HETEROCYCLES

Short Paper | Regular issue | Vol. 92, No. 2, 2016, pp. 353-360
Received, 5th December, 2015, Accepted, 6th January, 2016, Published online, 14th January, 2016.
DOI: 10.3987/COM-15-13386

■ Inhibition of NF-κB and Cellular Invasion by Novel Flavonoid Dismal in Ovarian Carcinoma Cells

Liyan Wang, Yinzhi Lin, Kulrawee Sidthipong, Jianqiang Tang, Mengjie Li, Takashi Koyano, Thaworn Kowithayakorn, Kengo Sumiyoshi, Tamami Ukaji, and Kazuo Umezawa*

Molecular Target Medicine, School of Medicine, Aichi Medical university, 1-1 Yazako-Karimata, Nagakute-shi, Aichi 480-1195, Japan

Abstract

In the course of our screening of NF-κB inhibitors, we isolated known flavonoids that inhibit LPS-induced NO production in mouse monocytic leukemia RAW264.7 cells. Since those flavonoids inhibited the NF-κB activity, we have evaluated the inhibitory activity of plant-derived novel flavonoid, desmal, on NF-κB, and found that it inhibits LPS-induced NO production and NF-κB. It also inhibited cellular invasion possibly by the decrease of NF-κB-dependent urokinase-type plasminogen activator. Thus, desmal was found to be a new NF-κB inhibitor having cellular anti-inflammatory and anti-metastatic activities.

Microorganisms and plants are useful sources of antibiotics, anticancer agents, and disease-related enzyme inhibitors. We are screening bioactive metabolites from microorganisms that may suppress inflammation and cancer. We have previously isolated protein-tyrosine kinase inhibitors, protein-tyrosine phosphatase inhibitors, and lipopolysaccharide function inhibitors.
Previously, we designed and synthesized dehydroxymethylepoxyquinomicin (DHMEQ) as a specific inhibitor of NF-κB.1, 2 DHMEQ showed various anti-inflammatory and anticancer activities in animal disease models.3 More recently, we have isolated 9-methylstreptimidone from a microorganism as a compound that inhibits LPS-induced NO production.4 Since the yield of this reported compounds by the producing organism is poor, we designed a simplified structure DTCM-glutarimide.5 This compound showed anti-inflammatory activity in animal heart transplantation model6 and ameliorated the animal model of inflammatory bowel disease.7 Therefore, we are continuing to screen new NF-κB inhibitors from microorganisms and plants. In the course of screening, we have isolated several flavonoids that inhibit LPS-induced NO production. In one hand, we have previously isolated novel flavonoid, desmal, as an inhibitor of protein-tyrosine kinase from a plant of Thailand.8 In the present research we found that novel flavonoid, desmal, inhibited NF-κB, and that it inhibited NO production in a macrophage-like cell line and cancer cell migration and invasion.
We screened Streptomyces culture filtrates for the inhibitors of LPS-induced NO production in mouse macrophage-like cell line RAW264.7 cells. The effect of the culture filtrates on cell death in each cell line was measured using the MTT. Effective culture filtrates without toxicity were selected. Microbial culture filtrates were kindly supplied from Meiji Seika Pharma Co., Ltd., Tokyo, Japan. Fermentation medium was extracted with aqueous acetone. The extract was filtered and evaporated in vacuo to remove the acetone, and the aqueous residue was extracted with ethyl acetate (pH 7.0). Among about 2000 ethyl acetate extract samples AFE72940 inhibited LPS-induced NO production without toxicity. The active substance was then purified and isolated from the obtained microbial culture solution as follows. The extract AFE72940 (2.96 g) was applied to a Sephadex LH-20 column chromatography (CC) eluted with CHCl3-MeOH (1 : 1) to give 8 fractions (Fr.1 – Fr.8), Fr.7 was then purified by HPLC with an ODS column eluted with MeOH : H2O (50 : 50 – 100 : 0, 0-20 min, 2.5 mL/min) to yield genistein (1) (2.5 mg, tR 16.3 min). Genistein 7-O-α-L-rhamnoside (2) (26.7 mg, tR 10.1 min) was obtained from Fr.6, daidzein 6-O-methyl-7-O-α-L-rhamnoside (3) (2.7 mg, tR 7.2 min) and daidzein 7-O-α-L-rhamnoside (4) (9.5 mg, tR 10.6 min) was obtained from Fr.4, by using the same HPLC method.

