Metabolomics of green tea catechins on vascular endothelial growth

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Metabolomics of green tea catechins on vascular endothelial growth factor-stimulated human endothelial cell survival Kai On Chu, Kwok Ping Chan, Sun On Chan, Tsz Kin Ng, Vishal Jhanji, Chi-Chiu Wang, and Chi Pui Pang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05998 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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Journal of Agricultural and Food Chemistry

Metabolomics of green tea catechins on vascular endothelial growth factor-stimulated human endothelial cell survival

Kai On Chu,a,b Kwok Ping Chan,a Sun On Chan,b Tsz Kin Ng,a Vishal Jhanji,a,# Chi Chiu Wang,b,c,d Chi Pui Pang.a,* a Department b

of Ophthalmology and Visual Sciences, the Chinese University of Hong Kong

School of Biomedical Sciences, the Chinese University of Hong Kong.

c Department d Li

of Obstetrics and Gynaecology, the Chinese University of Hong Kong

Ka Shing Institute of Health Science, the Chinese University of Hong Kong

#Current affiliation: Department of Ophthalmology, University of Pittsburgh School of Medicine, USA. *Correspondence: Prof. C. P. Pang (Tel: (852) 39433855; Fax: (852) 27159490; E-mail: [email protected])

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ABSTRACT

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Neovascularization causes serious oculopathy related to upregulation of vascular endothelial growth

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factor (VEGF) causing new capillary growth by endothelial cells. Green tea extract (GTE)

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constituents possess anti-angiogenesis properties. We used VEGF to induce human umbilical vein

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endothelial cells (HUVEC) and applied GTE, epigallocatechin gallate (EGCG), and different

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composition of purified catechins mixtures (M1 and M2) to evaluate the efficacy of inhibition and

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their underlying mechanisms using cell cycle analysis and untargeted metabolomics technique. GTE,

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EGCG, M1 and M2 induced HUVEC apoptosis by 22.1±2%, 20.0±0.7%, 50.7±8.5% and 69.8±4.1%

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respectively. GTE exerted a broad balanced metabolomics spectrum involving suppression of

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biosynthesis of cellular building blocks and oxidative phosphorylation metabolites but promoting

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biosynthesis of membrane lipids and growth factors. M2 mainly induced mechanisms associated with

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energy and biosynthesis suppression. Therefore, GTE exerted mechanisms involving both promotion

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and suppression activities, while purified catechins induced extensive apoptosis. GTE could be a

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more promising anti-neovascularization remedy for ocular treatment.

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KEYWORDS: metabolomics, green tea extract, catechins, HUVEC, apoptosis, proliferation


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INTRODUCTION

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Neovascularization is a common severe pathology in multiple eye diseases, including corneal

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neovascularization, age-related macular degeneration (AMD) and diabetic retinopathy that are often

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associated with vision decline or even blindness. Corneal neovascularization is the abnormal growth

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of capillaries blocking transmission, formation of corneal scar, lowering visual acuity, inflammation,

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and edema. Neovascular AMD is characterized by choroidal neovascularization in the macular

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region of the retina, which could be due to the oxidative damage of retinal pigment epithelial cells

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(RPE) and breakdown of Bruch’s membrane.1 Proliferative diabetic retinopathy (PDR) is associated

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with neovascularization in the vitreoretinal interface that eventually extends to the vitreous leading to

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visual loss as a result of tractional retinal detachment, vitreous hemorrhage, or neovascular

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glaucoma.2 One of the theories of neovascularization is the disequilibrium expression between

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pro-angiogenic and anti-angiogenic factors, which are related to upregulation of angiogenic factors

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including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and matrix

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metalloproteinases (MMP); or downregulation of anti-angiogenic factors like soluble VEGF

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receptor-2 (VEGFR-2), pigment epithelium derived factor (PEDF), angiostatin, and endostatin. Since

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VEGF has been shown to promote neovascularization, treatments antagonizing VEGF (anti-VEGF

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agents), including bevacizumab, ranibizumab, VEGF trap, siRNA and tyrosine kinase inhibitors,

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have been developed.3

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Catechins, particularly epigallocatechin gallate (EGCG), are the principal biologically active 3 ACS Paragon Plus Environment


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constituents in green tea extract (GTE) with anti-angiogenic,4 and anti-oxidative properties.5 They

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inhibit endothelial cell proliferation possibly through inhibition of VEGF receptor binding,6

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VEGFR-2 phosphorylation and expression, matrix metalloproteinase activity, gene expression,

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PI3-kinase activity, IL-8, and formation of receptor complex.7 EGCG ( 2, R2 > 0.6, Q2 >0.5.

