5-hydroxy-equol against hepatocellular carcinoma cells in

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1H-NMR-based metabolomics approach reveals metabolic mechanism of (-)-5-hydroxy-equol against hepatocellular carcinoma cells in vitro Li Gao, Ke-xin Wang, Nan-nan Zhang, Jia-qi Li, Xue-mei Qin, and Xiu-ling Wang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00853 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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Journal of Proteome Research

1

H-NMR-based metabolomics approach reveals metabolic

mechanism of (− −)-5-hydroxy-equol against hepatocellular carcinoma cells in vitro Li Gao, ‡*a Ke-xin Wang, ‡a, c Nan-nan Zhang, ‡b Jia-qi Li, a, c Xue-mei Qin,a Xiu-ling Wang b* a

Modern Research Center for Traditional Chinese Medicine, Shanxi University,

Taiyuan 030006, PR China. b

c

College of Life Sciences, Hebei Agricultural University, Baoding 071001, PR China.

College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,

China.

Note: ‡ Li Gao, Ke-xin Wang and Nan-nan Zhang contributed equally to this work.

To whom correspondence should be addressed: Li Gao* (E-mail: [email protected]); Tel & Fax: 86-351-7018379; Address: No.92 Wu Cheng Road, Taiyuan 030006, China Xiu-ling Wang* (E-mail: [email protected]); Tel: +86-312-7528259, Fax: +86-312-7528265; Address: Baoding 071001, PR China

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ABSTRACT 1

H-NMR-based metabolomics can rapidly detect metabolic shift under various

stimulus, thus it facilitated the dissection of the therapeutic mechanisms of compounds. (−)-5-hydroxy-equol is an isoflavone metabolite that be obtained by microbial biotransformation. In the current work, the effect of (‒)-5-hydroxy-equol on hepatocellular carcinoma cells and its mechanism have been explored based on 1

H-NMR-based

metabolomics

approach.

Our

results

revealed

that

(−)-5-hydroxy-equol can significantly inhibit the proliferation, migration and invasion of SMMC-7721 cells, and inhibit the proliferation of HepG2 cells. Metabolomics revealed that 17 differential metabolites involving in amino acid metabolism and energy metabolism were significantly changed inside and outside of the cells after treatment of (−)-5-hydroxy-equol. Specifically, (−)-5-hydroxy-equol at concentration of 30 µM significantly decreased the concentrations of pyruvate, glutamate and glucose. As glycometabolism is a crucial feature of cancer-specific metabolism, we further verified enzymes and proteins that closely relevant to glycometabolism. Our results indicated that (−)-5-hydroxy-equol modulated glycolysis in HCC through inhibition of activities of hexokinase, phosphofructokinase and pyruvate kinase, and the expression of pyruvate kinase M2. This study revealed that metabolomic analysis integrating with further verifications at the biochemical level can facilitate understanding the anti-HCC mechanisms of (−)-5-hydroxy-equol. Keywords: hepatocellular carcinoma / metabolomics / NMR / (‒)-5-hydroxy-equol / glycolysis

2


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INTRODUCTION Hepatocellular carcinoma (HCC) is one of the 10 most prevalent malignancies in the world, and it is ranked second in relation to mortality in digestive system malignancies.1-3 The delayed diagnosis and rapid expansion of tumor lead to a poor prognosis and high mortality of HCC patients.4, 5 The only drug approved by the FDA for the treatment of HCC is sorafenib.6 Evidence demonstrates that metabolic disorders are involved in the occurrence and development of HCC.7, 8 Several proteins and enzymes including hexokinase (HK), pyruvate kinase (PK) and pyruvate kinase M2 (PKM2) have been reported to be highly expressed in HCC.9-12 Metabolic reprogramming of tumor cells has been considered as a novel strategy for cancer therapy.13 Metabolomics can quantitatively analyze all metabolites in the organism, and provide a systematic platform that reflects the physiological or pathological phenotypes of cells after external disturbances.14 Nuclear magnetic resonance (NMR) allows samples to retain non-selective and non-destructive information in metabolomic studies, and it was regarded as a preferred platform for qualitative and quantitative analysis of metabolites.15 Recently, it has been demonstrated that inhibition of aerobic glycolysis is implicated in the anti-tumor effect of melatonin.16 Isoflavones present in soy have attracted considerable attention due to multiple biological activities, such as a reduced risk of cancer.17-19 However, soy isoflavones are extensively metabolized in the intestine before absorption. Currently, more attention has shifted to isoflavone metabolites which can exert stronger 3



