Astragalin Reduces Hexokinase 2 through Increasing miR-125b to

Jun 27, 2017 - Astragalin (ASG) can be found in a variety of food components. ASG exhibits cytotoxic effects on several different types of malignant c...
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Astragalin Reduces Hexokinase 2 through Increasing miR-125b to Inhibit the Proliferation of Hepatocellular Carcinoma Cells in Vitro and in Vivo Wei Li,†,‡ Ji Hao,†,‡ Lang Zhang,†,‡ Zhuo Cheng,†,‡ Xukun Deng,†,‡ and Guangwen Shu*,†,‡ †

School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, China 430074 National Demonstration Center for Experimental Ethnopharmacology Education, South-Central University for Nationalities, Wuhan, China 430074

‡

ABSTRACT: Astragalin (ASG) can be found in a variety of food components. ASG exhibits cytotoxic effects on several different types of malignant cells. However, its effects on hepatocellular carcinoma (HCC) cells and the underlying molecular mechanisms have remained to be fully elucidated. Here, we revealed that ASG remarkably suppressed the proliferation of HCC cells. In HCC cells, ASG inhibited glucose glycolysis and promoted oxidative phosphorylation, resulting in a surge of reactive oxygen species (ROS). Mechanistically, ASG suppressed the expression of hexokinase 2 (HK2). This event was indispensible for ASG-mediated metabolic reprogramming, ROS accumulation, and subsequent growth arrest. Our further investigations unveiled that ASG repressed HK2 expression via increasing miR-125b. In vivo experiments showed that gavage of ASG decreased the proliferation of Huh-7 HCC xenografts in nude mice and inhibited the growth of transplanted H22 HCC cells in Kunming mice. Declined HCC tumor growth in vivo was associated with boosted miR-125b and reduced expression of HK2 in tumor tissues. Collectively, our results demonstrated that ASG is able to suppress the proliferation of HCC cells both in vitro and in vivo. Inhibition of HK2 through upregulating miR-125b and subsequent metabolic reprogramming is implicated in the antiproliferative effects of ASG on HCC cells. KEYWORDS: astragalin, hepatocellular carcinoma, hexokinase 2, MiR-125b, reactive oxygen species

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INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common diagnosed malignant diseases and the third leading cause of cancer-related mortality worldwide.1 Surgical resection and liver transplantation are standard treatments of HCC. However, these approaches are only eligible for HCC patients in the early stages.2 The long-term prognosis of HCC is still dismal. Thus, searching for novel therapeutic options against HCC is imperative. Reprogramming of glucose catabolism from oxidative phosphorylation (OXPHOS) to aerobic glycolysis is one of the most prevalent and important features of HCC.3 In other words, even in the presence of sufficient oxygen, HCC cells prefer to produce ATP by glycolysis, giving rise to increased glucose uptake and lactate production. A series of previous studies demonstrated that aerobic glycolysis plays important roles in the survival, proliferation, and metastasis of HCC cells.4−6 The literature suggested that interference with the altered glucose metabolism can be a feasible strategy in the clinical management of HCC. Hexokinase (HK) catalyzes the first and irreversible step of glycolysis. Four isozymes of HK, known as HK1, 2, 3, and 4, have been identified in mammals until now.7 It has been well established for a long time that among these isozymes, HK2 is the major contributor to the metabolic alteration in HCC.8 Epidemiological survey displayed that levels of HK2 in clinical HCC tissues were positively associated with stages of this disease.9 Overexpression of HK2 was also reported as an independent predictor for the poor prognosis of HCC.10 By reducing HK2, resveratrol, a dietary polyphenol from grapes, © 2017 American Chemical Society

berries, peanuts, and other food sources, provokes apoptosis in HCC cells in vitro and inhibits their proliferation in mice.11 Similarly, a recent literature revealed that chrysin, a flavonoid widely exists in fruits, propolis, and honey, triggers HCC cell apoptosis by targeting HK2.12 Therefore, it is reasonable to consider HK2 to be a potential molecular target in the antiHCC therapy. Recently, active compounds from functional foods have received more and more concerns as a rich resource soil of potential anticancer drugs.13−16 Astragalin (ASG) is a natural product widely present in food components, such as persimmon leaves, mulberries, and lotus leaves. ASG is able to alleviate oxidative stress and inflammation.17,18 Cytotoxic effects of ASG on diverse types of cancer cells cultured in vitro can be detected.19 Astragalin heptaacetate, a derivative of ASG, was shown to impose cytotoxic impacts on human leukemia cells.20 However, effects of ASG on HCC cells and the molecular mechanisms through which ASG exerts its impacts have remained to be fully elucidated. Here, we unveiled that ASG inhibits the proliferation of HCC cells both in vitro and in vivo. ASG reduces the expression of HK2 in HCC cells, giving rise to inhibited glycolysis, upregulated OXPHOS, and accumulated reactive oxygen species (ROS). Our further studies demonstrated Received: Revised: Accepted: Published: 5961