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Markers were selected from S-Plot above 0.4 of the P(correlation), p < 0.05 by student t-test.

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Features with fold change more than 1.5 were chosen for further analysis.

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Metabolites were identified by accurate mass method. Metabolite mass (m/z) was searched

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through public database METLIN (http://metlin.scripps.edu/), HMDB (http://www.hmdb.ca/) with

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mass accuracy +/- 10 ppm with different adducts and confirmed by MS/MS fragmentation

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characteristics by public metabolomics database or commercial available standards. Some

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metabolites were further confirmed by GCMS with NIST database. The fold ratio was calculated as

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the intensity of the mass peak of treatment group divided by the intensity of the mass peak of VEGF

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group. When metabolites were highly expressed in the treatment group or minimal detected in the

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VEGF group, the fold changes can be very great. Then, the fold change was assigned as > 1000

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folds. On the other hand, when the metabolites were not detected in treatment group but detected in 10 ACS Paragon Plus Environment

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VEGF group, the fold change was assigned as < 0.01.

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Statistical analysis. The number of cells present in each phase of cell cycle was normalized by

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total gated cells in all phases as percentage. The percentages of each phase in each treatment were

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compared to that with VEGF treatment group by Dunnett’s t-test with the level of significance, p =

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0.05. Cell density was determined by the total cell count following gating divided by the volume

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used during flow cytometry analysis. Cell density in different treatment was compared with that in

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VEGF treatment Dunnett’s t-test. The fold changes of features/metabolites were calculated by the

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area response of treatment group divided by the area response of VEGF group. The area response of

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each feature in each treatment group was compared to that of VEGF group by Student t-test.

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RESULTS

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Cell cycle analysis showed that cell population in sub-G1 phase of negative control has higher

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percentage of population than VEGF group (8.0 ± 0.8%, p = 0.001) but lower in G0/G1 phase (69.6±

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1.3%, p = 0.02). No significant different of the percentage was found in S phase (6.7 ± 0.3%, p =

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0.25) but lower percentage in G2/M phase (15.7 ± 0.4%, p = 0.027) (Fig. 1a) (Table 1). A significant

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ascending trend of the cell populations in sub-G1 phase for GTE group (22.1 ± 2%, p = 8.8×10-5) ~

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EGCG group (20.0 ± 0.7%, p = 3.5×10-6) < M1 group (50.7 ± 8.5%, p = 6.7×10-4) < M2 group (69.8

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± 4.1%, p = 9.8×10-6), compared to the VEGF group (3.7 ± 0.3%). Bevacizumab treatment slightly

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increased in sub-G1 phase (4.2 ± 0.9%, p = 0.389). In contrast, a significant descending trend of cell 11 ACS Paragon Plus Environment


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population in G0/G1 phase was observed: GTE group (66.1 ± 2.0%, p = 0.006) ~ EGCG group (69.8

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± 1.4%, p = 0.031) > M1 group (41.5 ± 7.6%, p = 0.002) > M2 group (24.5 ± 4.2%, p = 3.9 ×10-5),

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compared to the VEGF group (72.4 ± 0.3%). Bevacizumab treatment showed only slight decrease in

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G0/G1 population (73.8 ± 0.7%, p = 0.033). On the other hand, significant reduction in S and G2/M

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phases was observed in the green tea catechin treatments (GTE: 5.7 ± 0.6%, p = 0.313 in S phase; 6.0

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± 0.5%, p = 3.5 × 10-5in G2/M phase; EGCG: 2.3 ±0.1%, p = 0.0001 in S phase; 8.0 ± 0.7%, p =

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0.0001 in G2/M phase; M1: 2.8 ± 0.3%, p = 0.0003 in S phase; 5.0 ± 0.7%, p = 3.7 ×10-5 in G2/M

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phase; and M2: 2.2 ± 0.2%, p = 0.0001 in S phase; 3.5 ± 0.3%, p = 1.1×10-5 in G2/M phase), but not

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bevacizumab treatment (6.4 ± 0.1%, p = 0.52 in S phase; 15.5 ± 0.1%, p = 0.02 in G2/M phase),

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compared to the VEGF treatment group (6.2 ± 0.4% in S phase; 17.6 ± 0.9% in G2/M phase;). (Table

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1) (Fig. 1a-b).