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anticarcinogenic activities than that of their precursor isoflavones. Equol, a metabolite of

isoflavone

daidzein,

can

exert

significant

antiproliferative

effect

on

hormone-related canc er cell lines20-22 as well as HCC23. Recently, we isolated a cock intestinal

bacterium,

which

converts

isoflavone

genistein

into

100%

(−)-5-hydroxy-equol.24 Although numerous studies have suggested the strong anticarcinogenic activity of equol, however, the effect of (‒)-5-hydroxy-equol on tumor cells and its metabolism mechanism has not yet been determined. In the present work, the anti-HCC mechanism of (‒)-5-hydroxy-equol were explored by applying 1H-NMR-based metabolomic approach. In addition, the changes of metabolites, enzymes and protein were further verified. Our study provided new insights into the metabolic mechanism of (‒)-5-hydroxy-equol for its anti-HCC effect. MATERIALS AND METHODS Materials (‒)-5-hydroxy-equol was obtained by using our previously established microbial bioconversion methods.24 High glucose DMEM (4.5 g/L), fetal bovine serum (FBS), 0.25% trypsin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). D2O was purchased from Norell (Landisville, NJ, USA). Sodium 3-trimethlysilyl [2,2,3,3-d4] propionate (TSP) was purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA). Primary antibodies for β-actin and PKM2 were obtained from Proteintech Group Co,. Ltd, (Chicago, Illinois, USA). The commercial kits for measuring pyruvate (PA), glutamate (Glu) content and PK 4


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activities were obtained from Suzhou Comin Biotechnology Co,. Ltd (Suzhou, China). The Elisa kits (48T×1) for measuring Human Glucose, Hexokinase (HK), Phosphofructokinase (PFK) activities were obtained from Andy Gene Biotechnology Co., Ltd (Beijing, China). Cell culture and treatment Human hepatocellular carcinoma SMMC-7721 cells were donated by Prof. Xiongzhi Wu (Tianjin Medical University Cancer Institute and Hospital, China). The cells were maintained in DMEM medium containing 10% FBS. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. When SMMC-7721 cells reached 80% confluency, the cells were treated with different concentrations of (‒)-5-hydroxy-equol. Cells and culture supernatant were collected after 24 h incubation for future analyses. Cell viability assay SMMC-7721 cells and HepG2 cells (5×104 cells/mL) were harvested and seeded in 96-well plates. After 24 h incubation, cells were exposed to different concentrations of (‒)-5-hydroxy-equol. After 24 h, 10 µL of MTT solution (5 mg/mL) was added and incubated for 4 hours, then the formazan crystals were dissolved in 100 µL of DMSO. The absorbance was measured by an Infinite M200 pro microplate reader (Tecan, Switzerland) at 570 nm. Wound-Healing Assay SMMC-7721 cells (5×104 cells/mL) were seeded at 6-well plates. When monolayers formed following overnight culture, the center of wound was created by 5



scratching with a sterile 10 µL pipette tip. After a wash in PBS, 1.5 mL of (‒)-5-hydroxy-equol (0, 20, 30 and 40 µM) were added, then cultured for 12, 24 or 36 h. The wound closures were photographed by inverted fluorescent microscope (Nikon, Ti-S, Japan), and were digitalized using NIS-Elements software. Cell Invasion Assay According to the literature25 with slight modification, the cell invasion assay was employed by using a Boyden chamber with an 8-µm micropore membrane in 24-well plate, and the filter membranes were coated with Matrigel (Coring, MA, USA). Then, 2 × 105 cells were seeded in the upper part of each chamber in 200 µL DMEM supplemented with 0.1% FBS, and 600 µL of (‒)-5-hydroxy-equol (0, 20, 30 and 40 µM, 10% FBS) were added to the lower compartments. After incubation for 36 h, non-invading cells were removed from membrane top layer. Afterwards, the invaded cells were fixed on the bottom of the membrane, and then stained with 0.1% (w/v) crystal violet. The representative fields were phtographed under an inverted microscope at 100× magnification. Cell collection for NMR analysis According to the results of cell experiments, we found that 30 µM (‒)-5-hydroxy-equol could significantly inhibit the proliferation, migration and invasion of SMMC-7721 cells (p＜0.001). Consequently, after treatment with 30 µM (‒)-5-hydroxy-equol for 24 h, cells were harvested by scraping and rinsed 2 times. The mixture was centrifuged at 1000 r/min for 5 min. The cells (3×107～4×107 cells per sample) were collected and frozen in liquid nitrogen to quench metabolic 6


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reactions. 10 mL of extracellular medium were centrifuged to collect supernatant, and the supernatant was frozen in liquid nitrogen to identify extracellular metabolites. Samples preparation for NMR analysis Samples were prepared according to the literature

26

with slight adjustments.