May 6, 2017 June 26, 2017 June 27, 2017 June 27, 2017 DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

Article

Journal of Agricultural and Food Chemistry

Figure 1. ASG inhibited HCC cell proliferation in vitro. (A) HepG2, Huh-7, and H22 HCC cells were treated by various concentrations of ASG. At the indicated time periods, viabilities of these cells were detected. (B) HL-7702 untransformed hepatocytes were treated by various concentrations of ASG. Cell viability was determined at the indicated time periods. (C−E) HCC cells were treated as indicated for 3 days and then subjected to BrdU cell proliferation assay (C) and FACS. Both representative FACS diagrams (D) and statistical results (E) were shown. *p < 0.05; **p < 0.01 versus vehicle treatment. been compared in previous literature.21 According to the expression patterns of marker genes, Huh-7 cells belong to the cholangiocellular type with expression of CK7/19, β-catenin, and CD34; HepG2 cells belong to a classical HCC type expressing CK18/19 and β-catenin. When implanted subcutaneously in immunocompromised mice, levels of Ki-67, a marker of proliferating cells, in HepG2 xenografts are higher than those in Huh-7 xenografts. H22 HCC cells are capable of growing subcutaneously in Kunming mice with intact immune system. HepG2 and Huh-7 cells stably overexpressing HK2 were produced by a lentiviral gene transduction system.22 Anit-miR-125b was transfected by Lipofectamine 2000 following the manufacturer’s instructions. MTT Cell Viability Assay and BrdU Cell Proliferation Assay. MTT cell viability assay was performed as described previously.23 Cell proliferation was determined by using the BrdU cell proliferation assay kit purchased from Cell Signaling Technology (Danvers, MA) according to the manufacturer’s instructions. Determination of Cell Cycle Distribution by Flow Cytometry Analysis (FACS). HCC cells were subjected to the indicated treatment. Then, cells were trypsinized, fixed by 70% ethanol, and incubated with RNaseA to remove intracellular RNA. Finally, samples were stained with 25 μg/mL propidium iodide and analyzed by a Becton-Dickinson FACSCalibur Flow Cytometer Instruction (Franklin Lakes, NJ).

that ASG suppresses the expression of HK2 by boosting microRNA-125b (miR-125b).

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MATERIALS AND METHODS

Reagents and Antibodies. ASG (>98% pure) was from Herbpurify Co., LTD (Chengdu, Sichuan, China). Dulbecco’s modified Eagle medium (DMEM) was from Hyclone (Logan, UT). Fetal calf serum (FCS), penicillin, streptomycin, and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). Matrigel for xenograft tumor transplantation was from BD Biosciences (Bedford, MA). Dichloro-dihydro-fluorescein diacetate (DCFH-DA), N-acetyl-L-cysteine (NAC), and methyl thiazolyl tetrazolium (MTT) were from Beyotime Biotechnology (Nantong, Jiangsu, China). Anti-miR-125b was bought from RiboBio Co., LTD (Guangzhou, Guangdong, China). Primary antibodies against HK2, Ki67, and β-actin were from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase-conjugated secondary antibodies were from Proteintech Group (Wuhan, Hubei, China). Cell Culture and Transfection. Human HepG2 HCC cells, Huh-7 HCC cells, HL-7702 untransformed hepatocytes, and murine H22 ascitic hepatoma cells were cultured in DMEM supplied with FCS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL). All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The characters of HepG2 and Huh-7 HCC cell lines have 5962

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Journal of Agricultural and Food Chemistry