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Morphology of the HUVECs with vehicle treatment showed a typical cuboidal cell shape

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(Fig.1c). With VEGF treatment, morphology of HUVECs was changed into elongated shape, and

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treatment with bevacizumab suppressed the VEGF-induced transformation in HUVECs. Similar

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suppressions of VEGF-induced transformation in HUVECs were also observed in GTE and EGCG

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treatments. In contrast, in M1 and M2 treatments, the cell numbers were reduced and round shaped

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cells were observed, indicating that the HUVECs underwent apoptosis. We applied cell density to

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semi- quantitatively estimate the effect of various treatments on the cellular status.18 (McCaffrey et

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al., 1988). Flow cytometry showed VEGF treatment induced the highest cell density. All treatments 12 ACS Paragon Plus Environment

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reduced the density in respect to the VEGF group to different extents: a) GTE suppressed 15.6%, b)

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EGCG suppressed 13.2%, c) M1 suppressed 33.4%, d) M2 suppressed 49.6%, and e) Bevacizumab

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suppressed 4.6%. Similar pattern, but with higher extent of suppression, was found in the drug only

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control groups, A decreased trend of cell population appeared in catechin treatment groups,

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GTE~EGCG> M1> M2 in both treatment and control. Bevacizumab gave the lowest suppression

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(38.4%) (Fig. 1d).

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The reproducibility (coefficient of variance, CV) of retention times for the three metabolites

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biomarkers was 0.03 – 1.1%, n = 12. CV of signaling for the three selected metabolites from the

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quality control (QC) samples in each batch of analysis was within 7.5 – 23.0%. All the ions in the

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QC samples were within 30% CV.

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OPLS-DA can differentiate VEGF treatment from other treatments (Supplementary Material,

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Fig. S1) except the negative control which has no significant biomarker being found. Metabolomics

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profiles showed that many metabolites were differentially expressed under different treatments

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(Supplementary Material, Table S1). Basically, 7 groups of metabolites were differentially expressed

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viz. nucleotides, amino acids, vitamins and coenzymes, antioxidant, membrane lipids, sugars, and

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phenolic acids metabolites. GTE affected all types of metabolites expressions (Supplementary

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Material, Table S1a) involving 7 pathways: purine, pyrimidine, phenylalanine, vitamin B6, cysteine

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and methionine, pantothenate and CoA, and glycophospholipid metabolism pathways analyzed by

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Metaboanalyst and Metscape (Fig. 2a, Supplementary Material, Fig. S2-S3). EGCG also influenced 7 13 ACS Paragon Plus Environment


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groups of metabolites (Supplementary Material, Table S1b). It mainly affected 4 pathways, viz.

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vitamin B6, valine and leucine, tyrosine, and glycerophospholipid metabolism (Fig. 2b,

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Supplementary Material, Fig. S4-S5). M1 mixture had metabolites expressed from 7 groups of

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metabolites (Supplementary Material, Table S1c). It mainly affected three pathways, viz.

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glycosylphosphotidylinositol, riboflavin, and pantothenate and CoA metabolism pathways (Fig. 2c,

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Supplementary Material, Fig. S6-S7). M2 mixture affected 7 groups of metabolites though with

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lesser number of metabolites found (Supplementary Material, Table S1d). It affected pantotheate and

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CoA metabolism pathways only (Fig. 3d, Supplementary Material, Fig. S8-S9). Bevacizumab

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affected only 3 groups of metabolites viz. amino acid metabolites, vitamin B6, and membrane lipid

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metabolites (Supplementary Material, Table S1e). It affected phenylalanine, tyrosine, and tryptophan

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biosynthesis, and to a less extent the vitamin B6 metabolism pathway (Fig. 3e, Supplementary

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Material, Fig. S10-11,).

