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Chrysin Inhibits Lymphangiogenesis in Vitro
Orawin Prangsaengtong Sirivan AthikomkulchaiJiuxiang XuKeiichi KoizumiAkiko InujimaNaotoshi ShibaharaYutaka ShimadaSarin TadtongSuresh Awale
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2016 Volume 39 Issue 4 Pages 466-472

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Abstract

The induction of lymphangiogenesis is an important process to promote cancer growth and cancer metastasis via the lymphatic system. Identifying the compounds that can prevent lymphangiogenesis for cancer therapy is urgently required. Chrysin, 5,7-dihydroxyflavone, a natural flavone extracted from Thai propolis, was used to investigate the effect on the lymphangiogenesis process of TR-LE, rat lymphatic endothelial cells. In this study, maximal nontoxic doses of chrysin on TR-LE cells were selected by performing a proliferation assay. The process of lymphangiogenesis in vitro was determined by cord formation assay, adhesion assay and migration assay. Chrysin at a nontoxic dose (25 μM) significantly inhibited cord formation, cell adhesion and migration of TR-LE cells when compared with the control group. We also found that chrysin significantly induced vascular endothelial growth factor C (VEGF-C) mRNA expression and nitric oxide (NO) production in TR-LE cells which was involved in decreasing the cord formation of TR-LE cells. In conclusion, we report for the first time that chrysin inhibited the process of lymphangiogenesis in an in vitro model. This finding may prove to be a natural compound for anti-lymphangiogenesis that could be developed for use in cancer therapy.

Cancer metastasis is the process by which cancer cells spread from the primary tumor to other parts of the body and then establish themselves in other tissues. Metastasis is responsible for most cancer deaths.1) Blood circulation and lymphatic circulation are the common routes for cancer metastasis. However, compared to blood vessels, lymphatic vessels seem to have a greater permeability for cancer metastasis due to the walls of lymphatic vessels being thinner and contain fewer tight junctions than those of blood vessels. In addition, lymphatic capillaries, absent basal lamina and lack associated pericytes led to an easy opening for macromolecular, such as cancer cells uptake into the lymphatic vessels. The prevention of newly lymphatic and blood vessels formation will improve ways to prevent cancer growth and metastatic disease.2)

The formation of lymphatic vessels from pre-existing endothelium is referred to lymphangiogenesis. The physiological functions of lymphatic vasculature is that it regulates the maintenance of tissue fluid homeostasis, immune cell trafficking and immune surveillance, and absorption of dietary fat and fat-soluble vitamins. The pathological conditions of promotion of lymphatic vasculature is found within several diseases, such as inflammation and tumor metastasis, while disruption in formation and function of the lymphatic network results in lymphedema and wound healing impairment.3) Lymphangiogenesis has been shown to be controlled by vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor receptor 3 (VEGFR-3), and other molecules such as hypoxia-inducible factor 1-alpha (HIF-1α), endothelial nitric oxide synthase (eNOS) and the heat shock protein 90 (Hsp90).2,4,5) Although, the molecular mechanisms of lymphangiogenesis which are currently investigated, when compared with knowledge of angiogenesis, are still obscure.6)

Therefore, the lymphatic endothelial cell line would help to clarify the processes or cell behaviors during lymphangiogenesis. We established a rat lymphatic endothelial cell line (TR-LE) from the thoracic duct of a transgenic rat, harboring a temperature-sensitive simian virus 40 (SV40) large T-antigen, for using in this study due to that they showed the markers of lymphatic endothelial cells and were able to form a continuous extensive network of thick cords and lumens.7)

Flavonoids are commonly found in plants, vegetables and fruits. Many studies have demonstrated flavonoids to be a scavenger of oxygen-derived free radicals and also possess anti-cancer, anti-inflammatory, anti-allergic and, anti-viral properties.8) Chrysin, 5,7-dihydroxyflavone (molecular formula: C15H10O4, molecular weight: 254.2375 g/mol) (Fig. 1), is a naturally occurring flavone extracted from plants, fruits, honey, and propolis. Chrysin has multiple bioactivities such as anti-diabetic, anti-estrogenic, anti-inflammatory, anti-allergic, anti-oxidant, anti-bacterial and antitumor activities. In vitro and in vivo models have shown that chrysin inhibits cancer growth and metastasis by induction of apoptosis, alteration of cell cycle and inhibition of angiogenesis and invasion through the modulation of multiple cell signaling pathways.9) However, the information between chrysin, lymphatic endothelial cells and lymphangiogenesis is not currently available.

