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. Author manuscript; available in PMC: 2022 Feb 10.
Published in final edited form as: Mol Carcinog. 2016 Jun 15;56(2):436–446. doi: 10.1002/mc.22506

Cinnamon Extract Reduces VEGF Expression Via Suppressing HIF-1α Gene Expression and Inhibits Tumor Growth in Mice

Keqiang Zhang 1,2, Ernest S Han 2, Thanh H Dellinger 2, Jianming Lu 1,2, Sangkil Nam 1, Richard A Anderson 3, John H Yim 2,*, Wei Wen 1,2,*
PMCID: PMC8830771  NIHMSID: NIHMS1773890  PMID: 27253180

Abstract

Although many anti-VEGF agents are available for cancer treatment, side effects of these agents limit their application for cancer treatment and prevention. Here we studied the potential use of a diet-based agent as an inhibitor for VEGF production. Using a VEGF reporter assay, our data showed that an extract from cinnamon (CE) was a potent inhibitor of VEGF production in human cancer cells and suggested inhibition might be mediated through the suppression of HIF-1α gene expression and protein synthesis. Furthermore, CE treatment was found to inhibit expression and phosphorylation of STAT3 and AKT, which are key factors in the regulation of HIF-1α expression, and significantly reduce angiogenesis potential of cancer cells by migration assay. Consistent with these results, we observed significant suppression of VEGF expression, blood vessel formation, and tumor growth in a human ovarian tumor model in mice treated with CE. Cinnamaldehyde, a major component in cinnamon, was identified as one active component in CE that inhibits VEGF expression. Taken together, our findings provide a novel mechanism underlying anti-angiogenic and anti-tumor actions of CE and support the potential use of CE in cancer prevention and treatment.

Keywords: cinnamon extract, VEGF, HIF-1α, angiogenesis, xenograft

INTRODUCTION

Vascular endothelial growth factor (VEGF) is one of the most critical and specific factors that stimulate angiogenesis [1]. The expression of VEGF in cancer cells is mainly regulated by hypoxia [2]. The primary regulator mediating the hypoxia response is a heterodimeric transcription factor, hypoxia-inducible factor-1(HIF-1), which consists of HIF-1β and HIF-1α. While HIF-1β is constitutively expressed, HIF-1α is highly regulated by the rate of protein synthesis, which is oxygen independent, and the rate of protein degradation, which is oxygen dependent [2]. In the presence of oxygen, the HIF-1α protein is hydroxylated, ubiquitinated, and subsequently degraded by the proteasome. Under hypoxic conditions, HIF-1α cannot be hydroxylated. Consequently, HIF-1α accumulates in the nucleus where it binds to the hypoxia response element (HRE) and activates transcription of target genes [2]. In many human cancers HIF-1α expression is elevated and linked to increased patient mortality [3-5]. In addition to hypoxia, VEGF expression in cancer cells is also modulated by growth factors, oncogenes, and other signaling molecules that increase HIF-1α protein synthesis via activation of the PI3K/AKT and STAT3 pathways [6-10]. Many transcription factor binding sites, including AP-1, Sp-1, STAT3, ERR, and CREB, have been identified within the VEGF promoter [6,11,12].

The biological function of VEGF is mainly mediated through binding to two receptor tyrosine kinases expressed on endothelial cells: VEGF receptor 1 (FLT-1/VEGFR1) and VEGF receptor 2 (KDR/FLK-1/VEGFR2) [1]. Inhibiting VEGF activity either by targeting VEGF ligand with a neutralizing antibody or by targeting VEGF receptors on endothelial cells with small molecule inhibitors [13] has led to the approval of numerous drugs by the U.S. Food and Drug Administration to treat several cancers, including colorectal and renal cancers [14]. However, most of these agents cause side effects, such as hypertension, bleeding, gastrointestinal perforation, and therefore cannot be used for cancer prevention [15]. A diet-based approach is an attractive alternative to reduce VEGF action given its demonstrated safety in humans [16,17]. Although the preventative effects of plant-based diets on cancer development and progression have been documented [18,19], their mechanisms of action on the VEGF pathway have not been well elucidated. While some diets inhibit angiogenesis through suppressing VEGF signaling on endothelial cells, others inhibit angiogenesis through blocking production of VEGF by cancer cells.

Cinnamon, the dry bark and twig of Cinnamomum spp, is one of the oldest and most commonly used spices worldwide and in traditional Chinese medicine [20]. Cinnamaldehyde and procyanidins are two major components found in cinnamon. While procyanidins are responsible for the antioxidant activity of cinnamon, cinnamaldehyde contributes to the fragrance and a variety of health benefits associated with cinnamon, including antimicrobial, anti-inflammatory, and anti-diabetic activities [21-24]. An extract from cinnamon (CE) was also shown to inhibit Tau aggregation associated with Alzheimer’s disease [25]. Recently the anti-tumor and anti-angiogenesis activity of CE has also been documented both in vitro and in vivo [26-29]. We previously showed that CE and its main component, procyanidins, significantly inhibited angiogenesis by suppressing VEGF receptor kinase activity on endothelial cells [26].

In this study, we identified and characterized diet-derived molecules that regulate VEGF production in cancer cells. Using a VEGF reporter cell line, we found that CE suppressed VEGF expression through inhibiting HIF-1α protein expression in human cancer cells, providing additional novel insight into the mechanisms of anti-tumor and anti-angiogenesis action of CE. In addition, we identified cinnamaldehyde as one active component in CE suppressing VEGF expression. Furthermore, we showed that CE inhibited angiogenesis and ovarian cancer growth in mice.

