scholar : PubMed/NCBI

8. 

Shankar S, Marsh L and Srivastava RK: EGCG inhibits growth of human pancreatic tumors orthotopically implanted in Balb C nude mice through modulation of FKHRL1/FOXO3a and neuropilin. Mol Cell Biochem. 372:83–94. 2013. View Article : Google Scholar : PubMed/NCBI

9. 

Inaba H, Nagaoka Y, Kushima Y, et al: Comparative examination of anti-proliferative activities of (-)-epigallocatechin gallate and (--)-epigallocatechin against HCT116 colorectal carcinoma cells. Biol Pharm Bull. 31:79–84. 2008. View Article : Google Scholar : PubMed/NCBI

10. 

Li ZG, Shimada Y, Sato F, et al: Promotion effects of hot water on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in F344 rats. Oncol Rep. 10:421–426. 2003.PubMed/NCBI

11. 

Kushima Y, Iida K, Nagaoka Y, et al: Inhibitory effect of (-)-epigallocatechin and (-)-epigallocatechin gallate against heregulin beta1-induced migration/invasion of the MCF-7 breast carcinoma cell line. Biol Pharm Bull. 32:899–904. 2009. View Article : Google Scholar : PubMed/NCBI

12. 

Huang CH, Tsai SJ, Wang YJ, Pan MH, Kao JY and Way TD: EGCG inhibits protein synthesis, lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells. Mol Nutr Food Res. 53:1156–1165. 2009. View Article : Google Scholar

13. 

Kim H and Sakamoto K: (-)-Epigallocatechin gallate suppresses adipocyte differentiation through the MEK/ERK and PI3K/Akt pathways. Cell Biol Int. 36:147–153. 2012. View Article : Google Scholar : PubMed/NCBI

14. 

Qin J, Xie LP, Zheng XY, et al: A component of green tea, (-)-epigallocatechin-3-gallate, promotes apoptosis in T24 human bladder cancer cells via modulation of the PI3K/Akt pathway and Bcl-2 family proteins. Biochem Biophys Res Commun. 354:852–857. 2007. View Article : Google Scholar : PubMed/NCBI

15. 

Shirakami Y, Shimizu M, Adachi S, et al: (-)-Epigallocatechin gallate suppresses the growth of human hepatocellular carcinoma cells by inhibiting activation of the vascular endothelial growth factor-vascular endothelial growth factor receptor axis. Cancer Sci. 100:1957–1962. 2009. View Article : Google Scholar

16. 

Tsang WP and Kwok TT: Epigallocatechin gallate up-regulation of miR-16 and induction of apoptosis in human cancer cells. J Nutr Biochem. 21:140–146. 2010. View Article : Google Scholar : PubMed/NCBI

17. 

Uesato S, Kitagawa Y, Kamishimoto M, Kumagai A, Hori H and Nagasawa H: Inhibition of green tea catechins against the growth of cancerous human colon and hepatic epithelial cells. Cancer Lett. 170:41–44. 2001. View Article : Google Scholar : PubMed/NCBI

18. 

Abou El Naga RN, Azab SS, El-Demerdash E, Shaarawy S, El-Merzabani M and Ammar el SM: Sensitization of TRAIL-induced apoptosis in human hepatocellular carcinoma HepG2 cells by phytochemicals. Life Sci. 92:555–561. 2013.PubMed/NCBI

19. 

Nishikawa T, Nakajima T, Moriguchi M, et al: A green tea polyphenol, epigalocatechin-3-gallate, induces apoptosis of human hepatocellular carcinoma, possibly through inhibition of Bcl-2 family proteins. J Hepatol. 44:1074–1082. 2006. View Article : Google Scholar

20. 

Bu X, Jia F, Wang W, Guo X, Wu M and Wei L: Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma cells. BMC Cancer. 7:2082007. View Article : Google Scholar : PubMed/NCBI

21. 

Tang C, Lu YH, Xie JH, et al: Downregulation of survivin and activation of caspase-3 through the PI3K/Akt pathway in ursolic acid-induced HepG2 cell apoptosis. Anticancer Drugs. 20:249–258. 2009. View Article : Google Scholar : PubMed/NCBI

22. 

Zhang Q, Tang X, Lu Q, Zhang Z, Rao J and Le AD: Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia- and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Mol Cancer Ther. 5:1227–1238. 2006. View Article : Google Scholar

23. 