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DISCUSSION

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Bevacizumab inhibited the VEGF-induced transformation in HUVECs though preventing

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VEGF from binding to VEGF receptor.19 The present bevacizumab dosage range is commonly used

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in clinical intraocular treatment.9,20 The cell culture protocol and test conditions using 50 µM EGCG

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in this study were referenced to our previous study on RPE and HUVEC cells 9,in which the EGCG

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ranged from 10 – 100µM and HUVEC cell viability at 50µM was not disrupted. The green tea 14 ACS Paragon Plus Environment

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catechins mixture M1 were based on the composition of major catechins present in GTE while

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mixture M2 were based on the dominant catechins found in ocular compartments in another previous

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study.12 The present dose may be higher in comparing to physiological plasma concentration, but it

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could be useful for treatment. In fact, green tea extract eye drop (0.75% w/v), containing about 5 mM

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EGCG, has been used for clinical trial of dry eye treatment without causing adverse effects.21 In a

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study involving neutrophils, EGCG at concentrations > 3.7 µM produced lower caspase activity and

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at > 6.25 µM caused less DNA fragmentation.22 Also, 100 µM EGCG reduced DNA fragmentation

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and caspase activation in the neutrophils to 75% and 25% respectively. EGCG concentrations up to

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100 µM thus could protect against apoptosis and DNA fragmentation, and EGCG at 50 µM did not

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cause in vitro toxicity of cells. Notably, 400 µM EGCG was required to suppress VEGF binding to

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HUVECs by 90%8 and 25 µM of EGCG reduced neutrophil migration by more than 90%.

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studies had used up to 100 µM EGCG to test lung cancer cells23 and colon adenocarcinoma cells24

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without affecting viability and apoptosis. EGCG at 50 µM, based on functional migration assay,9 was

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within the pharmacological range in extra-cellular fluid in in vitro studies up to 100 µM.24,25, In an in

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vivo endometriosis model 50 mg/kg EGCG i.p. was administered to mice.26Assuming the weight of a

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mouse to be 20 g and the blood volume 1.2 mL, the Cmax concentration of EGCG could reach 2.3

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µM27, which was lower than the in vitro concentration. Unlike the physiological concentration in

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plasma, the pharmacological concentration of EGCG for ocular treatment can be higher.21,24,25

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21

Other

We used bevacizumab as a positive control because it specifically inhibits VEGF binding to 15 ACS Paragon Plus Environment


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VEGF receptor to block the proliferative stimulation. It has been used in our previous

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reported studies. In the present study, we attempted to demonstrate the multi-targeting

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anti-proliferative effects of GTE and catechins that are different from specific blocking action of

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VEGF. Affecting mechanisms involved in the cellular inhibitory activities, green tea catechins have a

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wider spectrum of anti-proliferation than specific anti-VEGF treatment by bevacizumab. Although

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EGCG was found effective in suppressing VEGF receptor-2 activation possibly through binding of

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VEGF,8 the molecular mechanisms of anti-angiogenesis of catechins should be more extensive due

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to its multi-targeting properties.

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and other

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The population in different phases by bevacizumab was similar to vehicle control implying it

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did not induce cell apoptosis and affect the cell cycle of HUVECs (Fig. 1a-b). On the other hand, the

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apoptotic effect was observed in GTE and other catechins treatments, as more cells were induced to

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the sub-G1 population (GTE ~ EGCG> M1 > M2) (Fig. 1c) although GTE and EGCG have

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substantially less potency. Despite similar in composition of major catechins between GTE and M1,

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M1 showed stronger apoptotic effect than GTE (Table 1) (Fig. 1b) as higher proportion of cells

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treated with GTE remaining in G0/G1 phase and less proportion was present in sub-G1 phase. It was

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not caused by high dose of catechin because the major catechins contents were the same. This may

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be due to the protective effect of the other constituents other than the catechins in GTE. Similar

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findings showed green tea extract and oolong extract with similar catechin composition exerted

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different anti-hyperlipidemic effects on rats.28 However, we have not collected direct evidences to 16 ACS Paragon Plus Environment

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rule out the apoptotic effects as demonstrated by GTE and EGCG were not associated with the

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relatively high dosage of 32.5 μg/mL GTE or 50 μM EGCG. Further investigations under more study

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conditions are warranted.