Cells and culture supernatant were thawed in 4 °C. 2 mL of culture supernatant was taken for freeze-drying. For freeze–thaw cycle and ultrasonic disruption extraction, the cells were repeated freeze-thaw for 5 times. Next, we took chilled 1:2 methanol/H2O quenching solvent (1 mL) to the cell pellets for ultrasonic disruption using an ultrasonic processor for 5 min (sonicate 5 s, stop 9 s). After centrifuging at 13000 r/min for 10 minutes, the supernatant was collected and methanol aqueous solution (1 mL) was joined to the precipitate. The above steps were repeated, and the final supernatant was lyophilized. The lyophilized powders of cells and culture supernatants were joined to PBS containing 0.005% and 0.02% TSP, respectively, as well as 10% D2O. The precipitates were removed by centrifuging at 4 °C for 10 min at 13,000 r/min, and the supernatant was used for NMR measurement. NMR analysis and data preprocessing The 1H-NMR spectra of cells and culture supernatant were acquired on a Bruker 600-MHz AVANCE III NMR spectrometer (Bruker, Germany). A Nuclear Overhauser Effect

Spectroscopy

(NOESY,

RD–901–t1–901–tm–901–acquire) with

water

suppression was set for the samples analysis, and following parameters were used: spectral width 12,345.7 Hz, 65,536 spectral size points, 64 scans, and 1.0 s relaxation 7



delay. The spectra were processed using MestReNova software (version 10.0.1, Mestrelab Research, Santiago de Compostella, Spain). The baseline and phase pretreatment were set manually and the chemical shift of TSP was referenced at δ 0.00 ppm. Region (δ 4.7～5.0) distorted due to residual water was excluded. The spectral regions of δ 0.60 to 9.00 ppm were segmented at 0.01 ppm intervals and all data points were normalized for the further analysis. Multivariate data analysis The software package SIMCA-P version 13.0 (Umetrics AB, Umeå, Sweden) was performed for multivariate pattern recognition analysis. Principal component analysis (PCA) was performed on matrices of spectra from the mean-centered data to observe possible outliers. The model qualities of partial least squares discrimination analysis (PLS-DA) were assessed according to the parameters of model fitness (R2) and predictive ability (Q2) in a supervised manner followed with permutation tests (200 cycles). Orthogonal projection to latent structures discriminant analysis (OPLS-DA) was further performed using Pareto scaling. Then, the S-plot in which each point represents a metabolite signal was used to screen biomarkers. Besides, Variable importance in the projection (VIP) values together with t-test were performed to select potential biomarkers. Pathway analysis MetPA was used to analyze the potential metabolic pathway. MetaboAnalyst, an enrichment analysis tool, was utilized for assessment of potential biological roles. 8


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Metscape27

(http://metscape.ncibi.org./),

a

metabolic

network

analysis

and

visualization tool, was used to construct the metabolic network. Content determination of representative metabolites and PK 5×106 cells were added to 1 mL of the extract, and subjected to ultrasonic disruption. The suspension was subjected to centrifuge for 10 min at 8 000 r/min at room temperature, and the supernatant was collected for measurement. The contents of PA, Glu and PK activity were measured according to the manufacturer’s instructions and previous reports28. The detection principle of metabolites was based on extraction with a specific extract solution, then it reacted with chromogenic agent and the absorbance was measured. The detection of PK was based on the following principle: PK catalyzes the phosphoenolpyruvate (PEP) and ADP to generate pyruvate and ATP. Furtherly, lactate dehydrogenase catalyzes NADH and pyruvate to produce lactate and NAD+. The decrease of NADH can reflect PK activity. Enzyme-linked immunosorbent assay (ELISA) The kits were balanced 15-30 min in the room temperature. Ten standard wells were set, and 50 µL standard solution was added into each of the blank micro-wells, then testing sample was added. After closing plate with closure plate membrane, the enzyme marker plates were incubated for 30 min at 37 °C and then quickly washed 5 times. Enzyme marker solution 50 µL was added into each well (excluding the blank well). Finally, the absorbance was detected at 450 nm after 50 µL stop solution was added to stop the reaction. Western blot assay 9



