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. 2018 Nov 14;9(11):5682-5696.
doi: 10.1039/c8fo01397g.

Suppressing glucose metabolism with epigallocatechin-3-gallate (EGCG) reduces breast cancer cell growth in preclinical models

Ran Wei  1 , Limin Mao, Ping Xu, Xinghai Zheng, Robert M Hackman, Gerardo G Mackenzie, Yuefei Wang
Affiliations

Suppressing glucose metabolism with epigallocatechin-3-gallate (EGCG) reduces breast cancer cell growth in preclinical models

Ran Wei et al. Food Funct. .

Abstract

Numerous studies propose that epigallocatechin-3-gallate (EGCG), an abundant polyphenol in green tea, has anti-cancer properties. However, its mechanism of action in breast cancer remains unclear. This study investigated the capacity of EGCG to suppress breast cancer cell growth in vitro and in vivo, characterizing the underlying mechanisms, focusing on the effect of EGCG on glucose metabolism. EGCG reduced breast cancer 4T1 cell growth in a concentration- (10-320 μM) and time- (12-48 h) dependent manner. EGCG induced breast cancer apoptotic cell death at 24 h, as evidenced by annexin V/PI, caspase 3, caspase 8 and caspase 9 activation. Furthermore, EGCG affected the expression of 16 apoptosis-related genes, and promoted mitochondrial depolarization. EGCG induced autophagy concentration-dependently in 4T1 cells by modulating the levels of the autophagy-related proteins Beclin1, ATG5 and LC3B. Moreover, EGCG affected glucose, lactate and ATP levels. Mechanistically, EGCG significantly inhibited the activities and mRNA levels of the glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), and lactic dehydrogenase (LDH), and to a lesser extent the activity of pyruvate kinase (PK). In addition, EGCG decreased the expression of hypoxia-inducible factor 1α (HIF1α) and glucose transporter 1 (GLUT1), critical players in regulating glycolysis. In vivo, EGCG reduced breast tumor weight in a dose-dependent manner, reduced glucose and lactic acid levels and reduced the expression of the vascular endothelial growth factor (VEGF). In conclusion, EGCG exerts an anti-tumor effect through the inhibition of key enzymes that participate in the glycolytic pathway and the suppression of glucose metabolism.

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Conflicts of interest

There are no conflicts to declare.

Figures

Figure 1:
Figure 1:. EGCG inhibited 4T1 breast cancer cells growth.
A: EGCG reduced 4T1 cell growth in a time- and concentration-dependent manner. Cell viability was determined in 4T1 cells after 12, 24 and 48 h of incubation with EGCG. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control). B: Differential cytotoxic effect in 4T1 cells compared with that of mouse breast normal epithelial cells (HC11) after treatment with escalating concentrations of EGCG for 24 h. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control).
Figure 2:
Figure 2:. EGCG induced cell death by apoptosis in 4T1 breast cancer cells.
A: 4T1 cells treated with EGCG for 24 h and 48 h were stained with Annexin V/propidium iodide, and the percentage of apoptotic cells was determined by flow cytometry. B: Results are expressed as the mean ± SD. (*p<0.05, **p<0.01 vs. control). C: EGCG activated caspase activity in 4T1 cells. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control). D: EGCG promoted mitochondrial depolarization after 24 h incubation. Representative images (x400) of 4T1 cells treated with either vehicle (control) or EGCG and stained for JC-1 fluorescence.
Figure 3:
Figure 3:. EGCG affected the expression of apoptosis-related proteins.
Microarray heat map of differentially expressed genes (as indicated) levels within control cells or cells treated with EGCG at various concentrations (20–240 μM) for 24 h. Heat map results are shown as the mean fold changes (log2) of the relative mRNA expression level for each protein (2−△△t).
Figure 4:
Figure 4:. EGCG affected autophagy in 4T1 breast cancer cells.
A: 4T1 cells lysates were analyzed for Beclin1, ATG5 and LC3B by immunoblotting. Loading control: β-actin. Bands were quantified and results are expressed as percentage of control. (*p<0.05, **p<0.01 vs. control). B: Representative images (x400) of 4T1 cells treated with either vehicle (control) or EGCG and stained for mCherry-GFP-LC3B. C: Autophagosome formation was depicted using transmission electron microscope in 4T1 cells treated with or without EGCG for 24 h. Representative images (x8000, x30000) are shown.
Figure 5:
Figure 5:. Effect of EGCG on glucose uptake, lactic acid levels and ATP production.
A: EGCG decreased the uptake of glucose. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control). B: EGCG reduced lactic acid levels. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control). C: EGCG reduced intracellular ATP levels in 4T1 cells. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control). D: Immunoblots of HIF1α and GLUT1 in 4T1 cell protein extracts incubated with or without EGCG for 24 h. Loading control: β-actin. Bands were quantified and results are expressed as percent control (*p<0.05, **p<0.01 vs. control).
Figure 6:
Figure 6:. EGCG inhibited the activity and the mRNA expression of glycolysis-related enzymes.
A: Enzymatic activity of HK, PFK, PK and LDH was determined in 4T1 cells treated with EGCG for up to 24 h. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control). B: mRNA expression of HK2, PFK1, PKM2 and LDHA was measured by qPCR in 4T1 cells treated with EGCG. Results are expressed as percent control. (*p<0.05, **p<0.01 vs. control).
Figure 7:
Figure 7:. EGCG inhibited the growth of breast cancer in Balb/c mice.
A: Effect of EGCG on tumor weight at sacrifice. Results are expressed as the mean ± SD (*p<0.05, **p<0.01 vs. control). B: Mice body weight over time for control and EGCG treated groups. Results are expressed as the mean ± SD. C: H&E staining of isolated tumors. Representative images (x100, x400) of tumor tissues treated with either vehicle (control) or EGCG and stained for H&E. D: Immunofluorescence staining for VEGF expression of tumor tissues. Representative images (×200) of tumor tissues treated with or without EGCG and stained for VEGF. E: Glucose and lactic acid levels in tumors. Results are expressed as the mean ± SD. (*p<0.05, **p<0.01 vs. control).
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