Figure 2. ASG induced metabolic reprogramming in HCC cells. (A,B) HepG2, Huh-7, and H22 HCC cells were incubated with 11 and 33 μM ASG for 2 days. Then, rates of glucose consumption (A) and lactate production (B) were determined. (C,D) Enzymatic activities of complex I (C) and IV (D) in cell lysate were detected. (E) The indicated HCC cells were incubated with 11 and 33 μM ASG. After 2 days, cells were further incubated with 50 ng/mL oligomysin, and intercellular levels of ATP were detected at the indicated time periods. **p < 0.01 versus vehicle treatment. Determination of Reactive Oxygen Species (ROS) and Malondialdehyde (MDA). Levels of ROS in cultured HCC cells were quantified by DCFH-DA staining followed by FACS as described previously.24 Levels of ROS in transplanted HCC tumor tissues were quantified by fluorescence spectrophotometry analysis as described previously.25 Levels of MDA were quantified by the assay kit purchased from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Cell Metabolic Analysis. Rate of glucose consumption and lactate production were determined by using the methods as described previously.26 Levels of glucose and lactate were measured by kits bought from Sigma-Aldrich (Stockholm, Sweden). Enzymatic activities of mitochondrial complex I and IV were measured by kits from Abcam (Cambridge, UK). ATP contents were quantified by the kit from Beyotime Biotechnology (Nantong, Jiangsu, China). Enzymatic activity of HK was measured by the HK activity assay kit from SigmaAldrich (Stockholm, Sweden). Immunoblotting and Immunohistochemical Examination. Immunoblotting analysis was performed as described previously.27 Briefly, cells were homogenized in cell lysis buffer (Beyotime, Jiangsu, China). Then, protein samples (30 μg per lane) were separated by SDS-PAGE and transferred to PVDF membranes. After blotting with corresponding primary and secondary antibodies, protein bands on the membrane were visualized by enhanced chemiluminescence. The density values of immunoreactive bands were analyzed by ImageJ software. The density value for each protein was normalized to that for β-actin and expressed as the fold increase relative to the control. Immunohistochemical examination was performed as described previously.28 RNA Extraction, Reverse Transcription, and Real-Time PCR. Quantification of mRNA was performed as described previously.29 Primers sequences are listed as follows (5′ to 3′). HK2 (human): forward,

GAG CCA CCA CTC ACC CTA CT; reverse, CCA GGC ATT CGG CAA TGT G. GAPDH (human): forward, GGA GCG AGA TCC CTC CAA AAT; reverse, GCC ATC ACG CCA CAG TTT C. HK2 (mouse): forward, CTA AGG GGT TCA AGT CCA GTG G; reverse, AGA CCA ATC TCG CAG TTC TGA. GAPDH (mouse): forward, GCA GTG GCA AAG TGG AGA TT; reverse, GGA GAC AAC CTG GTC CTC AG. Levels of miR-125b was detected by stem-loop real-time PCR as described previously,30 using U6 snRNA as an internal control. Animals and Animal Experimentations. Male Kunming mice and athymic nude mice (6-week old, 18−22 g) were bought from the Center of Experimental Animals, Institute of Health and Epidemic Prevention (Wuhan, Hubei, China). Animals were maintained under standard specific pathogen free conditions and had free access to sterilized food and water ad libitum. All of the animal experimental procedures in our current study had been approved by the Experimental Animal Care and Use Committee of South-Central University for Nationalities (Permission number: Scuec-aec-013). To establish the xenograft tumor model in nude mice, 1 × 106 of Huh-7 HCC cells in 0.2 mL of serum-free DMEM containing 50% Matrigel were subcutaneously transplanted into the dorsal flanks of animals. Tumor-bearing nude mice were randomly divided into groups. Each group contained 5 mice. When volumes of xenograft tumors reached approximately 200 mm3, ASG was gavaged at the indicated dosages once a day, using the vehicle (0.9% NaCl) as the negative control. Tumor volumes were monitored every 3 days by the formula: tumor volume = 0.5 × length × width × width. Drug treatment lasted for 3 weeks. To establish the in vivo HCC cell proliferation model in Kunming mice, 1 × 106 of H22 cells were subcutaneously injected into the dorsal flanks of the animals. Tumor-bearing mice were randomly divided into groups, with 5 mice for each group. Drug administrated 5963