Zhang G, Wang Y, Zhang Y, et al: Anti-cancer activities of tea epigallocatechin-3-gallate in breast cancer patients under radiotherapy. Curr Mol Med. 12:163–176. 2012. View Article : Google Scholar : PubMed/NCBI

24. 

Park SB, Bae JW, Kim JM, Lee SG and Han M: Antiproliferative and apoptotic effect of epigallocatechin-3-gallate on Ishikawa cells is accompanied by sex steroid receptor downregulation. Int J Mol Med. 30:1211–1218. 2012.PubMed/NCBI

25. 

De Amicis F, Perri A, Vizza D, et al: Epigallocatechin gallate inhibits growth and epithelial-to-mesenchymal transition in human thyroid carcinoma cell lines. J Cell Physiol. 228:2054–2062. 2013.PubMed/NCBI

26. 

Dang TP: Notch, apoptosis and cancer. Adv Exp Med Biol. 727:199–209. 2012. View Article : Google Scholar : PubMed/NCBI

27. 

Datta SR, Brunet A and Greenberg ME: Cellular survival: a play in three Akts. Genes Dev. 13:2905–2927. 1999. View Article : Google Scholar : PubMed/NCBI

28. 

Roos WP and Kaina B: DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. 332:237–248. 2013. View Article : Google Scholar : PubMed/NCBI

29. 

Shimizu M, Shirakami Y, Sakai H, et al: EGCG inhibits activation of the insulin-like growth factor (IGF)/IGF-1 receptor axis in human hepatocellular carcinoma cells. Cancer Lett. 262:10–18. 2008. View Article : Google Scholar : PubMed/NCBI

30. 

Kaufmann R, Henklein P, Henklein P and Settmacher U: Green tea polyphenol epigallocatechin-3-gallate inhibits thrombin-induced hepatocellular carcinoma cell invasion and p42/p44-MAPKinase activation. Oncol Rep. 21:1261–1267. 2009. View Article : Google Scholar

31. 

Chen XL, Wang Q, Cao LQ, et al: Epigallocatechin-3-gallate induces apoptosis in human hepatocellular carcinoma cells. Zhonghua Yi Xue Za Zhi. 88:2524–2528. 2008.(In Chinese).

32. 

Roomi MW, Monterrey JC, Kalinovsky T, Rath M and Niedzwiecki A: Comparative effects of EGCG, green tea and a nutrient mixture on the patterns of MMP-2 and MMP-9 expression in cancer cell lines. Oncol Rep. 24:747–757. 2010.PubMed/NCBI

33. 

Zhang Y, Owusu L, Duan W, et al: Anti-metastatic and differential effects on protein expression of epigallocatechin-3-gallate in HCCLM6 hepatocellular carcinoma cells. Int J Mol Med. 32:959–964. 2013.PubMed/NCBI

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2014-March
Volume 44 Issue 3

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Spandidos Publications style
Shen X, Zhang Y, Feng Y, Zhang L, Li J, Xie Y and Luo X: Epigallocatechin-3-gallate inhibits cell growth, induces apoptosis and causes S phase arrest in hepatocellular carcinoma by suppressing the AKT pathway. Int J Oncol 44: 791-796, 2014
APA
Shen, X., Zhang, Y., Feng, Y., Zhang, L., Li, J., Xie, Y., & Luo, X. (2014). Epigallocatechin-3-gallate inhibits cell growth, induces apoptosis and causes S phase arrest in hepatocellular carcinoma by suppressing the AKT pathway. International Journal of Oncology, 44, 791-796. https://doi.org/10.3892/ijo.2014.2251
MLA
Shen, X., Zhang, Y., Feng, Y., Zhang, L., Li, J., Xie, Y., Luo, X."Epigallocatechin-3-gallate inhibits cell growth, induces apoptosis and causes S phase arrest in hepatocellular carcinoma by suppressing the AKT pathway". International Journal of Oncology 44.3 (2014): 791-796.
Chicago
Shen, X., Zhang, Y., Feng, Y., Zhang, L., Li, J., Xie, Y., Luo, X."Epigallocatechin-3-gallate inhibits cell growth, induces apoptosis and causes S phase arrest in hepatocellular carcinoma by suppressing the AKT pathway". International Journal of Oncology 44, no. 3 (2014): 791-796. https://doi.org/10.3892/ijo.2014.2251