Exponentially growing SMMC-7721 cells were harvested by trypsinization. After centrifugation, the pellet was resuspended in RIPA lysis buffer containing 1% PMSF. Equal amounts of protein (50 µg) were subjected to 10% SDS-PAGE for separation and electrophoretically transferred onto PVDF membranes. Then the membranes were incubated with the primary antibodies PKM2 (dilution 1: 400) and β-actin (dilution 1: 1000) overnight at 4 °C. After incubation with fluorescent secondary antibody, fluorescent scanner was used to detect the signals of protein. Statistical Analysis Results were presented as mean ± SEM from at least three independent repetitions. One-way ANOVA followed by Dunnett post hoc test was used to compare more than two groupss, and t-test was used to compare the control and treatment groups. Obtained p-values were corrected by Benjamini-Hochberg false discovery rate (FDR).29 Results were considered to be statistically significant if p value < 0.05. RESULTS (‒)-5-hydroxy-equol inhibits the proliferation of SMMC-7721 cells and HepG2 cells To explore whether (‒)-5-hydroxy-equol had antiproliferative effects on SMMC-7721 cells and HepG2 cells, the cells were treated with different concentrations of (‒)-5-hydroxy-equol. The results of MTT assay showed that (‒)-5-hydroxy-equol significantly inhibited the proliferation of SMMC-7721 cells and HepG2 cells in a dose-dependent manner. After 24 h incubation, the cell viabilities of SMMC-7721 cells were 94.47 %, 82.44 %, 70.02 %, 59.51 % and 54.92 % after 10


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exposure to (‒)-5-hydroxy-equol at concentrations of 10, 20, 30, 40 and 50 µM, respectively (Fig. 1A), and the cell viabilities of HepG2 cells were 92.64 %, 86.68 %, 78.98 %, 68.59 % and 58.92 % after exposure to (‒)-5-hydroxy-equol at concentrations of 10, 20, 30, 40 and 50 µM, respectively (Fig. 1B). (‒)-5-hydroxy-equol inhibits the migration and invasion of SMMC-7721 cells The effects of (‒)-5-hydroxy-equol on migration and invasion in SMMC-7721 cells were investigated. By using wound-healing assay, the results showed that (‒)-5-hydroxy-equol at 20, 30 and 40 µM significantly suppressed the migration of SMMC-7721 cells at 12, 24, and 36 h (Fig. 2). By using cell invasion assay, we found that 30 and 40 µM (‒)-5-hydroxy-equol dramatically inhibited the invasion of SMMC-7721 cells (Fig. 3). These results reveal that (‒)-5-hydroxy-equol displayed significant inhibitory activities on the migration and invasion of SMMC-7721 cells. Metabolomic study of SMMC-7721 cells Typical 1H-NMR spectra of cell and culture supernatant from the control and (‒)-5-hydroxy-equol treatment group were shown in Fig. 4. The metabolites were identified by matching the chemical shifts from the Chenomx NMR suite, and those recorded in literature, Biological Magnetic Resonance Data Bank (BMRB), and the Human Metabolome Database (HMDB) (Table S-1). A total of 41 endogenous metabolites (cell: 37, culture supernatant: 22) were obtained and subjected to further multivariate pattern recognition analysis. Some amino acids, a range of amines, choline, organic acids, together with other metabolites were contained in the spectra. (‒)-5-hydroxy-equol induced metabolomic changes in SMMC-7721 cells and