MATERIALS AND METHODS

Cinnamon Extract (CE)

Ground cinnamon (Cinnamomum zeylanicum) powder (Frontier Natural Products Co-op in Norway, IA) was dissolved in water (70°C, 1 h) and centrifuged (13,000 rpm, 10 min) to remove insoluble ingredients. The supernatant was filtered, transferred to a new tube, and lyophilized under vacuum. The dry pellet was weighed, resuspended in water, and characterized using HPLC/UV detection as described previously [26,30]. A water-based cinnamon extract, prepared and characterized by Dr. John Lew’s group (University of California, Santa Barbara), was also used to confirm some of the results in this study [25].

Reagents and Antibodies

Cinnamaldehyde, cycloheximide (CHX), deferoxamine mesylate (DFX), MG132, and anti-β-actin antibody were purchased from Sigma. Other antibodies were against: phospho-AKT, phosph-STAT3, and total STAT3, from Cell Signaling Technology; total AKT, from Santa Cruz Biotechnology; and HIF-1α, from BD Biosciences.

Cell Culture

As described previously [31], MDA-MB-231 human breast cancer cells (ATCC) were cultured in RPMI1640 medium containing 10% FBS; U251 human glioma cells (kindly provided by Dr. Karen Aboody at Beckman Research Institute of the City of Hope, CA) and SKOV3 human ovarian cancer (ATCC) were cultured in DMEM medium supplemented with 10% FBS; and HUVEC (Human Umbilical Vascular Endothelial Cells, Clonetics, Lonza) were cultured in EGM-2 medium (Lonza) containing 10% FBS (FBS;Gibco BRL). For cell culture under hypoxia, cells were grown (37 °C) in a chamber containing 1%O2, 5% CO2, and 94% N2. Hypoxia can also be induced in normoxia using 250 μM DFX to mimic hypoxic conditions.

VEGF-Luc Reporter Cell Line and Luciferase Reporter Assay

The reporter cell line U251/VEGF-Luc was established as described previously [31]. Transfected U251 cells and reporter cells were plated the day before treatment then treated with CE. Luciferase activity was determined as described previously [31].

Quantitative Real-Time PCR

Total RNA was extracted from cell lines using Qiagen RNeasy Min Kit. Quantitative real-time PCR (qRT-PCR) was performed using the ABI Prism 7900HT Sequence Detection System with SYBR Green PCR Master Mix, as described previously [31].

Quantification of VEGF

An ELISA-based bead multiplex assay (Luminex Corp., Austin, TX, USA) was used to measure VEGF levels in conditioned medium using a human cytokine kit from Invitrogen. Quantikine human VEGF Elisa kit from R&D system was used to measure human VEGF levels in the ascites and tumor extract. The conditioned medium from U251/VEGF-Luc cells and MDA-MB-231 cells were collected as described previously [31].

Immunoblot

Total cell extract was prepared in Laemmli sample buffer, electrophoresed on SDS gels, transferred to polyvinylidene difluoride (PVDF, Millipore) membrane and immunoblotted with various antibodies, as described previously [31].

Migration Assay

A modified Boyden chamber assay was used to assess endothelial cell migration, as described previously [31]. Briefly, HUVEC cells (1 × 105) were plated in EBM-2 medium containing 0.05% FCS in the upper chamber of the transwell (8-μm pore, Costar). Serum free DMEM, containing conditioned medium (50%) from tumors either treated with vehicle (water), CE (32 μg/ml), or cinnamaldehyde (10 μg/ml), were added to the lower chamber of the transwell. After incubation (5 h), cells were fixed and stained, and non-migrated cells were removed by cotton swap. The number of migrated cells was quantified by counting cells using an Olympus microscope, 40× objective.

Tumor Formation in Mice

SCID mice, 6–8 wk old, were housed under pathogen-free conditions, according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. All studies followed approved institutional experimental animal care and use protocols.

SCID mice were inoculated intraperitoneally with SKOV3 human ovarian cancer cells (5 × 106 cells/mice). Two weeks after inoculation, mice were individually gavage fed daily with cinnamon suspension (0.3 mg/g) (n = 5) or 300 μl of water control (n = 7) for 4 wk then sacrificed. Tumors and ascites were collected at the end of treatment. The ascites volume was assayed, and tumors were weighed, fixed in formalin, embedded in paraffin, and sectioned for immunohistochemistry analysis. Blood vessels in the tumor section were visualized using an anit-CD31 antibody (BD Bioscience) as described previously [32]. Sections were photographed at ×200 magnification using an Olympus AX70 microscope (Melville, NY). Vessel density (average five fields) was determined by Image-Pro software.

Statistical Analysis

Data were presented as mean ± S.D. Comparisons between two groups were determined by Student’s t-test. One way ANOVA followed by Dunnett’s post hoc test was used for multiple comparisons against the control group. Each assay was repeated 2–4 times; P < 0.05 was considered statistically significant.