Thus we obtained 4 reported genistein (1), genistein 7-O-α-L-rhamnoside (2), daidzein 6-O-methyl-7-O-α-L-rhamnoside (3), and daidzein 7-O-α-L-rhamnoside (4) (Figure. 1A). The inhibitory activity and toxicity of these isoflavones are shown in Figures. 1B and 1C, respectively. Genistein (1) and its glycoside (2) showed the potent inhibitory activity at the nontoxic concentrations. Daidzein 7-O-α-L-rhamnoside (4) showed weaker inhibitory activity, and daidzein 6-O-methyl-7-O-α-L-rhamnoside (3) did not show prominent activity. Anti-inflammatory activities of genistein9 and daidzein10 were already reported. We previously isolated a novel flavone desmal (Figure 1A) from a Thai plant Desmos chinensis as an inhibitor of protein-tyrosine kinase.8 Its other biological activity has not been reported. Since genistein and its glycosides showed the inhibitory activity, we tested the inhibitory activity of desmal. The cytotoxicity is shown in Figure 2A, in which desmal of 10 μg/mL is not toxic at all, and that of 30 μg/mL slightly decreases the viability. As shown in Figure 2B, desmal inhibited the NO production at 10-30 μg/mL. NO is produced by inducible NO synthase (iNOS) which is dependent on NF-κB. Then, we studied the effect on LPS-induced NF-κB activation. As a result, it inhibited the activation at 10-30 μg/mL (Figure 2C).

We previously reported that NF-κB inhibitor DHMEQ inhibited cellular invasion in ovarian clear cell carcinoma ES-2 cells.11 Then, we studied the effect of desmal on cellular migration and invasion in ES-2 cells. ES-2 cells were shown to be more resistant than RAW264.7 cells to desmal, and desmal below 50 μg/ml mL was not toxic (Figure 3A). Desmal inhibited the migration (Figure 3B) and invasion (Fig. ure 3C) at the nontoxic concentrations. Urokinase-type plasminogen activator (uPA) is one of the enzymes that accelerate cellular invasion. Desmal was found to lower the uPA mRNA expression (Figure 3D). The uPA expression was measured by PCR as described before.11 UK122 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), a uPA inhibitor, did not decrease the viability of cells at 30 μM in 24 h (data not shown). It inhibited the invasion at 30 μM, as shown in Fig. ure 3E. UK122 and desmal did not show the additive effect, indicating that desmal is likely to inhibits the invasion via suppression of uPA (Figure 3E).
In conclusion, we obtained 4 reported isoflavones, genistein (1), genistein 7-O-α-L-rhamnoside (2), daidzein 6-O-methyl-7-O-α-L-rhamnoside (3), and daidzein 7-O-α-L-rhamnoside (4) from Streptomyces. The inhibitory activity against NO production and toxicity in mouse monocytic leukemia RAW264.7 cells are evaluated. Compounds (1) and (2) showed potent inhibitory activity at the nontoxic concentrations. Desmal, which is a new flavonoid isolated from the leaves of Thai plant Desmos chinensis, is thus evaluated for NO production and NF-κB inhibitory activities.. It is known to inhibit protein-tyrosine kinase, but other biological activity has not been reported. SinceAs the result, dDesmal it inhibits NF-κB with similar activity with genistein., Iit may be useful as a chemical ligand for the mechanistic study of inflammation and cancer progression. Desmal is known to inhibit protein-tyrosine kinase, and this is the first report on NF-κB inhibitory activity.

Experimental
Materials. Desmal was isolated from the leaves of Desmos chinensis collected in Khon Kaen, Thailand, as described before.8

Cell culture. Murine macrophage-like cell line RAW264.7 was purchased from Riken Cell Bank, Tsukuba, Japan. The cells were cultured in RPMI-1640 medium (Sigma-Aldrich Co., St Louis, MOSigma Chemical Co.) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, and 5% fetal bovine serum (FBS; Invitrogen, Carlsbad, CAUSA), and maintained at 37 °C in humidified 95% air, 5% CO2 atmosphere. Human ovarian clear cell carcinoma ES-2 cells (ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2.

Cell viability. Cell viability was evaluated by the MTT assay. Cell suspensions at a density of 3 x 105 cells per ml mL were seeded in a 96-well plate and incubated for 1 h. Then each flavonoid at different concentrations were added and further incubated for 24 h. MTT solution was added to each well and incubated for 1 h. Then, the culture medium was replaced by DMSO to dissolve formazan crystals formed. Absorbance was measured at 570 nm with a microplate reader.