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Figure 3. Accumulation of ROS is essential for ASG-induced growth arrest in HCC cells. (A−C) HepG2, Huh-7, and H22 HCC cells were treated with 11 and 33 μM ASG for 2 days. Then, levels of ROS were determined by FACS. Both representative FACS diagrams (A) and statistical results (B) were shown. Levels of MDA in cell lysates were also detected (C). (D,E) HCC cells were treated as indicated for 3 days and then subjected to cell viability assay (D) and BrdU cell proliferation assay (E). **p < 0.01 versus vehicle treatment. started 24 h later and lasted for 10 days. ASG was gavaged at the indicated dosages once a day. The vehicle group received 0.9% NaCl. Tumor volumes were monitored every 2 days. At the end of animal experimentations, mice were anesthetized by inhalation of diethyl ether and subjected to blood collection. Then, all of the animals were euthanized. The livers and xenograft tumors were resected immediately and subjected to further analysis. Determination of Parameters of Liver Functions. Liver weight index was calculated by the following formula: weight index = organ weight (g)/body weight (g) × 1000. Serums were separated from blood by centrifugation at 5000g for 10 min. Activities of aspartate aminotransferase (ALT) and alanine aminotransferase (AST) in the serum were measured by an automatic biochemical analyzer (Sysmex, Japan). Statistical Analysis. Data shown here are either representatives or statistics (mean value ± standard deviation) of results from at least 3 independent experiments or all of the mice from each group. Statistical significance of the data was analyzed by one-way analysis of variance (ANOVA). Differences were considered as statistically significant when p < 0.05 and indicated by “∗” and were considered as statistically very significant when p < 0.01 and indicated by “∗∗”.

Consistently, as shown by BrdU incorporation assay, ASG does-dependently inhibited HCC cell proliferation (Figure 1C). To evaluate whether ASG induced apoptosis or cell cycle arrest in HCC cells, we performed FACS. We unveiled that the ratios of HCC cells at the G1 phase were dramatically increased by ASG. Sub-G1 DNA is an indicator of apoptotic cells. Upregulation of the ratios of Sub-G1 DNA in HCC cells could also be observed in response to ASG (Figure 1D,E). These observations indicated that induction of cell cycle arrest and apoptosis contribute to the antiproliferative effect of ASG on HCC cells. ASG Repressed Glycolysis and Promoted OXPHOS in HCC Cells. As shown in Figure 2A,B, ASG suppressed rates of glucose consumption and lactate production. In addition, catalytic activities of respiratory complex I and IV were considerably elevated by ASG (Figure 2C,D). To confirm ASG-induced metabolic shift in HCC cells, cells were treated with oligomysin, an inhibitor of OXPHOS, and levels of intracellular ATP were determined at different time periods. In control cells, levels of ATP remained almost unchanged in response to oligomysin within 2 h. These data indicated that glycolysis, which is insensitive to oligomysin, was the major resource of ATP in control HCC cells. In HCC cells pretreated with ASG for 2 days, levels of ATP could be considerably downregulated by oligomysin (Figure 2E), suggesting that ratios of ATP from glycolysis were decreased.

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RESULTS ASG Suppressed the Proliferation of HCC Cells in Vitro. As shown in Figure 1A, proliferations of 3 different kinds of HCC cells were suppressed by ASG in a dose-dependent manner. Especially, the antiproliferative impact of ASG on human HL-7702 untransformed hepatocytes was undetectable (Figure 1B). 5964

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Figure 4. ASG suppressed HK2 expression in HCC cells. (A−D) HepG2, Huh-7, and H22 HCC cells were treated by the indicated concentrations of ASG for 2 days. Then, protein levels of HK2 were detected by immunoblotting (A), and densities of immunoreactive bands of HK2 were normalized to those of β-actin (B). Levels of HK2 mRNA were determined by reverse transcription and real-time PCR (C). Effects of ASG on enzymatic activity of HK in HCC cells were examined (D). **p < 0.01, compared to vehicle treatment.