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We have previously reported EGCG level can affect oxidative stress, apoptotic, and

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anti-inflammatory potency to the eye.12 In this study, EGCG, the major catechin component of GTE,

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exerted similar effects on the phases as GTE indicating they has similar suppression effects. M2

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mixture, contained only EGCG, GCG, ECG, and GC, caused the highest apoptosis that indicated

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purified catechins mixtures could possess higher apoptotic potency. VEGF reduced the proportion of

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HUVEC in apoptosis but increased it in G0/G1 and G2/M phases comparing to the vehicle control;

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bevacizumab and vehicle control groups showed similar level of cell cycle (Table 1) (Fig. 1b);

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whereas the green tea catechin treatments showed reduction in G0/G1, S and G2/M phase but

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increase in sub-G1 phase indicating catechins have effects on arresting the cell cycle from division or

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induce apoptosis (Fig. 1b). Gallate derivatives of catechins reportedly promoted cancer cells to

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apoptosis.29 The apoptotic effect can be further supported by the trend of the cell density under

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different treatments, VEGF+GTE ~ VEGF+EGCG >VEGF+M1>VEGF+M2 (Fig. 1c-1d). Lower

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cell density suggests more cells undergoing apoptosis during incubation. Similar pattern but even

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lower density in the drug only controls is due to lack of promoting effects from VEGF. Bevacizumab

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reversed the cell to normal status without affecting the cell cycle. It suggests the inhibition

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mechanism of bevacizumab is different from that of green tea catechins. 17 ACS Paragon Plus Environment


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Metaboanalyst and Metscape analysis demonstrated different treatments produced different

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metabolomic profiles and pathways (Fig. 2-3). GTE suppressed metabolic pathways mainly

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associated with nucleotides, nucleotide sugars, amino acids, and pantothenic acid synthesis. But it

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activated the pathways related to vitamin B6, glycerophopholipids and related lipids, and

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antioxidants production (Supplementary Material, Fig. S2-S3). Suppressions of nucleotides and

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amino acids metabolism were associated with growth inhibition, but upregulation of vitamin B6 and

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glycerophospholipid metabolisms promoted cellular viability.30 Accordingly, GTE suppressed

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cellular energy production while maintaining cellular integrity and activity. Apparently

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contradictory, these metabolism pathways are interconnected (Supplementary Material, Fig. 2a). The

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resultant effect is to provide a balance in cellular environment. Metabolic profiling showed

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suppression of co-factors, vitamin B3,31 vitamin B5,32 and tetrahydro-L-biopterin33 and unregulated

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oxidative phosphorylation metabolites, increased D-glycerate phosphate and decreased succinic acid,

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resulting in inefficient mitochondrial oxidative phosphorylation that lead to growth inhibition and

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apoptosis34 (Supplementary Material, Table S1a). GTE also affected signaling for proliferation.

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Downregulation of D-4-O-Methyl-myo-inositol, which is a secondary messenger and mediator of

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phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) for lipid synthesis and cell

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proliferation, would inhibit proliferation. Flavonoids accumulating on the membrane surface can

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affect membrane binding.35 On the other hand, GTE upregulated vitamin B6 (Supplementary

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Material, Fig. S2b), and therefore reduced degradation of medium and long chain fatty acids. Thus, 18 ACS Paragon Plus Environment

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cell membranes are protected. GTE, as an antioxidant, also upregulated antioxidant precursors like

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methionine and cysteine,36 and L-ascorbic acid phosphate37 (Supplementary Material, Table S1a).

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Our findings therefore indicated balanced regulation of cellular proliferation and activities.

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EGCG shared some similar effects as by GTE (Fig. 2). It enhanced vitamin B6 expression (Fig.

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Supplementary Material, Fig. S4-S5) but suppressed glycerophospholipid pathway. Metabolic

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profiles of nucleotide metabolites showed elevations level of secondary messenger cyclic nucleotides

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(Supplementary Material, Table S1b). EGCG could increase cAMP level through 67 kDa laminin

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receptor activation38 and suppress ceramides, surface signaling molecules,39 causing proliferation

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inhibition. EGCG, as a pro-oxidant,13 elevated glutathione, amino acids like Ser-TrpOH, and fatty

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acids oxidation, and reduced organoseleno antioxidants, such as phosphoroselenoic acid and

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methylselenopyruvate. The prooxidation caused destruction of membrane resulting growth inhibition

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and even apoptosis.40 However, increased putrescine, which is involved cellular protection and

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proliferation,41 and elevation of vitamin B6 and NAD, which are related to growth promotion and

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redox protection, help to maintain cellular integrity. Also, increased riboflavin metabolite,

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5-Amino-6-(5'-phosphoribosylamino)-uracil, supports pyridoxine and NAD regeneration.42 Similar

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to GTE, EGCG also exerts both protective and inhibitory effects but through different pathways. It

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protects cells from damage possibly through expression of vitamin B6, NAD, B2 and putrescine but