RESULTS

Effects of CE on VEGF Production

We previously established a VEGF reporter cell line U251 to identify diet-derived inhibitors that regulate VEGF expression [31]. The expression of the reporter gene, luciferase, in this cell line is under the human VEGF promoter containing a 2.1 kb fragment. We found that an extract from cinnamon (CE) inhibited hypoxia-induced VEGF reporter activity in a dose-dependent manner (Figure 1A), with little effect on cell numbers (Figure 1B).

Figure 1.

Figure 1.

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CE inhibits VEGF expression in cells. (A) and (B) CE inhibits luciferase activity of VEGF reporter without reducing cell viability. Human glioma U251 cells expressing luciferase reporter under the human VEGF promoter were incubated (24 h, under hypoxia) with increasing concentrations of CE then assayed for luciferase activity and cell viability. Data are expressed as the ratio to control treated with vehicle under normoxia. *P < 0.05, versus control treated with vehicle under hypoxia. (C) CE inhibits VEGF mRNA expression. U251 cells were incubated (16 h) with CE (32 μg/ml) under both normoxia and hypoxia, either in the presence of 1% oxygen or 250 μM DFX that mimics hypoxia conditions. qRT-PCR was used to determine VEGF mRNA expression. Data are expressed as the ratio to control treated with vehicle under normoxia. *P < 0.05, versus control. (D) U251 cells were incubated with CE (32 μg/ml) for indicated times in the presence of DFX then assayed for mRNA by qRT-PCR. Data are expressed as the ratio to the control treated with vehicle at each time point. *P < 0.05, versus control treated with vehicle at each time point. (E) and (F) U251 and MDA-MB-231 cells were incubated (24 h) with CE (32 μg/ml) in serum free medium either in the presence of 1% oxygen or 250 μM DFX. After incubation, conditioned media were collected and analyzed for the presence of VEGF by Luminex multiplex assay. Data are normalized to cell numbers and medium volume. *P < 0.05 versus control treated with vehicle.

We next determined whether VEGF mRNA expression was also suppressed by CE. Quantitative real-time PCR (qRT-PCR) was used to compare VEGF mRNA expression in U251 human glioma cells. Our data suggested that CE inhibited VEGF mRNA levels under both normoxia (20% oxygen) and hypoxia, either in the presence of 1% oxygen or 250 μM DFX, a hypoxiamimetic agent (Figure 1C). The inhibitory effect of CE on VEGF mRNA production increased with time, beginning after 8 h (Figure 1D).

To determine whether VEGF protein expression was also inhibited by CE, we treated U251 human glioma with CE in the presence of 1% O2 or 250 μM DFX. Conditioned media were collected and VEGF protein levels were assessed by Luminex multiplex assay. CE treatment significantly reduced VEGF levels in the cells. To understand whether a similar result can be achieved in other cancer cells, we studied this inhibitory activity in MDA-MB-231 human breast cancer cells and found treatment of CE led to a decreased level of VEGF under hypoxia (Figures 1E and F).

Effects of CE on HIF-1α Expression

To further elucidate the underlying mechanisms mediating reduced VEGF expression by CE, we examined the effects of CE on the expression of HIF-1α, a major regulator of VEGF expression. HIF-1α is often over-expressed in many human cancers, including breast and ovarian cancer cells [3-5]. U251 cells were treated with CE and analyzed for HIF-1α expression by Western blot. Our data showed that, in the presence of 1% O2 or 250 μM DFX, CE significantly decreased HIF-1α protein levels in U251 (Figure 2A). However, decreased HIF-1α expression could be caused by the increased degradation and/or decreased synthesis of HIF-1α protein.

Figure 2.

Figure 2.

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CE inhibits HIF-1α expression. (A) U251 cells were incubated with increasing concentrations of CE under hypoxia, either in the presence of 1% oxygen or treated (16 h) with DFX. Whole-cell lysates were analyzed by immunoblotting with antibody against HIF-1α. β-actin was used as a loading control. (B) CE has little effect on HIF-1α degradation. Cells were first incubated (4 h) in the presence of DFX (250 μM) then treated with CHX (10 μM) in the presence or absence of CE (32 μg/ml) for indicated times. Whole-cell lysates were analyzed for HIF-1α protein by immunoblotting. (C) CE inhibits HIF-1α protein synthesis. Cells were pretreated (1 h) with CE (32 μg/ml) prior to the addition of proteasome inhibitor MG-132 (10 μM) then incubated for indicated times. Whole-cell lysates were analyzed by immunoblotting for the presence of HIF-1α. (D) and (E) Relative levels of HIF-1α protein were determined by measuring the density of the HIF-1α protein band after normalization to β-actin. Data are expressed as a ratio to control at time zero. Results are representative of 2–4 preparations.

To determine whether CE affects protein degradation, cycloheximide (CHX), a protein translation inhibitor, was used to prevent de novo HIF-1α protein synthesis. Without new protein synthesis, the HIF-1α level would be expected to decline rapidly. U251 cells were treated with DFX (4 h) to induce HIF-1α expression and were then incubated with CHX either in the presence or absence of CE for various times. As expected, in the presence of CHX, HIF-1α levels rapidly declined. The degradation rate of HIF-1α in the presence of CE was comparable to that observed in the absence of CE, suggesting that CE had little effect on protein degradation and that reduced HIF-1α expression occurred most likely through a different mechanism (Figures 2B and D).