ASG Suppressed HK2 Expression through Elevating MiR-125b. HK2 is a target of miR-125b.31 ASG considerably elevated miR-125b in HCC cells (Figure 6A). Transfection of anti-miR-125b abrogated the repressive effects of ASG on HK2 (Figure 6B−D). Consistently, inhibition of miR-125b significantly diminished ASG-induced metabolic reprogramming (Figure 6E−G). The antiproliferative effects of ASG on HCC cells were in turn dose-dependently crippled by anti-miR-125b (Figure 6H,I). These results indicated that ASG suppresses HK2 expression by the induction of miR-125b. Effects of ASG on Huh-7 Xenograft Tumor Growth in Nude Mice. To understand whether ASG was capable of suppressing the proliferation of HCC cell in vivo, an HCC xenograft tumor model was established. ASG significantly inhibited the growth of xenograft Huh-7 HCC tumors (Figure 7A). Moreover, ASG reduced the ratio of Ki-67 positive cells in xenograft tumors (Figure 7C), which supported the in vivo anti-HCC effects of ASG. To assess toxic impacts of ASG on normal hepatocytes of tumor-bearing mice, liver indexes and serum parameters of liver function were measured at the end of the study. Few changes in any of these indexes were detected (Figure 7B). In addition, we detected effects of ASG on miR-125b/HK2 cascade in HCC cells in vivo. In xenograft tumors, ASG dosedependently elevated miR-125b (Figure 7E); mRNA and protein levels of HK2, as well as the catalytic activity of HK, were coordinately suppressed (Figure 7D,F). Moreover, in Huh-7 HCC xenograft tumors, ASG dramatically upregulated levels of MDA and ROS (Figure 7G). These results indicated that ASG modulates miR-125b/HK2 cascade and provokes growth arrest in HCC cell in vivo. Effects of ASG on Transplanted H22 Cell Proliferation in Kunming Mice. To confirm the anti-HCC impacts of ASG in vivo, murine H22 HCC cells were transplanted into Kunming mice subcutaneously. Tumor-bearing mice received ASG through gavage. ASG inhibited the proliferation of transplanted tumors without detectable toxic effects on normal

Collectively, these data indicated that ASG provokes a reprogramming of glucose metabolism from glycolysis to OXPHOS. ASG Induced Growth Arrest through Generation of ROS. Reduced glycolysis and elevated OXPHOS have the potential to upregulate intracellular ROS. To test this hypothesis, we performed FACS. Levels of ROS were boosted by ASG in a does-dependent manner (Figure 3A,B). Levels of MDA, an indicator of oxidative stress, were coordinately increased in response to ASG (Figure 3C). NAC is a scavenger of ROS. MTT cell viability assay and BrdU cell proliferation assay showed that NAC dosedependently abrogated ASG-induced HCC cell growth arrest (Figure 3D,E). These results suggested that accumulation of ROS acts as an important mediator in the antiproliferative effects of ASG on HCC cells. ASG Suppressed HK2 Expression in HCC Cells. HK2 is one of the key enzymes affecting the rate of glycolysis. In HCC cells, protein levels of HK2 were strongly downregulated by ASG (Figure 4A,B). Furthermore, mRNA levels of HK2 were dramatically declined in response to ASG (Figure 4C). Reduced expression of HK2 was associated with a substantial decrease in the catalytic activity of HK (Figure 4D). These results indicated that ASG inhibits the expression of HK2 in HCC cells. Overexpression of HK2 Abrogated ASG-Induced Metabolic Reprogramming. To understand the role of reduced expression of HK2 in ASG-induced HCC cell growth arrest, HK2 was stably transfected into HepG2 and Huh-7 cells (Figure 5A). Overexpression of HK2 effectively antagonized ASG-mediated inhibition of catalytic activity of HK (Figure 5B). In HK2 overexpression cells, rates of glucose consumption and lactate production as well as catalytic activities of respiratory complex I and IV were insensitive to ASG (Figure 5C−F). In control HCC cells, ASG gave rise to accumulation of ROS, elevation of MDA, and inhibition of cell proliferation. These effects were abrogated by the overexpression of HK2 (Figure 5G−J). 5965

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Figure 5. Reduction of HK2 was required for ASG-mediated metabolic reprogramming and growth arrest. (A) Human HK2 encoding gene was stably transfected into HepG2 and Huh-7 HCC cells. Levels of HK2 protein in the indicated cells were detected (left). Density values of immunoreactive bands of HK2 were normalized to those of β-actin (right). (B−D) The indicated HCC cells were treated by 33 μM ASG. After 2 days, enzymatic activity of HK in cell lysates was determined (B); rates of glucose consumption (C) and lactate production (D) were also detected. (E−H) Activities of complex I (E) and IV (F) in cell lysates were determined. Levels of ROS (G) and MDA (H) were also quantified. (I,J) The indicated HCC cells were incubated with ASG for 3 days and then subjected to cell viability assay (I) and BrdU cell proliferation assay (J). **p < 0.01 versus the indicated control.