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induce growth inhibition or even apoptosis by pro-oxidation and suppression of membrane signaling

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molecules. 19 ACS Paragon Plus Environment


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The purified catechin mixture M1 contains major catechins of GTE but it exhibits different

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metabolomics profile. Unlike GTE, M1 suppressed both vitamins B2 and B5 pathways without

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affecting vitamin B6 (Supplementary Material, Fig. S6-S7). Since vitamins B2 and B5 are essential

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for maintaining normal oxidative phosphorylation, fatty acids and glutathione production, their

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suppression may affect cell integrity.43 Downregulation of glycosylphosphatidylinositol (GPI),

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ceramide and choline could affect cell signaling leading to quiescence and even cause apoptosis.

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Depression of nucleoside metabolites, 7-methylinosine and 2-aminoadenosine, indicates decreased

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DNA replication and ATP production44 (Supplementary Material, Table S1a). Downregulated

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methylselenopyruvate, metabolite of Se-methyl-selencystine, indicates prooxidation.45

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upregulated oxidized and degraded amino acids and fatty acids metabolites also indicated stress and

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structure destruction. Although M1 has similar catechins composition as GTE, its action should be

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mainly demoting biological repairing activities. It explains why M1 caused more serious apoptosis

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and growth inhibition.

Many

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Catechin mixture M2 contained EGCG, GC, GCG, and ECG. Suppression of pantothenic acid

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pathway suggests reduction of oxidative phosphorylation (Supplementary Material, Fig. S8-S9).

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Metabolic profiling revealed decreased adenosine and increased catabolites of purine, inosine and

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hypoxanthine, xanthosine 5'-pyrophosphate, implying retarded DNA synthesis and even

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degradation.46 In addition, elevation of oxidized amino acids catabolites, guanidinosuccinic acid and

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asp-ser-OH, also indicated extensive protein destruction. Besides, guanidinosuccinic acid is a uremic 20 ACS Paragon Plus Environment

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toxin.47 Antioxidants, methylselenopyruvate, and pantothenic acid and triphosphoric acid decrease

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supports oxidative stress and suppressed energy production. It is notable that polyphenol metabolites

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were found in the cell but none of them was intact catechin molecule. In fact, some studies indicated

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chronic effects of EGCG were due to its metabolites instead of EGCG.48 However, intact catechin

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molecules may be metabolized after exerting their biological effect.

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Bevacizumab mainly suppressed biosynthesis of amino acids, mainly phenylalanine, tyrosine,

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and tryptophan, rather than nucleotide metabolism. It may have a minor suppression on vitamin B6

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metabolism (Supplementary Material, Fig. S10-S11). It also increases polyunsaturated fatty acids

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expression that may alter the membrane structure to prevent regulatory kinase from binding to

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initiate proliferation.49 The metabolic profiles obtained in this study showed large metabolic

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regulatory differences caused by green tea catechins.

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In summary, different catechin mixtures exhibit different metabolic profiles although they

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contain same amount of EGCG. The biological effects of catechin mixtures are related to the balance

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of inhibitory and promoting activities (Fig. 4). GTE contains multiple components including

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dominant catechins and many minor phenolic substances. Its cellular effects involve both promotion

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and suppression of cellular activities. EGCG promoted cellular activities by enhancing vitamin B6,

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but it cause deterioration of membrane lipids and signaling possibly through pro-oxidation. EGCG

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and GTE exhibited similar effects on cell cycle but the inhibition mechanisms are different. EGCG

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caused membrane lipids degradation and pro-oxidation with little effect on nucleotide and energy 21 ACS Paragon Plus Environment


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production. M1 has similar catechins composition as GTE but gave different metabolomics profiles.

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It suppressed vitamin B2, signaling molecules, DNA synthesis, energy production, and membrane

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lipid production; induced pro-oxidation resulting extensive apoptosis and proliferation inhibition.

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Some promotion activities found in EGCG may be counteracted by other catechins whereas other

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constituents in GTE would neutralize the counteracting effect. Therefore, GTE constituents other

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than catechins play a crucial role in maintaining homeostasis. M2 exerted higher destructive and

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stressed effects, and induced strong apoptotic potency through reduction of nucleotide biosynthesis,

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energy production, and inducing oxidative stress. Since these catechins are dominantly present in

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ocular tissues following GTE administration,10 giving purified mixture may cause serious side effects

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due to endothelial cell apoptosis. In order to give a similar strength of anti-proliferation as

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bevacizumab, the present GTE preparations were tested as potential candidate for ocular

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neovascularization treatment rather than purified EGCG and catechin mixtures due to its moderate

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apoptotic effects and balanced homeostasis according to the cell cycle and metabolic mechanisms.