To understand whether CE inhibited HIF-1α expression via suppression of HIF-1α protein synthesis, the proteasome inhibitor, MG132, was used to prevent HIF-1α degradation. In the presence of MG-132, HIF-1α rapidly accumulated over a period of 1 h under normoxia. However, once cells were treated with CE, HIF-1α protein accumulation was significantly reduced (Figures 2C and E), suggesting CE inhibited HIF-1α protein synthesis.

We then questioned whether CE affected HIF-1α mRNA expression to directly affect HIF-1α protein synthesis. To address this question, qRT-PCR was used to measure HIF-1α expression. HIF-1α mRNA levels were significantly decreased in the presence of CE under both normoxia and hypoxia conditions in U251 cells (Figure 3A). In contrast, there was no difference in mRNA expression of the housekeeping gene HPRT between CE treated and control cells, suggesting that the inhibitory effect of CE on HIF-1α gene expression was specific (Figure 3B). The inhibitory effect of CE on HIF-1α mRNA expression was also observed in a dose-dependent manner in MDA-MB-231 cells (Figure 3C).

Figure 3.

Figure 3.

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CE inhibits HIF-1α mRNA expression. (A) and (B) U251 cells were incubated (16 h) with CE (32 μg/ml) under normoxia, 1% oxygen, or in the presence of DFX (250 μM) then harvested and examined for HIF-1α and HPRT mRNA expression by qRT-PCR. Data are normalized to β-actin and expressed as the ratio to control treated with vehicle. *P < 0.05, versus control treated with vehicle. (C) MDA-MB-231 cells were incubated with increasing concentrations of CE under normoxia and hypoxia. Data are normalized to β-actin and represented as the ratio to the control treated with vehicle alone under normoxia. *P < 0.05, versus control under normoxia; #P < 0.05, versus control under hypoxia.

Effects of CE on Expression and Phosphorylation of STAT3 and AKT

Previous studies have shown that STAT3 plays a critical role in the regulation of HIF-1α expression, both at the RNA and protein synthesis levels [9,10,33-35]. To address whether the STAT3 pathway was involved in the inhibition of HIF-1α expression, U251 cells were incubated with various amounts of CE and the effects of CE on STAT3 phosphorylation were evaluated. The phosphorylation of STAT3, as well as total STAT3, was significantly reduced in the presence of CE (Figure 4).

Figure 4.

Figure 4.

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CE inhibits expression and phosphorylation of STAT3 and AKT. U251 cells were incubated (16 h) with increasing concentrations of CE in the presence of DFX that mimics hypoxia conditions. Cells were harvested and whole-cell lysates were analyzed by immunoblotting. β-actin level was used as a loading control. Results are representative of 2–4 preparations.

STAT3 has been shown to regulate HIF-1α expression through the AKT pathway, as knockdown of STAT3 in cancer cells results in reduced AKT expression [9]. We then asked whether AKT was involved in the inhibition of HIF-1α gene expression by CE treatment. To address this question, the effects of CE on phosphorylation and expression of AKT were investigated. CE treatment dramatically inhibited both phosphorylation and expression of AKT (Figure 4). Taken together, our results implicate the STAT3/AKT pathway in the inhibition of HIF-1α expression by CE.

Active Components in CE

Procyanidins and cinnamaldehyde are two major components found in the CE. We next investigated whether these components could suppress VEGF expression. We previously demonstrated that procyanidins could block kinase activity of VEGF receptor on endothelial cells [30]. Here, we found little effect of procyanidins on VEGF expression when cells were treated with trimeric (MW = 864) and tetrameric (MW = 1,152) procyanidins isolated from CE using HPLC (Figure 5A). In contrast, cinnamaldehyde was found to suppress VEGF reporter gene expression, VEGF mRNA and protein expression (Figures 5B-D), consistent with the results from a very recently published study [36]. Taken together, our study implicated the potential of cinnamaldehyde as one major component in CE suppressing VEGF expression by cancer cells.

Figure 5.

Figure 5.

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Cinnamaldehyde inhibits VEGF expression. (A) Oligomeric procyanidins have little effect on VEGF expression. U251 VEGF reporter cell lines were treated (24 h) with trimeric and tetrameric procyanidins then assayed for luciferase activity. Data are presented as the ratio to vehicle-treated control. (B) Cinnamaldehyde inhibits VEGF reporter expression. VEGF reporter cells were incubated with increasing concentrations of cinnamaldehyde under hypoxia then assayed for luciferase activity. Data are normalized to cell numbers expressed as the ratio to control without treatment. *P < 0.05, versus control under normoxia; #P < 0.05, versus control under hypoxia. (C) Reporter cells were treated (16 h) with cinnamaldehyde (10 μg/ml), under hypoxia, then subjected to qRT-PCR for VEGF mRNA expression. Data are expressed as the ratio to control without treatment. *P < 0.05, versus control. (D) VEGF reporter cells were incubated with cinnamaldehyde (10 μg/ml) and assayed for VEGF protein expression. VEGF concentration was normalized to cell numbers and medium volume. Data are expressed as the ratio to untreated control under normoxia. *P < 0.05, versus control treatment.