hepatocytes of the host (Figure 8A,B). The ratio of Ki-67 positive HCC cells in transplanted tumors was also declined by ASG (Figure 8C). In transplanted H22 cells, ASG dose-dependently increased levels of miR-125b (Figure 8E). The expression levels of HK2 and the catalytic activity of HK were in turn decreased in response to ASG (Figure 8D,F). Levels of MDA and ROS were also boosted in response to ASG (Figure 8G). These results provided more evidence supporting that ASG suppresses the expression of HK2 by boosting miR-125b and induces growth arrest in HCC cells in vivo.

consistent with a preliminary screening showing that ASG had the capacity of reducing the viability of HepG2 HCC cells.32 Furthermore, we revealed that ASG inhibited HCC cell proliferation in vivo with undetectable toxic effects on livers of tumor-bearing mice. These results have an important implication for considering ASG to be a novel functional food ingredient applied as an adjuvant reagent in clinical HCC treatment. Though the anticancer capacity of ASG is supported by an array of independent studies, the knowledge concerning the molecular mechanisms underlying its tumor-inhibitory effects is still limited. In lung cancer cells, ASG provokes caspasedependent cell death via repressing NF-κB signaling.33 Our current investigation reported that in HCC cells, ASG inhibits the proliferation of HCC cells through suppressing HK2 expression and subsequent accumulation of ROS. These results

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DISCUSSION In this study, we first unveiled that ASG induced growth arrest in HepG2, Huh-7, and H22 HCC cells in vitro. Our findings are 5966

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Figure 6. Augment of miR-125b played an important role in ASG-mediated inhibition of HK2 expression. (A) HepG2, Huh-7, and H22 HCC cells were incubated as indicated for 2 days. Then, levels of miR-125b were determined. (B−D) HepG2 and Huh-7 cells were transfected with 30 (indicated by “+”) or 100 (indicated by “++”) nM anti-miR-125b, using 100 nM scrambled control RNA as the negative control. After 1 day, cells were incubated with 33 μM ASG for 2 days. Then, levels of HK2 protein were determined (B). The densitometry analysis of relative protein levels of HK2 were also performed (C). The enzymatic activity of HK in cell lysates was also quantified (D). (E−G) Rates of glucose consumption (E), rates of lactate production (F), and levels of ROS (G) were also determined. (H,I) Effects of anti-miR-125b on ASG-induced growth arrest were detected by cell viability assay (H) and BrdU cell proliferation assay (I). *p < 0.05; **p < 0.01 versus the indicated control.

indicated that the mechanisms underlying ASG-mediated anticancer effects vary in different kinds of cancer cells. ASG can be commonly found in food components. For instance, a previous study showed that about 3.0 mg of ASG can be prepared from 80 mg of crude extract from dried lotus leaves. In this report, the yield of crude extract from the raw plant material was 135 g/3 kg = 4.5%.34 Therefore, according to their study, it is reasonable to estimate the mass content of ASG in dried lotus leaves as 0.17%. In our in vivo experiment, the

highest dosage of ASG is 20 mg/kg for mice. On the basis of the notion of animal surface area, this dosage is equivalent to approximately 1.63 mg/kg for human.35 Thus, a person weighing 65 kg needs to take 106 mg of ASG per day. To get this daily dose of ASG, 62 g of dried lotus leaves should be consumed, which is in fact a feasible dosage. ASG is kaempferol-3-O-β-D-glucopyranoside. A previous study showed that the IC50 (the concentration of a compound at which cell viability was reduced by 50%) of kaempferol in 5967

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Figure 7. ASG suppressed the proliferation of Huh-7 xenografts in nude mice. Nude mice bearing Huh-7 HCC cells were treated with the indicated dosages of ASG through gavage for 3 weeks. (A) Volumes of tumors were monitored every 3 days. (B) At the end of drug treatment, serum activities of ALS and AST were determined and liver weight indexes were calculated. (C−G) Mice were sacrificed and xenograft tumors were dissected and subjected to immunohistochemical staining for Ki-67 (C) and HK2 (D). Levels of miR-125b (E), HK2 mRNA (F, left), HK activity (F, right), MDA content (G, left), and ROS content (G, right) in xenograft tumors were determined. *p < 0.05; **p < 0.01, compared to vehicle treatment.