11



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culture supernatant Multivariate pattern recognition analysis was conducted to obtain changes in metabolites after treatment with (‒)-5-hydroxy-equol. A clear separation was observed between the control and (‒)-5-hydroxy-equol groups in PCA scatter plot, which was generated by PC1 (77.4 %), PC2 (12.7 %) in the cell and PC1 (75.8 %), PC2 (10.3 %) in the culture supernatant (Fig. 5A, F). As shown in Fig. 5 B, G, the PLS-DA models (cell: R2X = 0.629, Q2 = 0.981; culture supernatant: R2X = 0.734, Q2 = 0.983) indicate an excellent predictive ability in which R2 and Q2 values from the permutation model are lower than original ones, and the Q2 line intercepts a negative value at the Y axis. Then supervised statistical method OPLS-DA was further used to determine the potential biomarkers associated with (‒)-5-hydroxy-equol treatment (Fig. 5C, H). The corresponding S-plot (Fig. 5D, I), in combination with variable importance in the projection (VIP) (Fig. 5E, J) were utilized for seeking some endogenous metabolites contributing to the separation. Then independent t-test was also used to compare the statistical significance of these metabolites. Compared with control group, the abundance levels of 12 metabolites were significantly changed in cells after treatment with (‒)-5-hydroxy-equol, including higher levels of glycerophosphocholine, ethanolamine, taurine, fumarate, leucine, acetate and lower levels of pyruvate, glutamate, glutamine, adenosine monophosphate, creatine, glycine; 5 metabolites were

significantly

changed

in

culture

supernatant

after

treatment

with

(‒)-5-hydroxy-equol, including higher levels of dimethylamine and lower levels of isoleucine, lactate, phosphocholine and taurine. As a result, these metabolites could be 12


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significantly regulated and the metabolic disorders were ameliorated in SMMC-7721 cells after (‒)-5-hydroxy-equol treatment (Fig. 6, Table 1). Correlation analysis between differential metabolites Spearman’s correlation analysis was performed to explore the relationships between differential metabolites after (‒)-5-hydroxy-equol treatment. Combined with Table S1, it was found that there was a strong positive correlation among glutamine, glycine, isoleucine and dimethylamine, but had a strong negative correlation with ethanolamine and C-taurine. The positive correlation among ethanolamine, C-taurine and PCc is high (Fig. 7). Metabolic pathway analysis Pathway enrichment analysis was performed for differential metabolites between (‒)-5-hydroxy-equol treatment and control group. The results of the MetPA pathway analysis (Fig. 8A) were consistent with the metabolite enrichment results (Fig. 8B). The analysis results of 24 pathways were obtained from MetPA. Combined with the Holm P value, FDR (false discovery rate) and Impact value, we found that 5 metabolic pathways were significantly affected by (‒)-5-hydroxy-equol including pyruvate metabolism, taurine and hypotaurine metabolism, d-glutamine and d-glutamate metabolism, glycine, serine and threonine metabolism and alanine, aspartate and glutamate metabolism. The metabolite network was constructed by Metscape to better understand the relationship between differential biomarkers (Fig. 9). Effects of (‒)-5-hydroxy-equol on the levels of pyruvate and glutamate 13



In order to identify the changes of metabolites between control and (‒)-5-hydroxy-equol groups, 2 metabolites were selected for content determination. Pyruvate participates in 5 enriched pathways including pyruvate metabolism, taurine and hypotaurine metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism and arginine and proline metabolism. Glutamate participates in d-glutamine and d-glutamate metabolism. As shown in Fig. 10 A, B, the results of the metabolic analysis were consistent with content determination, which suggested that (‒)-5-hydroxy-equol can significantly decrease the levels of pyruvate and glutamate in the cell. Effects of (‒)-5-hydroxy-equol on the glucose level Metabolomics analysis reveals that (‒)-5-hydroxy-equol significantly affected the glycometabolism, therefore the mechanism of (‒)-5-hydroxy-equol on glycolysis pathway was further examined. The results suggested that glucose level was significantly reduced after treatment with (‒)-5-hydroxy-equol compared with the control group (Fig. 10 C). Effects of (‒)-5-hydroxy-equol on the HK, PFK and PK activities The metabolic status of tumor cells was different from the corresponding normal cells. The high level of glycolysis ensures the survival and proliferation of tumor cells. The activities of HK, PFK and PK were further evaluated. As shown in Fig. 11 A, C, we found that levels of HK and PK were significantly reduced after (‒)-5-hydroxy-equol treatment. Furthermore, the activity of PFK decreased following exposure to 30 µM (‒)-5-hydroxy-equol, but no significant difference was observed 14