Effect of CE on Angiogenesis Potential of Tumor Cells In Vitro

Given that VEGF is one of the most critical and specific factors stimulating angiogenesis, we next investigated whether CE inhibited angiogenesis by targeting VEGF expression in vitro. To address this question, we used an endothelial cell migration assay to evaluate the activity of conditioned medium derived from tumor cells treated either with CE or water. U251 cells were incubated with either CE, cinnamaldehyde, or vehicle control. The conditioned media from these cells were collected and subjected to migration assay. As shown in Figure 6A, more endothelial cells were found to be able to migrate in response to conditioned medium derived from cells treated with vehicle control than CE or cinnamaldehyde, suggesting that CE, as well as cinnamaldehyde, can inhibit angiogenesis by suppressing VEGF expression in cancer cells.

Figure 6.

Figure 6.

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CE inhibits angiogenesis. (A) CE treatment reduced the capability of tumor conditioned medium to induce endothelial cell migration. Conditioned media from cells treated (24 h) with CE (32 μg/ml), cinnamaldehyde (10 μg/ml), or vehicle control were placed in the bottom chamber of a transwell with HUVEC placed on the top wells. Data are represented as the ratio to the control medium from cells treated with vehicle. *P < 0.05, versus control treatment. (B) CE inhibits tumor growth and ascites formation. Mice were inoculated intraperitoneally with SKOV3 cells and fed via oral gavage with either cinnamon suspension (0.3 mg/g) or water. The ascites volume and weight of tumor nodules were compared between CE and control treated groups. *P < 0.05, versus control treatment. (C) CE inhibits VEGF expression by ovarian tumor. The expression of VEGF mRNA in the tumor was analyzed by qRT-PCR. Data are presented as the ratio to the vehicle control. The expression of VEGF protein in the tumor tissue and ascites were determined by ELISA assay. Data are normalized to total protein content. *P < 0.05, versus control treatment. (D) CE inhibits blood vessel formation. Blood vessels in the tumor were stained with antibody against CD31. Vessel density was quantified and compared between CE treated and water treated group. Scale bar: 50 μm. *P < 0.05, versus control treatment.

Effect of CE on VEGF Expression and Angiogenesis in Human Tumor in Mice

To further investigate the anti-angiogenesis and anti-tumor activity of CE, we evaluated the effect of CE on VEGF expression, angiogenesis, and tumor growth in mice. Mice were inoculated intraperitoneally with SKOV3 human ovarian cells and treated with either CE or vehicle. Our data suggested CE treatment led to a substantial reduction in tumor burden and ascites volume (Figure 6B) but had no detectable effect on body weight and behavior of mice bearing SKOV3 (data not shown). To determine whether CE treatment suppressed VEGF expression in tumors, we assayed VEGF mRNA expression by qRT-PCR and protein expression by ELISA. Both VEGF mRNA and protein expression were significantly decreased in CE treated tumors (Figure 6C). VEGF protein expression in ascites was also reduced (Figure 6C), consistent with our in vitro results. To address whether CE had any effect on tumor angiogenesis, we evaluated blood vessel formation in the tumor by immunostaining with an antibody against CD31. CE treatment led to a significant reduction in blood vessel count in the tumor (Figure 6D). Taken together, our results implicated CE as a potent inhibitor of VEGF expression and angiogenesis.

DISCUSSION

Angiogenesis, the formation of new blood vessels, is an important target for cancer prevention [16,17,37]. Although many anti-angiogenesis agents are available for cancer treatment, their side effects limit their application for cancer prevention [15]. Our goal is to investigate the potential of a diet-based agent as an angiogenesis inhibitor. Previously, we demonstrated an extract from cinnamon, as well as its main component, procyanidins, was a potent inhibitor of angiogenesis through suppressing VEGF receptor kinase activity on endothelial cells [26]. In this study, we identified additional anti-angiogenesis activity of CE and demonstrated its anti-tumor activity in a mouse xenograft model.

We found CE and another main component, cinnamaldehyde, effectively inhibited cancer cell VEGF expression through suppression of HIF-1α gene expression and protein synthesis. Thus, components of cinnamon have dual effects on angiogenesis and tumor cell growth—while cinnamaldehyde prevents the expression of VEGF by cancer cells, procyanidins inhibit the action of VEGF kinase activity on endothelial cells. As a result, CE potently inhibited angiogenesis and tumor growth in mice.

An ovarian tumor model was used to study the anti-tumor activity of CE in mice due to lack of treatment for late stage ovarian cancer and an important role of VEGF in ovarian cancer growth and ascites formation [38]. Ovarian cancer is the most deadly gynecological malignancy in women. The late stage of ovarian cancer is usually associated with peritoneal dissemination and ascites formation. Unlike most solid tumors that are surrounded by stroma, human ovarian cancer is often surrounded by ascites, a unique tumor-friendly microenvironment. Ascites are an abnormal accumulation of fluid in the peritoneal cavity due to lymphatic obstruction and increased vessel permeability. VEGF has been shown to be involved in ovarian cancer progression and ascites formation. Several drugs targeting VEGF have shown varying success [39], however, they are often associated with toxicity. In this study, we found that CE was able to inhibit both tumor growth and ascites formation with little toxicity.

But it remains to be determined whether CE could be used to treat or prevent tumors from growing in humans. One of the main concerns is whether cinnamon can circulate in vivo with sufficient concentration to be effective. Although this information is currently unknown, it may become available soon since a clinical trial is currently being conducted to investigate the bioavailability of cinnamon in healthy human subjects. The bioavailability of cinnamaldehyde has been investigated in rats and the results suggest a sustained high local concentration of cinnamaldehyde in vivo [40]. Recently, cinnamon has been investigated for its anti-diabetic activity. The effective dose for cinnamon is about 100 μg/ml in vitro and between 50 mg/kg and 300 mg/kg in mice [24,41]. These doses are comparable to the doses that we used in our study. Several anti-diabetic studies of cinnamon in patients have shown that cinnamon may help improve blood sugar level when 500 mg–6 g cinnamon was given each day [42,43]. Taken together, these studies suggest that an effective dose of cinnamon might be achievable for anti-cancer activity in human patients.