HepG2 HCC cells in vitro is 84.7 μM.36 On the basis of our current study, the IC50 value of ASG in HepG2 HCC cells in vitro is less than 30 μM, suggesting a much stronger antiproliferative activity of ASG against HCC cells. It is generally believed that before the absorption of ASG in the digestive tract, glucose moiety of ASG is removed enzymatically. In this way, ASG should be absorbed as an aglycone, kaempferol. However, a previous literature showed that ASG given through gavage can be absorbed in its intact form.37 In their study, rats were given total flavonoids extract from mulberry leaves through gavage. The mass content of ASG was 24.9 mg/g in this extract, but kaempferol was undetectable. Although both kaempferol and ASG could be detected in the blood of animals,

and their peak concentrations (Cmax) in serum both appeared within 20 min after oral administration, the Cmax of ASG was 10 times more than that of kaempferol. In other words, much more ASG was absorbed in its intact form than aglycone form. On the one hand, the cytotoxic effect of kaempferol on HCC cells was weaker than ASG. On the other hand, the absorbed kaempferol was less than 10% of the absorbed ASG. It is thus unlikely that kaempferol, the aglycone form of ASG, contributed to the in vivo effects observed in our current study. In addition, another independent study also demonstrated that ASG can be absorbed in its intact form.38 Moreover, in our current study, dosages of ASG used both in vitro and in vivo are comparable to those recruited in the literature already 5968

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Figure 8. ASG suppressed the proliferation of transplanted H22 HCC cells in Kunming mice. Kunming mice bearing H22 HCC tumors were treated as indicated through gavage for 10 days. (A) Volumes of tumors were monitored every 2 days. (B) At the end of the treatment, serum parameters of liver functions were examined and liver weight indexes were calculated. (C−G) Mice were sacrificed and transplanted tumors were dissected out and subjected to immunohistochemical staining using the primary antibodies against Ki-67 (C) and HK2 (D). Levels of miR-125b (E), HK2 mRNA (F, left), HK activity (F, right), MDA content (G, left), and ROS content (G, right) in transplanted tumors were determined. *p < 0.05; **p < 0.01, compared to vehicle treatment.

published in which the in vivo findings are considered to be explained by the results obtained from the in vitro study.18,33,39 Nevertheless, the detailed pharmacokinetic process of ASG deserves further investigation. ROS constitute a group of incomplete reducing products of oxygen, majorly generated as byproducts of OXPHOS in mitochondrions. Accumulation of ROS leads to oxidative stresses. In response to ASG, activities of mitochondrial complex I and IV in HCC cells increased, elevating intracellular ROS. Conversely, low activities of complex I and IV protect pancreatic cancer cells exposed to chemotherapeutic reagents from accumulation of ROS.40 Similarly, SB3 is a serine protease

inhibitor frequently overexpressed in various kinds of malignancies, including HCC. Through inhibiting mitochondrial complex I, SB3 alleviates the rapid rise of intracellular ROS prompted by anti-HCC reagents.41 These data implied a possibility that enhanced OXPHOS is potential to result in a surge of ROS in cancer cells. Many types of anticancer reagents inhibit the proliferation of cancer cells by elevating ROS. For instance, induction of ROS plays a pivot role in Calebin-Ainduced cell cycle arrest in colon cancer cells.42 ROS also mediates PX-12- or volasertib-induced growth arrest in HCC cells.43,44 Here, ROS scavenger NAC significantly abrogated the 5969

DOI: 10.1021/acs.jafc.7b02120 J. Agric. Food Chem. 2017, 65, 5961−5972

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Journal of Agricultural and Food Chemistry

For a brief summary, in this study, we found that ASG suppressed the proliferation of HCC cells. In HCC cells, ASG inhibited glucose glycolysis and promoted OXPHOS, resulting in a surge of intracellular ROS. ROS scavenger NAC dosedependently abrogated ASG-mediated growth arrest. ASG suppressed the expression of HK2. Reduction of HK2 was indispensible for ASG-mediated metabolic reprogramming and subsequent growth arrest. Further studies unveiled that ASG repressed HK2 expression via increasing miR-125b. In vivo experiments showed that gavage of ASG reduced the proliferation of Huh-7 HCC xenografts in nude mice and inhibited the growth of transplanted H22 HCC cells in Kunming mice. Declined HCC tumor growth in vivo was associated with upregulated miR-125b and reduced HK2 in tumor tissues. Our findings collectively revealed that ASG induces metabolic alteration and growth arrest in HCC cells through modulating miR-125b/HK2 cascade.