Fig. 1. Structure of Chrysin

In this study we found the effect of chrysin that extracted from Thai propolis10) suppressed cord formation ability, cell adhesion and cell migration ability of human lymphatic endothelial cells which were involved in the induction of VEGF-C mRNA expression and nitric oxide (NO) production. This finding revealed the function of chrysin as an anti-lymphangiogenesis.

MATERIALS AND METHODS

Preparation of Extracts

Thai propolis was harvested from a hive of Apis mellifera collected in Chiangmai province, Thailand, in August 2006. A voucher specimen (SWU 0212) was deposited at the Faculty of Pharmacy, Srinakharinwirot University, Nakhon Nayok province, Thailand.10) The propolis (1 kg) was extracted by sonication with methanol (2 L, 90 min×3) at room temperature. After removal of methanol, the methanol extract (517 g) was yielded. Part of the methanol extract (140 g) was subjected to silica gel column chromatography using MeOH–CH2Cl2 gradient system and gave 16 fractions. Chrysin (732 mg) was isolated by crystallization from fraction 8 (9.5 g), 2% MeOH–CH2Cl2 eluate, and was identified by comparison of physical and spectroscopic data with authentic standard and literature values.1012) Chrysin was dissolved in dimethyl sulfoxide (DMSO) for further experiments.

TR-LE Cells

TR-LE cells, a conditionally immortalized temperature sensitive rat lymphatic endothelial cell line, were maintained on 10 μg/mL fibronectin (Iwaki Glass, Tokyo, Japan) pre-coated dishes in HuMedia-EG2 (Kurabo, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (ICN Biomedicals, Inc., Aurora, OH, U.S.A.) at 33°C, a permissive temperature with 5% CO2. The cells were transferred to 37°C with 5% CO2 and then cultured for 48 h before the experiments was performed.7)

Proliferation Assay

TR-LE cells (2×104/50 μL/well) were seeded in a 96-well plate and allowed to adhere for 2 h. Then 50 μL of 10% FBS containing medium with various doses of chrysin were added to each well. At 24 h of incubation, 10 μL of WST-8 proliferation assay kit (DOJINDO, Kumamoto, Japan) was added and incubated for 2 h at 37°C with 5% CO2. Absorbance was measured at 450 nm to determine % cell viability.4,13)

Cord Formation Assay

TR-LE cells (3×102 cells/well) were seeded on 96 well plates that were pre-coated with Matrigel (10 mg/mL) and allowed to polymerize at 37°C. The cells were cultured with and without chrysin containing medium for 2, 4, and 6 h. At each time point, cells were fixed with a 4% paraformaldehyde fixative and stained using Mayer’s hematoxylin (Muto Pure Chemical, Tokyo, Japan). The cord network was photographed and cord length was measured using an Angiogenesis Image Analyzer Program (Kurabo).4,13)

Cell Adhesion Assay

Cells were (2×104 cells/well) were seeded on 96-well plate, which pre-coated with 100 μg/mL of Matrigel. Cells were cultured in chrysin containing medium at 37°C for 30 min. Non-adherent cells were washed with phosphate buffered saline (PBS). Attached cells were fixed with a 4% paraformaldehyde fixative for 30 min and stained with 20% formalin for 30 min and stained 4% paraformaldehyde fixative and stained using Mayer’s hematoxylin (Muto Pure Chemical). Cells were photographed and then counted. This method was described previously with some modification.5,13)

Cell Migration Assay

Cell migration was assayed using Falcon cell culture PET inserts with a pore size of 8 μm and a 24-well format (No. 353097; Falcon, Franklin Lakes, NJ, U.S.A.). The lower surface of filters was coated with 5 μg/mL Matrigel. Cells were added to the upper compartment of the chamber and 600 mL normal culture medium was added to the lower compartment. The chambers were incubated for 24 h at 37°C. Cells that had not migrated were removed with cotton swabs. The migrated cells on the filter were fixed with methanol, stained with Mayer’s hematoxylin and 1% eosin Y solution (Muto Pure Chemical), photographed and counted.5)

Real-Time Polymerase Chain Reaction (PCR)