Due to the proven safety in human use, cinnamon has an advantage over most current anti-VEGF agents. Without toxicity, cinnamon has the potential to be used chronically to improve its anti-tumor efficacy in cancer treatment and prevention. As anti-VEGF agents have been shown to be more effective when combined with chemotherapy agents, cinnamon may also be able to enhance anti-tumor activity of other anti-cancer agents without causing additional toxicity. Future study is needed to explore this potential.

Elevated HIF-1α expression in tumors is associated with increased angiogenesis and tumor growth [3-5]. HIF-1α expression is mainly regulated by protein degradation and protein synthesis [2], but HIF-1α expression can also be controlled at the mRNA level [44]. STAT3 is an important transcription factor regulating HIF-1α expression both at the protein and mRNA levels, through modulating AKT expression [9,33,34]. In this study, our results showed that CE inhibited phosphorylation and expression of STAT3 and AKT, suggesting that the reduced expression of HIF1α and VEGF by CE might be mediated in part via inhibiting STAT3/AKT pathway. However, the precise mechanism by which CE inhibits HIF-1α expression remains to be further elucidated.

HIF-1α has become an important therapeutic target for drug discovery, with many agents developed to suppress its expression. Flavonoids, including epigallocatechins-3-gallate (EGCG), soy, curcumin, resveratrol, apigenin, chrysin, and grape seed extract (GSE), have also been shown to inhibit VEGF expression, mainly through promoting HIF-1α degradation [31,45-53]. Here we show, HIF-1α expression was suppressed by CE through inhibiting HIF-1α mRNA expression.

Although our results suggest that CE inhibits angiogenesis via suppressing VEGF expression and function, it remains to be investigated whether CE inhibits angiogenesis via pathways other than VEGF. Our preliminary results have shown that CE also reduced mRNA expression of other angiogenesis factors, such as IL-8 and bFGF. However, additional study is needed to further explore these possibilities.

Taken together, this study has uncovered a novel activity of cinnamon extract—suppression of VEGF and HIF-1α expression—and further demonstrates that CE is a potent, natural angiogenesis inhibitor that could be used as a safe agent for cancer prevention and treatment.

ACKNOWLEDGMENTS

This work was supported by Stop Cancer Foundation, Concern Foundation, and Markel Friedman Fund (W. Wen). The core facilities used in this study were supported by NCI P30CA033572. We thank Drs. John Lew, Dylan Peterson and Donald Graves for providing the CE and for their suggestions and discussions. We thank Drs. Silvia da Costa and Chris Gandhi for critical reading of this manuscript and members of the Clinical Immunobiology Correlative Studies laboratory Core, Pathology Core, and Animal Facility at the Beckman Research Institute of City of Hope for their technical assistance.

Abbreviations:

CHX

cycloheximide

DFX

deferoxamine mesylate

ELISA

enzyme-linked immunosorbent assay

CE

cinnamon extract

HIF-1

hypoxia-inducing factor

Luc

luciferase

qRT-PCR

quantitative real time PCR

SF

serum free

VEGF

vascular endothelial growth factor

REFERENCES

  • 1.Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–676. [DOI] [PubMed] [Google Scholar]
  • 2.Semenza GL. Targeting Hif-1 for cancer therapy. Nat Rev Cancer 2003;3:721–732. [DOI] [PubMed] [Google Scholar]
  • 3.Schindl M, Schoppmann SF, Samonigg H, et al. Overexpression of hypoxia-inducible factor 1{alpha} is associated with an unfavorable prognosis in lymph node-positive Breast cancer. Clin Cancer Res 2002;8:1831–1837. [PubMed] [Google Scholar]
  • 4.Bachtiary B, Schindl M, Potter R, et al. Overexpression of hypoxia-inducible factor 1{alpha} indicates diminished response to radiotherapy and unfavorable prognosis in patients receiving radical radiotherapy for cervical cancer. Clin Cancer Res 2003;9:2234–2240. [PubMed] [Google Scholar]
  • 5.Hui EP, Chan ATC, Pezzella F, et al. Coexpression of hypoxia-inducible factors 1{alpha} and 2{alpha}, carbonic anhydrase IX, and vascular endothelial growth factor in nasopharyngeal carcinoma and relationship to survival. Clin Cancer Res 2002;8:2595–2604. [PubMed] [Google Scholar]
  • 6.Mizukami Y, Kohgo Y, Chung DC. Hypoxia inducible factor-1 independent pathways in tumor angiogenesis. Clin Cancer Res 2007;13:5670–5674. [DOI] [PubMed] [Google Scholar]
  • 7.Karni R, Dor Y, Keshet E, Meyuhas O, Levitzki A. Activated pp60c-Src leads to elevated hypoxia-inducible factor (HIF)-1alpha expression under normoxia. J Biol Chem 2002;277:42919–42925. [DOI] [PubMed] [Google Scholar]
  • 8.Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL. HER2 (neu) signaling increases the rate of hypoxia-Inducible factor 1{alpha} (HIF-1{alpha}) synthesis: Novel mechanism for HIF-1-Mediated vascular endothelial growth factor expression. Mol Cell Biol 2001;21:3995–4004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Xu Q, Briggs J, Park S, et al. Targeting Stat3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways. Oncogene 2005;24:5552–5560. [DOI] [PubMed] [Google Scholar]
  • 10.Niu G, Wright KL, Huang M, et al. Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene 2002;21:2000–2008. [DOI] [PubMed] [Google Scholar]
  • 11.Pages G, Pouyssegur J. Transcriptional regulation of the Vascular Endothelial Growth Factor gene-a concert of activating factors. Cardiovasc Res 2005;65:564–573. [DOI] [PubMed] [Google Scholar]
  • 12.Arany Z, Foo S-Y, Ma Y, et al. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1[agr]. Nature 2008;451:1008–1012. [DOI] [PubMed] [Google Scholar]
  • 13.Noble MEM, Endicott JA, Johnson LN. Protein kinase inhibitors: Insights into drug design from structure. Science 2004;303:1800–1805. [DOI] [PubMed] [Google Scholar]
  • 14.Ellis LM, Hicklin DJ. VEGF-targeted therapy: Mechanisms of anti-tumour activity. Nat Rev Cancer 2008;8:579–591. [DOI] [PubMed] [Google Scholar]
  • 15.Kamba T, McDonald DM. Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer 2007;96:1788–1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bhat TA, Singh RP. Tumor angiogenesis—A potential target in cancer chemoprevention. Food Chem Toxicol 2008;46:1334–1345. [DOI] [PubMed] [Google Scholar]
  • 17.Albini A, Noonan DM, Ferrari N. Molecular pathways for cancer angioprevention. Clin Cancer Res 2007;13:4320–4325. [DOI] [PubMed] [Google Scholar]
  • 18.Adlercreutz HMY, Clark J, Höckerstedt K, et al. Dietary phytoestrogens and cancer: In vitro and in vivo studies. J Steroid Biochem Mol Biol 1992;41:331–337. [DOI] [PubMed] [Google Scholar]
  • 19.Aggarwal BB, Shishodia S. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol 2006;71:1397–1421. [DOI] [PubMed] [Google Scholar]
  • 20.Jarvill-Taylor KJ, Anderson RA, Graves DJ. A hydroxychalcone derived from cinnamon functions as a mimetic for insulin in 3T3-L1 adipocytes. J Am Coll Nutr 2001;20:327–336. [DOI] [PubMed] [Google Scholar]
  • 21.Anderson RA. Chromium and polyphenols from cinnamon improve insulin sensitivity. Proc Nutr Soc 2008;67:48–53. [DOI] [PubMed] [Google Scholar]
  • 22.Singh G, Maurya S, deLampasona MP, Catalan CAN. A comparison of chemical, antioxidant and antimicrobial studies of cinnamon leaf and bark volatile oils, oleoresins and their constituents. Food Chem Toxicol 2007;45:1650–1661. [DOI] [PubMed] [Google Scholar]
  • 23.Imparl-Radosevich J, Deas S, Polansky MM, et al. Regulation of PTP-1 and insulin receptor kinase by fractions from cinnamon: Implications for cinnamon regulation of insulin signalling. Horm Res 1998;50:177–182. [DOI] [PubMed] [Google Scholar]
  • 24.Cao H, Polansky MM, Anderson RA. Cinnamon extract and polyphenols affect the expression of tristetraprolin, insulin receptor, and glucose transporter 4 in mouse 3T3-L1 adipocytes. Arch Biochem Biophys 2007;459:214–222. [DOI] [PubMed] [Google Scholar]
  • 25.Peterson DW, George RC, Scaramozzino F, et al. Cinnamon extract inhibits tau aggregation associated with alzheimer's disease in vitro. J Alzheimers Dis 2009;7:585–597. [DOI] [PubMed] [Google Scholar]
  • 26.Lu J, Zhang K, Nam S, Anderson RA, Jove R, Wen W. Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling. Carcinogenesis 2010;31:481–488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Schoene NW, Kelly MA, Polansky MM, Anderson RA. Water-soluble polymeric polyphenols from cinnamon inhibit proliferation and alter cell cycle distribution patterns of hematologic tumor cell lines. Cancer Lett 2005;230:134–140. [DOI] [PubMed] [Google Scholar]