antiproliferative effects of ASG, indicating the critical involvement of ROS in the antiproliferative effects of ASG. Glycolysis and OXPHOS are two metabolic pathways producing ATP. In untransformed cells, only approximately 10% of ATP is generated from glycolysis, but in malignant cells, this percentage raises up to around 60%.45 This notion was supported by our observations here that in control HCC cells, levels of intracellular ATP were insensitive to oligomysin, an inhibitor of OXPHOS. ASG repressed HK2 expression in HCC cells, leading to a reduction of glycolysis. Interestingly, the change of basal levels of intracellular ATP in response to ASG was unobvious. A reasonable explanation to these data is that decrease of glycolysis gave rise to a compensatory increase of OXPHOS. This hypothesis could be supported by our results that ASG elevated the activities of mitochondrial complex I and IV, which in turn resulted in a surge of ROS. Similarly, it has been reported previously that activating OXPHOS elevates ROS production in HCC cells.46 In our study, repressing glycolysis by reducing HK2 boosted intracellular ROS, and overexpression of HK2 remarkably abolished ASG-mediated ROS accumulation. Likewise, jolkinolide B, a natural diterpenoid, suppresses HK2 expression in mouse melanoma cells, leading to downregulated glycolysis and elevated production of ROS.47 The synthetic compound, 3-bromopyruvate, is an inhibitor of HK2. It suppresses glycolysis and induced overproduction of ROS in human nasopharyngeal carcinoma cells.48 Taken together, these findings implied that suppression of glycolysis by targeting HK2 can be a feasible strategy to provoke accumulation of ROS in malignant cells. MiRNAs are endogenous small noncoding RNA molecules which negatively regulate the expression of protein-coding genes at the post-transcriptional level. Dysregulation of miRNA is involved in the occurrence and development of HCC.49 MiR125b expression is strongly reduced in HCC cells and negatively correlated with the prognosis of HCC.50,51 Previous studies showed that an increase of miR-125b through transfection suppressed the proliferation of HCC cells.52 These observations suggested that boosting miR-125b expression appears to be a reasonable approach in the clinical control of HCC. However, little is known about the chemical entities with the capacity of increasing miR-125b expression in HCC cells. Here, we revealed that ASG upregulated levels of miR-125b, which in turn attenuated the expression of HK2 in HCC cells. Conversely, anti-miR-125b abrogated the inhibitory effects of ASG on HK2 expression and cell proliferation. These data indicated that upregulation of miR-125b is a crucial molecular event mediating ASG-induced metabolic reprogramming and subsequent growth arrest in HCC cells. Similar to our results, induction of miR-326 is involved in cancer cell death induced by resveratrol;53 phenethyl isothiocyanate suppresses prostate cancer cell invasion by increasing miR-194.54 These phenomena imply a possibility that modulating the expression of miRNA plays important roles in the anticancer effects of natural products. It has been demonstrated that deficiency of p53 signaling and enhanced DNA methylation of miR-125b promoter are two contributors to the declined expression of miR-125b in HCC cells.55 ASG is a glycoside derivative of kaempferol which is able to activate p53 and inhibit DNA methylation in cancer cells.56,57 Therefore, it is reasonable to speculate that activation of p53-dependent transcription and demethylation of miR-125b promoter have the potential to be implicated in ASG-induced expression of miR-125b in HCC cells.

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AUTHOR INFORMATION

Corresponding Author

*Phone/fax: + 86 27 6784 1196. E-mail: shuguangwen@whu. edu.cn. ORCID

Guangwen Shu: 0000-0002-7332-6365 Funding

This project was supported by the Research Project of Higher Education from the Hubei Provincial Department of Education (Grant No. 2015195) and the Innovation and Entrepreneurship Training Program Founded by South-Central University for Nationalities (Grant XCX17093). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ASG, astragalin; HCC, hepatocellular carcinoma; HK, hexokinase;; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species

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