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(Fig. 11 B). Effects of (‒)-5-hydroxy-equol on the expression of PKM2 In order to further explore the mechanism of (‒)-5-hydroxy-equol in hepatoma tumorigenesis, we determined the expression of key protein PKM2 in glycolysis pathway. The results showed that (‒)-5-hydroxy-equol treatment significantly down-regulated the expression of PKM2 (Fig. 12). DISCUSSION In contrast to (−)-5-hydroxy-equol (the microbial metabolite of genistein), (−)-equol (the microbial metabolite of daidzein) differs from (−)-5-hydroxy-equol only in the lack of a single hydroxy group at the C5 position in ring A of the phenylbenzopyrone structure. Numerous studies have demonstrated that equol can exert strong anticarcinogenic activities.20-22 Our previous study also showed that equol induced apoptosis of HCC cells (SMMC-7721) through both the intrinsic and the endoplasmic reticulum stress pathway.23 However, the underlying mechanisms of (‒)-5-hydroxy-equol resistance to HCC were still a riddle. Metabolomics measures metabolic changes after a given stimulus, thus it is helpful for unravelling drug mechanisms.30 However, few studies were focused on quantitative determination of these metabolites. We confirmed the consistency of the content determination and metabolic analysis results by testing the same metabolite independently, and further verified enzymes and proteins that closely related to metabolic process. In our research, we found that (‒)-5-hydroxy-equol could significantly inhibit the 15



proliferation, migration and invasion of SMMC-7721 cells, and inhibit the proliferation of HepG2 cells. Metabolomic results revealed that (‒)-5-hydroxy-equol significantly regulated the metabolites levels in SMMC-7721 cells and culture supernatant. A set of metabolic pathways affected by (‒)-5-hydroxy-equol were discovered, which include pyruvate metabolism, taurine and hypotaurine metabolism, d-glutamine and d-glutamate metabolism and other metabolic pathways. Changes in amino acid metabolism Increasing evidence suggests the correlations between amino acid metabolic disorders and tumor.31, 32 Amino acids play a vital role for building blocks of proteins and as intermediates in metabolism.33 Hepatoma has the characteristics of rapid proliferation and fast metabolism, which requires a large number of amino acids to synthesize nucleic acids and proteins.31 Tumor cells has a strong demand for energy, which leads to a high rate of glutamate uptake.34 Therefore, glutamate level was high in normal hepatoma cells, whereas intracellular glutamate levels were significantly decreased after (-)-5-hydroxy equol treatment. Changes in energy metabolism In the course of cell proliferation and survival, the maintenance of energy was necessary. Glycine plays a vital role in energy storage. Studies have shown that the consumption of glycine and the expression of proteins in the mitochondrial glycine biosynthetic pathway are closely related to the rapid proliferation of cancer cells.35, 36 After treatment with (‒)-5-hydroxy-equol, the glycine level was significantly decreased, which suggests that (‒)-5-hydroxy-equol attenuates energy metabolic 16


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disorders in HCC. Fumarate, an important intermediate of TCA cycle, is a metabolite associated with energy metabolism.37 The accumulation of fumarate can cause epithelial-to-mesenchymal-transition (EMT), which is related with the occurrence, invasion and metastasis of cancer.38 It is worth to noticing that we strangely observed a significant increase in fumarate, an important intermediate in TCA cycle, after treatment with (‒)-5-hydroxy-equol. While the changes in other TCA intermediates have not been observed. We surmised that the accumulated fumarate was not mainly from succinic acid catalyzed by succinate dehydrogenase in TCA cycle. Some other metabolic pathways might be involved in the accumulation of fumarate. For example, in urea cycle, fumarate can be biosynthesized from argininosuccinate by argininosuccinase.39 The definite reason needs to be explored further. Changes in glycometabolism The rapid conversion of pyruvate into lactate and alanine was necessary for fast glucose consumption by tumors, which was known as aerobic glycolysis or the Warburg effect.18, 31 Warburg effect is a highlighted characteristic for tumor-specific metabolism.30, 40, 41 Given that tumor cells display special roles in glycometabolism and they relies on glycolysis to obtain energy and sustain survival, it is of great significance to discuss the effect of (‒)-5-hydroxy-equol on glycometabolism. Our study has shown that (‒)-5-hydroxy-equol group had significantly lower levels of pyruvate, a glycolysis product, as compared with control group. Pyruvate is one of the most critical intermediate metabolites, and its reduction is clearly detrimental to the proliferation of tumors. Tumor cells can be supplemented by extracellular uptake of 17