  • 28.Kwon H-K, Jeon WK, Hwang J-S, et al. Cinnamon extract suppresses tumor progression by modulating angiogenesis and the effector function of CD8+ T cells. Cancer Lett 2009;278:174–182. [DOI] [PubMed] [Google Scholar]
  • 29.Bansode RR, Leung T, Randolph P, Williams LL, Ahmedna M. Cinnamon extract inhibits angiogenesis in zebrafish and human endothelial cells by suppressing VEGFR1, VEGFR2, and PKC-mediated MAP kinase. Food Sci Nutr 2013;1:74–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Anderson RA, Broadhurst CL, Polansky MM, et al. Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity. J Agric Food Chem 2004;52:65–70. [DOI] [PubMed] [Google Scholar]
  • 31.Lu J, Zhang K, Chen S, Wen W. Grape seed extract inhibits VEGF expression via reducing HIF-1alpha protein expression. Carcinogenesis 2009;30:636–644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wen W, Lu J, Zhang K, Chen S. Grape seed extract inhibits angiogenesis via suppression of the vascular endothelial growth factor receptor signaling pathway. Cancer Prev Res 2008;1:554–561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yu H, Jove R. The STATs of cancer-new molecular targets come of age. Nat Rev Cancer 2004;4:97–105. [DOI] [PubMed] [Google Scholar]
  • 34.Lee H, Herrmann A, Deng JH, et al. Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer Cell 2009;15:283–293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Rius J, Guma M, Schachtrup C, et al. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature 2008;453:807–811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Bae W-Y, Choi J-S, Kim J-E, Jeong J-W. Cinnamic aldehyde suppresses hypoxia-induced angiogenesis via inhibition of hypoxia-inducible factor-1α expression during tumor progression. Biochem Pharmacol 2015;1:41–50. [DOI] [PubMed] [Google Scholar]
  • 37.Folkman J. Tumor angiogenesis: Therapeutic implications. N Engl J Med 1971;285:1182–1186. [DOI] [PubMed] [Google Scholar]
  • 38.Vaughan S, Coward JI, Bast RC Jr., et al. Rethinking ovarian cancer: Recommendations for improving outcomes. Nat Rev Cancer 2011;11:719–725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Han ES, Wakabayashi M, Leong L. Angiogenesis inhibitors in the treatment of epithelial ovarian cancer. Curr Treat Options Oncol 2013;14:22–33. [DOI] [PubMed] [Google Scholar]
  • 40.Zhao H, Xie Y, Yang Q, et al. Pharmacokinetic study of cinnamaldehyde in rats by GC-MS after oral and intravenous administration. J Pharm Biomed Anal 2014;89:150–157. [DOI] [PubMed] [Google Scholar]
  • 41.Kim SH, Hyun SH, Choung SY. Anti-diabetic effect of cinnamon extract on blood glucose in db/db mice. J Ethnopharmacol 2006;104:119–123. [DOI] [PubMed] [Google Scholar]
  • 42.Medagama AB. The glycaemic outcomes of Cinnamon, a review of the experimental evidence and clinical trials. Nutr J 2015;14:108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Anderson RA, Zhan Z, Luo R, et al. Cinnamon extract lowers glucose, insulin and cholesterol in people with elevated serum glucose. J Tradit Complement Med 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Page EL, Robitaille GA, Pouyssegur J, Richard DE. Induction of hypoxia-inducible factor-1alpha by transcriptional and translational mechanisms. J Biol Chem 2002;277:48403–48409. [DOI] [PubMed] [Google Scholar]
  • 45.Fu B, Xue J, Li Z, Shi X, Jiang B-H, Fang J. Chrysin inhibits expression of hypoxia-inducible factor-1{alpha} through reducing hypoxia-inducible factor-1{alpha} stability and inhibiting its protein synthesis. Mol Cancer Ther 2007;6:220–226. [DOI] [PubMed] [Google Scholar]
  • 46.Fang J, Zhou Q, Liu L-Z, et al. Apigenin inhibits tumor angiogenesis through decreasing HIF-1{alpha} and VEGF expression. Carcinogenesis 2007;28:858–864. [DOI] [PubMed] [Google Scholar]
  • 47.Zhang Q, Tang X, Lu Q, Zhang Z, Rao J, Le AD. Green tea extract and (−)-epigallocatechin-3-gallate inhibit hypoxia-and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Mol Cancer Ther 2006;5:1227–1238. [DOI] [PubMed] [Google Scholar]
  • 48.Guo Y, Wang S, Hoot DR, Clinton SK. Suppression of VEGF-mediated autocrine and paracrine interactions between prostate cancer cells and vascular endothelial cells by soy isoflavones. J Nutr Biochem 2007;18:408–417. [DOI] [PubMed] [Google Scholar]
  • 49.Zhang Q, Tang X, Lu QY, Zhang ZF, Brown J, Le AD. Resveratrol inhibits hypoxia-induced accumulation of hypoxia-inducible factor-1{alpha} and VEGF expression in human tongue squamous cell carcinoma and hepatoma cells. Mol Cancer Ther 2005;4:1465–1474. [DOI] [PubMed] [Google Scholar]
  • 50.Raina K, Singh RP, Agarwal R, Agarwal C. Oral grape seed extract inhibits prostate tumor growth and progression in TRAMP mice. Cancer Res 2007;67:5976–5982. [DOI] [PubMed] [Google Scholar]
  • 51.Sartippour MR, Shao Z-M, Heber D, et al. Green tea inhibits vascular endothelial growth factor (VEGF) induction in human Breast cancer cells. J Nutr 2002;132:2307–2311. [DOI] [PubMed] [Google Scholar]
  • 52.Fang J, Xia C, Cao Z, Zheng JZ, Reed E, Jiang B-H. Apigenin inhibits VEGF and HIF-1 expression via PI3 K/AKT/p70S6K1 and HDM2/p53 pathways. FASEB J 2005;19:342–353. [DOI] [PubMed] [Google Scholar]
  • 53.Ajaikumar BK, Parmeswaran D, Preetha A, et al. Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX-2, MMP-9, VEGF and CXCR4 expression in an orthotopic mouse model. Int J Cancer 2009;125:2187–2197. [DOI] [PubMed] [Google Scholar]

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