We evaluated the mRNA expression of VEGF-C in TR-LE by a real-time PCR. The total RNA was extracted from cultured using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.). For each sample, 0.5 μg of total RNA was reverse transcribed into cDNA using the Prime Script RT reagent kit (Perfect Real Time) (TaKaRa, Dalian, China). Real-time PCR analysis was performed using the LightCycler Nano System (Roche Diagnostics, Mannheim, Germany) using FastStart Essential DNA Green Master (Roche Diagnostics) according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The relative quantification of mRNA expression was calculated as a ratio of the target gene to GAPDH. The primer sequences were as follows: VEGF-C sense, 5′-GCC AAT CAC ACT TCC TGC CG-3′, and VEGF-C antisense, 5′-GTG ATG TAG TAG CTG CAT GAT CG-3′; GAPDH sense, 5′-GTG AGG TGA CCG CAT CTT CT-3′, and GAPDH antisense, 5′-TGG AAG ATG GTG ATG GGT TT-3′.4,7,14)

Griess Assay

Detection of NO release was measured in the culture medium using the colorimetric Griess assay. Nitrite, a stable end product of NO, accumulation was determined by mixing equal volumes of cell culture medium and Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine, and 5% H3PO4). Absorbance (A540) was measured using a microplate reader.15)

Statistical Analysis

Statistical significance was assessed by one-way ANOVA, with Dunnett’s test and by Student’s t-test. p-Values of less than 0.05 were considered to be significant.

RESULTS

Toxicity Effect of Chrysin on TR-LE Cells

To find out the nontoxic doses of chrysin on TR-LE cells, cells were exposed to various concentrations of chrysin (0–200 μM) for 24 h and then performed proliferation assay to determine % cell viability. Figure 2 shows that chrysin at concentration 50, 100 and 200 μM were toxic to cells by decreasing % cell viability by 74.5, 66.3, and 70.8%, respectively (*p<0.05, **p<0.01) when compared with the control. The result indicated that chrysin at concentration 0–25 μM had no harmful effect on the cells. A maximal nontoxic dose of chrysin at 25 μM was selected for use in the following studies. The concentration of DMSO used in these studies was 0.2%.

Fig. 2. Cytotoxicity Effect of Chrysin on TR-LE Cells

To identify non-toxic doses of chrysin, TR-LE cells were incubated with various doses (0–200 μM) of chrysin in a 96 well plate for 24 h. Cell viability was determined by proliferation assay after treatment. The maximal non-toxic dose was chosen for further experiments. A representation experiment is shown as the mean±S.D. for three wells (* p<0.05, ** p<0.01) compared with solvent control (0.2% DMSO) (ANOVA with Dunnett’s test). Similar results were observed from three independent experiments.

Non-toxic Dose of Chrysin Decreased Cord Formation Ability of TR-LE Cells

To investigate the effect of chrysin on lymphangiogenesis in vitro, we evaluated the cord length of TR-LE cells on 10 mg/mL Matrigel which incubated chrysin at 25 μM, a maximal non-toxic dose by using cord formation assay. The pattern of cord formation of TR-LE cells that incubated in normal culture medium started to change morphology and formed cord at 2 h and the length of the cord network was increased to their maximum levels at 4 h. The cords were slightly declined at 6 h of incubation (Figs. 3A, B). After exposure to 25 μM chrysin, TR-LE cells decreased their cord formation ability on Matrigel which was observed from 4 h by 87.7%, and significantly decreased cord formation ability of TR-LE cells at 6 h by 57.3% when compared with 100% of control (0.025% DMSO) (*p<0.05) (Fig. 3C).

Fig. 3. Effect of Chrysin on Cord Formation of TR-LE Cells

TR-LE cells were resuspended in a culture medium containing 25 μM chrysin and 0.025% control DMSO before seeding on Matrigel-coated dishes. After 2, 4, 6 h of incubation, cord formation of TR-LE cells were photographed (A), the whole length of the cord was measured by Angiogenesis Image Analyzer program and represented in arbitrary units (B). The cord lengths were calculated to be percentage compared with 100% control in each time point (C). A representation experiment is shown as the mean±S.D. for three wells (* p<0.05) (B) compared with solvent control (0.025% DMSO) that was calculated to be 100% at each time point (Student’s t-test). The similar results were observed from three independent experiments.