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Further work on more combinations, different controls and in vivo studies are warranted.

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ACKNOWLEDGMENT

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The green tea extract, Theaphenon® E, was kindly supplied by Prof. Yukihiko Hara from Tea

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Solutions, Hara Office Inc. (Shizuoka, Japan).

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CONFLICT OF INTEREST

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We have no conflict of interest in this study. 22 ACS Paragon Plus Environment

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SUPPORTING INFORMATION

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Metabolomics analyzed by OPLS-DA (Fig. S1), pathways analysis following different treatments

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(Fig. S2 – Fig. S11), and metabolomics profiles influenced by different treatments (Table S1).



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FUNDING This work was supported by a block grant of the University Grants Committee Hong Kong; a Health 28 ACS Paragon Plus Environment

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and Medical Research Fund (Project No. 12130791 to T.K.N. and 12130811 to C.P.P) and a Research Grant Council General Research Fund to S.O.C. (Project No.: CUHK14113815).



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FIGURE CAPTIONS Fig. 1 Cell cycle populations after different treatments. (a) Cytometric histogram in different phase of cell cycle following different treatment on VEGF induced HUVEC by flow cytometry. Zone A: apoptosis; B: Go/G1; C: S phase; D: G2/M. (b) Comparison of percentage of cell population in different phase of cell cycle following different treatment on VEGF induced HUVEC by flow cytometry. Zone A: apoptosis; B: Go/G1; C: S phase; D: G2/M. (c) Photographs show the shape and population of the cells under different treatments. (d) The chart shows cells density under different treatments. Vehicle CTL: negative control, cells treated with vehicle only; VEGF: cells treated with VEGF only; VEGF+GTE: cells treated with GTE with VEGF; VEGF+EGCG: cells treated with EGCG with VEGF; VEGF+M1: cells treated with M1 mixture (8 purified catechins with similar composition to GTE) with VEGF; VEGF+M2: cells treated with M2 mixture (4 catechins – GC, EGCG, GCG, and ECG with composition according to GTE) with VEGF; VEGF+Bevacizumab: cells treated with 312.5 µg/mL Bevacizumab with VEGF; GTE: cells treated with GTE only; EGCG: cells treated with EGCG only; M1: cells treated with M1 only; M2: cells treated with M2 mixture only; Bevacizumab: cells treated with 312.5µg/mL Bevacizumab only. Cell population by VEGF treatment was compared with each treatment by student t-test. * - p < 0.05.

Fig. 2 Significant pathways from Metaboanalyst were verified by Metscape following bevacizumab treatment on VEGF induced HUVEC. Diagrams show the affected (a) vitamin B6 metabolism 30 ACS Paragon Plus Environment

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biosynthesis, and (b) tyrosine metabolism pathways. Large hexagon indicates highly expressed. Small hexagon indicates under expressed.

Fig. 3 Metabolites are analyzed and identified by Metaboanalyst. Red and green colour box show significant matched pathways according to p values by pathway enrichment analysis and pathway impact values by pathway topology analysis. Red colour indicates upregulation of the pathway while green colour indicates down-regulation as verified by Metscape. Yellow colour indicates the pathway not found in the treatment.

Fig. 4 Schematic diagram showing the promotion and suppression of biological activities of various pathways by different catechins mixtures.



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Table 1. Percentage of Population of HUVEC in Different Cell Cycle Phase under Different Treatments. Vehicle CTL – negative control with vehicle only; VEGF – cells treated with 20 ng/mL VEGF; VEGF+GTE – cells treated with VEGF and GTE; VEGF+EGCG – cells treated with VEGF and EGCG at the same level as GTE; VEGF+M1 – cells treated with VEGF and eight purified catechin compounds at the same level as GTE; VEGF+M2 – cells treated with VEGF and four purified catechin compounds at the same levels as GTE; and VEGF+Avastin – cells treated with VEGF and bevacizumab at 312.5 μg/mL. The percentage are shown as mean±standard derivation, n = 6. * - significant difference comparing to VEGF treatment, p

Metabolomics of green tea catechins on vascular endothelial growth

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