Measurement of NO production. RAW264.7 cells were cultured in a 96-well plate at a density of 3 x 105 cells per mlmL. The cells were treated with Streptomyces culture filtrate or the indicated flavonoid for 30 min. LPS 10 ng/ml mL was added and incubation was continued for 24 h. Then, the supernatants were collected, and nitrite was measured colorimetrically after adding 50 µL of Griess reagent consisting of 0.1%, N-naphthyl-ethylenediamine 2HCl, l% sulfanilamide, and 2.5% H3PO4 to a 50 µL sample of supernatants. Sodium nitrite was used as the standard. The absorbance of the mixture was measured at 570 nm with a microplate reader.

Measurement of NF-κB activity in cultured RAW264.7 cells. RAW264.7 cells in complete medium (2.5 x 106 cells) were grown in 60-mm dishes. The next day, the cells were treated with the desired concentrations of desmal for 30 min and stimulated with 10 ng/mL LPS for 2 h. The nuclear extracts were prepared with a Nuclear Extract Kit (Active Motif Japan, Tokyo, Japan). Then, the DNA binding activity of NF-ĸB in nuclear extracts was measured with the TransAM NF-ĸB p65 Transcription Factor Assay Kit (Active Motif, Japan).

Wound healing assay. Cells in 24-well plates were allowed to reach confluence before the surface was uniformly scratched across the center of each well with a pipette tip. The wells were then rinsed twice with serum-free media to remove floating cells and growth media, after which the cells were cultured in serum-free media for 24 h. The initial wounded area and movement of the cells into the scratched area were recorded. Experiments were performed in triplicate in three independent experiments.

Cell invasion assay. ES-2 cells were suspended in 500 µL of serum-free medium containing desmal or the DMSO and seeded into the upper chambers coated with BD Matrigel Basement Membrane Matrix (Corning Inc., Corning, NY). The lower chambers were filled with 750 µL of medium containing 10% FBS and incubated for 24 h at 37 °C in a humidified CO2 incubator. Then, after fixation of the invading cells, non-invading cells remaining on the upper surface were removed by wiping with a cotton swab. Invading cells attached to the underside were stained with Diff-Quick solution (Sysmex, Kobe, Japan), and counted.


Acknowledgments
This work was financiallyalso supported by grant (A20) from Chubu Advanced Medicine Consortium of Nagoya University. This work was also financially supported in part by JSPS Kakenhi Grant Number 26350975 and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, which is for Aichi Medical University 2011-2015 (S1101027). This work was also supported by grant (A20) from Chubu Advanced Medicine Consortium of Nagoya University. This work was also supported by Science and Technology Project of Shenzhen city, Shenzhen Bureau of Science, Technology and Information under Grant JCYJ20130408172946974.

References

1. N. Matsumoto, A. Ariga, S. To-e, H. Nakamura, N. Agata, S. Hirano, J. Inoue, and K. Umezawa, Bioorg. Med. Chem. Lett., 2000, 10, 865. CrossRef
2. M. Yamamoto, R. Horie, M. Takeiri, I. Kozawa, and K. Umezawa, J. Med. Chem., 2008, 51, 5780. CrossRef
3. T. Ukaji and K. Umezawa, Advances Adv. in Biological Biol. Regul.ation, 2014, 56, 108. CrossRef
4. Z. Wang, M. Igarashi, Y. Ikeda, R. Horie, and K. Umezawa, Heterocycles, 2006, 69, 377. CrossRef
5. Y. Ishikawa, M. Tachibana, C. Matsui, R. Obata, K. Umezawa, and S. Nishiyama, Bioorg. Med. Chem. Lett., 2009, 19, 1726. CrossRef
6. S. Shibasaki, K. Yamashita, R. Goto, K. Wakayama, Y. Tsunetoshi, M. Zaitsu, R. Igarashi, S. Haga, M. Ozaki, K. Umezawa, and S. Todo, Transplantation, 2013, 95, 542. CrossRef
7. M. El-Salhy, K. Umezawa, O.-H. Gilja, J. G. Hatlebakk, D. Gundersen, and T. Hausken, The Scientific World, 2014, Article ID 813804 (8 pages).
8. H. Kakeya, M. Imoto, Y. Tabata, J. Iwami, H. Matsumoto, K. Nakamura, T. Koyano, and K. Umezawa, FEBS Lett., 1993, 320, 169. CrossRef
9. J. N. Davis, O. Kucuk, and F. H. Sarkar, Nutr. Cancer, 1999, 35, 167. CrossRef
10. M. Hamalainen, R. Nieminen, P. Vuorela, M. Heinonen, and E. Moilanen, Mediators Inflamm., 2007, 2007, 45673.
11. N. Miyanishi, Y. Suzuki, S. Simizu, Y. Kuwabara, K. Banno, and K. Umezawa, Biochem. Biophys. Res. Commun., 2010, 403, 154 CrossRef