Non-toxic Dose of Chrysin Decreased Adhesion and Migration Ability of TR-LE Cells

The process of lymphangiogenesis in vitro initiated from cell adhesion to extracellular matrix, this was then followed by migration for forming cord networks.16) We investigated the effect of chrysin in the adhesion of TR-LE cells on the well that pre-coated with 100 μg/mL Matrigel and performed cell adhesion assay. Figure 4 shows that chrysin at concentration 25 μM significantly inhibited cell adhesion ability by 66.0% suppression (**p<0.01) when compared with the 100% control (0.025% DMSO). Following, the effect of chrysin on chemotactic migration of TR-LE cells was determined by using transmigration chambers and performed cell migration assay. Cells were migrated to the lower part of the chamber which was pre-coated with 5 μg/mL Matrigel. The density of migrated cells was shown in Fig. 5A. The cell migration ability of TR-LE cells was significantly suppressed to 78.8% when cultured with 25 μM of chrysin for 24 h of incubation (*p<0.05) compared with 100% control (0.025% DMSO) (Fig. 5B).

Fig. 4. Effect of Chrysin on TR-LE Cell Adhesion

TR-LE cells were resuspened in culture medium which contained 25 μM chrysin and 0.025% control DMSO before seeding on 96-well plates which were pre-coated with 100 μg/mL Matrigel. After 30 min of incubation, nonadherent cells were washed away. Adherent cells that had performed adhesion assay and photographed (A) were then counted. The data is shown as the mean±S.D. for three wells (** p<0.01) (B) compared with the solvent control (0.025% DMSO) that had been calculated to be 100% (Student’s t-test). Similar results were observed from three independent experiments.

Fig. 5. Effect of Chrysin on TR-LE Cell Migration

Serum-starved TR-LE cells were seeded on the upper part of filters which had been pre-coated with 5 μg/mL Matrigel on the lower surface of the filters. The culture medium that contained 25 μM chrysin and DMSO (0.025%) was added to the lower compartment of the chambers. After 24 h of incubation, non-migrated cells on the upper surface of the filter were removed and cells that had migrated to the lower surface of the filter by performing migration assay were photographed (A) and counted. The data is expressed as the mean±S.D. for three wells (** p<0.01) (B) compared with the solvent control (0.025% DMSO) that was calculated to be 100% (Student’s t-test). Similar results were observed from three independent experiments.

Non-toxic Dose of Chrysin Enhanced VEGF-C mRNA Expression and NO Production of TR-LE Cells

Vascular endothelial growth factor C (VEGF-C) is a protein that is a member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. The main function of VEGF-C is the promotion of lymphangiogenesis and mediates tumor cell dissemination and the formation of lymph node metastasis. VEGF-C stimulate eNOS-derived NO production which plays a causal role in VEGF-C-induced lymphangiogenesis, lymphatic hyperplasia, and lymphatic metastasis in vivo.17) Chrysin, which inhibited cord formation of TR-LE cells in this study, may correlate with VEGF-C mRNA expression and NO production. TR-LE cells after treatment with 25 μM chrysin and 0.025% control DMSO shows the significantly increasing of VEGF-C mRNA levels at 12 and 24 h of incubation (Fig. 6A) and also significantly increasing NO production levels (Fig. 6B) at 24 h of incubation (**p<0.01). These results could be concluded that chrysin enhanced VEGF-C mRNA expression and NO production in TR-LE cells.

Fig. 6. Effect of Chrysin on VEGF-C mRNA Expression and NO Production of TR-LE Cells

TR-LE cells were treated in culture medium which contained 25 μM chrysin and 0.025% control DMSO for 12 and 24 h. Then cells were subjected to real-time PCR and Griess assay. (A) VEGF-C mRNA levels. (B) NO production levels. Similar results were obtained in three independent experiments; ∗ p<0.05, ∗∗ p<0.01 compared with the control (Student’s t-test).

DISCUSSION

This present study shows that chrysin, 5,7-dihydroxyflavone (Fig. 1) extracted from Thai propolis (Western honeybee; Apis mellifera)10) significantly inhibited cord formation, cell adhesion and cell migration in TR-LE cells, a conditionally immortalized rat lymphatic endothelial cell line.