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lactate and alanine into pyruvate. However, after treatment with (‒)-5-hydroxy-equol, the level of lactate was significantly decreased, indicating that (‒)-5-hydroxy-equol might play anti-HCC role by blocking the glycolysis through regulation of intermediate metabolites including pyruvate and lactate. Some studies have shown that active intervention in glycolysis levels determines the fate of tumor cells. 42, 43 Compared with normal cells, cancer cells partially ferment glucose into lactate even without hypoxia to support their rapid proliferation and expansion.44 Since tumor cells rely on glycolysis for survival and proliferation, effective interference with this process is a potentially effective strategy for killing cancer cells.45 The metabolomics results suggest that (‒)-5-hydroxy-equol could ameliorate disturbed glycometabolism. To further explore the potential mechanisms of (‒)-5-hydroxy-equol on glycolysis, the level of glucose, the activities of relevant enzyme HK, PFK and PK and the expression of PKM2 were determined. The first step in glucose metabolism is the transportation of glucose into the cell through glucose

transporters

glucose-6-phosphate

(GLUTs). (G-6-P)

In by

cells, HK.

glucose Next,

is

G-6-P

phosphorylated

to

is

to

converted

fructose-6-phosphate (F-6-P) by phosphoglucose-isomerase (PGI). PFK is a kinase that catalyzes the ATP-dependent phosphorylation of F-6-P.46 PK, an enzyme involved in glycolysis, catalyzes phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP, and the large amount of pyruvate converted to lactate, resulting in increased level of lactate.10 The PKM2 is important for cancer metabolism, which was overexpressed in cancers.47 After treatment with (‒)-5-hydroxy-equol, the glucose level, HK and PK 18


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activities and PKM2 expression were significantly decreased. Therefore, these results further reveal that (‒)-5-hydroxy-equol decreased the glycolysis level in hepatoma cells. Based on the metabolites in the 5 metabolic pathways regulated by (‒)-5-hydroxy-equol, we further reconstructed related metabolic network by utilization of metscape. The network revealed that (‒)-5-hydroxy-equol exerted anti-HCC effect might through acting on multiple metabolites and enzymes. CONCLUSION In this research, the anti-HCC effect of (‒)-5-hydroxy-equol and its metabolic mechanism was investigated. The results revealed that (‒)-5-hydroxy-equol evidently inhibited the proliferation, migration and invasion of SMMC-7721 cells, and inhibited the proliferation of HepG2 cells. 1H-NMR based-metabolomic results suggest that (‒)-5-hydroxy-equol could regulate amino acid metabolism, glycometabolism and energy metabolism. Content determination suggested that (‒)-5-hydroxy-equol significantly decreased the levels of pyruvate and glutamate, which was consistent with metabolomics results. Additionally, (‒)-5-hydroxy-equol could significantly decrease the level of glucose, the activities of HK, PFK and PK and PKM2 expression involved in glycolysis. The results would provide the scientific basis for elucidation of the metabolic mechanisms of (‒)-5-hydroxy-equol. ASSOCIATED CONTENT Supporting information The supporting information associated with this article is available free of charge, in 19



the online version, ACS website http://pubs.acs.org: Table S-1. Chemical shift and multiplicity of identified metabolites from SMMC-7721 cells (C) and culture supernatant (S). Author Contributions L.G. and X.-l.W. designed the experiments. K.-x.W. and N.-n.Z. performed the experimental work and wrote the manuscript. L.G., X.-l.W. and J.-q.L. provided oversight. L.G., X.-l.W. revised the manuscript. Acknowledgments This project was supported by Applied and Fundamental Research Plan in Hebei Province of China (No. 16962504D), Base Program of Joint training graduate student of Shanxi Province (No. 2016JD05), Science and Technology Innovation Team of Shanxi Province (No. 201605D131045-18), and Key laboratory of Effective Substances Research and

Utilization in TCM

of Shanxi province (No.

201705D111008-21). Conflict of Interest The authors have no conflicts of interest, either real or potential, associated with this work. References 1.

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FIGURE

Figure 1. Effect of (‒)-5-hydroxy-equol on viabilities of SMMC-7721 cells (A) and HepG2 cells (B). All values are expressed as mean ± SEM (n=6). *p

5-hydroxy-equol against hepatocellular carcinoma cells in

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