Chrysin has the multiple bioactivities in vitro and in vivo models that are involved in cancer treatment, such as induction of apoptosis and inhibition of cancer metastasis through the modulation of multiple pathways.9,11,12) In addition, chrysin inhibits angiogenesis by suppression of endothelial cell proliferation and migration in human umbilical vascular endothelial cells (HUVEC).1821) However, the potency of chrysin on lymphangiogenesis is not currently available. The chrysin at non-toxic concentration 25 μM (Fig. 2) was chosen to observe the effect on the cord formation ability of TR-LE cells. We performed cord formation assay by seeding TR-LE cells on Matrigel, which mimics extracellular matrix that derives from a mouse tumor which is rich in extracellular matrix proteins and growth factors.22) In control group, TR-LE cells, after seeding on Matrigel, were adhered to extracellular matrix for cell stabilization, survival, and migration toward their targets, which is important for sprouting and finding each other, leading to changed morphology to form cord networks.16) The maximal length of networks can be observed at 4 h, and then slightly declined at 6 h of incubation (Fig. 3). Chrysin significantly inhibited the cord formation ability of TR-LE cells at 6 h on Matrigel. The length of cords was shorten (Fig. 3) when compared with control. The lymphatic endothelial cell behaviors such as cell adhesion and migration which are important processes for lymphangiogenesis in vitro16) also observed after chrysin treatment. We found that chrysin significantly inhibited cell adhesion (Fig. 4) and cell migration abilities (Fig. 5). The decreasing of these behaviors led to decreased ability of cord formation of TR-LE cells on Matrigel (Fig. 3).

Chrysin has been reported that inhibited angiogenesis and metastatic growth in several mechanisms such as inhibited VEGF expression via inhibition of signal transducer and activator of transcription (STAT) 3 activation and down-regulation of VEGF through the phosphorylation of extracellular signal-regulated kinase (ERK)/AKT in lipopolysaccharide (LPS) and interleukin-6 (IL-6)-induced angiogenesis models.11,12,20) In addition, chrysin also reported to inhibit cell migration and tube formation ability of HUVECs which induced by several stimulators such as VEGF and IL-6 through suppression of the sIL-6R/gp130/Janus kinase (JAK) 1/STAT3/VEGF signaling pathway.18,20,21,23) However, the mechanisms of chrysin on lymphatic endothelial cells have not been proved.

Lymphangiogenesis has been shown to be controlled by VEGF-C, VEGF-D, VEGFR-32) and also involved in the interaction between eNOS and Hsp90 which is possible to produce NO.5) The main function of VEGF-C is promotion of lymphangiogenesis and mediates tumor cell dissemination and the formation of lymph node metastasis. In this study, we found that chrysin significantly increased VEGF-C mRNA levels and NO production in TR-LE cells at 12 h (Fig. 6A) and 24 h (Fig. 6B) of incubation, respectively, while cord formation ability on Matrigel after treatment with chrysin significantly decreased from 6 h (Fig. 3B) to 12 and 24 h without recovery (data not shown) when compared with control. It has been reported that eNOS plays a crucial role in lymphangiogenesis in vivo by mediating VEGF-C signaling and lymphatic metastasis.17) eNOS-mediated cord formation by human lymphatic endothelial cells by interaction between eNOS and Hsp90 promotes the activation of eNOS and the release of NO.17) However, the production of NO can protect against cellular damage and also act as cytotoxicity to cells from reactive oxygen species which are depending on the relative amounts of each which are present in the target cell or its environment at a particular time.24,25) It has been reported that chrysin stimulated endothelial NO production from endothelial cells which were isolated from rat aorta.26) The increasing of NO could promote oxidative stress in TR-LE cells lead to endothelial cell dysfunction, alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-kappaB (NF-κB).26) The chrysin mainly inhibits cord length elongation due to the inhibitory effects on adhesion and migration in TR-LE cells (Figs. 35). NF-κB act as a transcriptional regulation of endothelial cell adhesion molecules such as E-selectin and intercellular adhesion molecule 1 (ICAM-1) which are surface expressions of adhesion molecules on endothelial cells and play a role during cell adhesion to extracellular matrix. Chrysin significantly attenuated these surface adhesion molecules by inhibition their transcription factor NK-κB function led to decreasing adhesion and migration abilities of endothelial cells.2729) This study provides novel targets of chrysin for anti-lymphangiogenic therapy. However, the details of chrysin on oxidative stress in TR-LE cells require further proof in vitro and also in vivo.

In conclusion, chrysin mainly inhibits cord length elongation because of inhibitory effects on adhesion and migration in TR-LE cells which are involved in VEGF-C mRNA expression and NO production. This is the first information taken from the study of chrysin, which has the potential to inhibit lymphangiogenesis in vitro.

Acknowledgments

The authors wish to acknowledge the Faculty of Pharmacy, Srinakharinwirot University, Nakornnayok, Thailand for the research funding (Grant number 317/2557). We also acknowledge Dr. Chuda Chittasupho, the Research Center for Drug Discovery and Development, Faculty of Pharmacy, Srinakharinwirot University, Thailand and the Department of Kampo Diagnostics, University of Toyama, Japan for supplying the facilities and equipment.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2016 The Pharmaceutical Society of Japan
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