Effect of Okinawa Propolis on PAK1 Activity, Caenorhabditis elegans

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Effect of Okinawa Propolis on PAK1 activity, Caenorhabditis elegans Longevity, Melanogenesis, and Growth of Cancer Cells Nozomi Taira, Binh Cao Quan Nguyen, Pham Thi Be Tu, and Shinkichi Tawata J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01785 • Publication Date (Web): 23 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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Journal of Agricultural and Food Chemistry

Effect of Okinawa Propolis on PAK1 activity, Caenorhabditis elegans Longevity, Melanogenesis, and Growth of Cancer Cells

Nozomi Taira,1 Binh Cao Quan Nguyen,1 Pham Thi Be Tu,1 and Shinkichi Tawata2,*

1

Department of Bioscience and Biotechnology, The United Graduate School of Agricultural

Sciences, Kagoshima University, Kagoshima 890-0065, Japan 2

Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the

Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0213, Japan

*

Corresponding author: (S.T.) Tel: +81-98-895-8803. Fax: +81-98-895-8734.

E-mail: [email protected].

1

ACS Paragon Plus Environment


1

ABSTRACT: Propolis from different areas has been reported to inhibit oncogenic/ageing

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kinase PAK1, which is responsible for a variety of conditions, including cancer, longevity and

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melanogenesis. Here, crude extract of Okinawa propolis (OP) was tested against PAK1

4

activity, Caenorhabditis elegans (C. elegans) longevity, melanogenesis, and growth of cancer

5

cells. We found that OP blocks PAK1 and exhibits anti-cancer activity in A549 cell (human

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lung cancer cell) line with IC50 values of 6 µg/mL and 12 µg/mL, respectively. Most

7

interestingly, OP (1 µg/mL) significantly reduces reproduction and prolongs the lifespan of C.

8

elegans by activating HSP-16.2 gene, as shown in the PAK1-deficient strain. Furthermore, OP

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inhibits melanogenesis in a melanoma cell line (B16F10), by downregulating intracellular

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tyrosinase activity with an IC50 of 30 µg/mL. Our results suggest that OP demonstrated life

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span extending effect C. elegans, anticancer and antimelanogenic effects via PAK1

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inactivation, therefore, this can be a potent natural medicinal supplement against

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PAK1-dependent diseases.

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KEYWORDS: PAK1, Okinawa propolis, longevity, Caenorhabditis elegans, melanogenesis,

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cancer

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INTRODUCTION

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A major oncogenic/ageing kinase, PAK1 [p21 (RAC/CDC42)-activated kinase 1], is

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accountable for a number of conditions including cancer, type 2 diabetes, Alzheimer’s disease,

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obesity, hypertension, and a number of inflammatory and infectious diseases.1 Furthermore,

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biochemical and genetic studies published in the last decade, have proven the essential role of

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PAK1 in the malignant transformation of normal cell induced by RAS.1–3 Recently, by using a

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model organism (C. elegans), it has been demonstrated that downregulation of PAK1 leads to

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extended healthy life span, increased stress-resistance, and reduced fertility.4 In addition,

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genes involved in regulating lifespan were identified in this oranism.5–7 PAK1 is also

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accountable for the growth and progression of over 70% malignancies in human including

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neurofibromatosis, breast and prostate cancers, and RAS-induced pancreatic and colon

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cancers.8,9

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Propolis, collected from different parts of the plant and exudates by honeybees is a

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natural antibiotic. It has been used to treat or alleviate several maladies in traditional medicine

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since ancient Egyptian time. Previous studies have shown that propolis possesses anti-cancer,

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anti-inflammatory and anti-oxidant activities.10–14 There is no information about the

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toxicological profile of Okinawa propolis (OP). However, Brazilian, New Zealand, and

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European propolis have been reported reasonably non-toxic, with a no-effect level in mouse.15

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The vegetation at the site of propolis collection widely determine the ingredients present in it.

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For example, caffeic acid phenethyl ester (CAPE) present in the propolis from New Zealand

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(namely Bio30) is the major anti-cancer ingredient responsible for downregulating the

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Rac-PAK1 signaling pathway.16 Green propolis (GP) from Brazil contains Artepillin C (ARC),

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which blocks PAK1 and prevents the growth of lung cancer cells (A549 cell line).8 However,

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propolis obtained from semi-tropical areas in the Pacific such as Okinawa, Taiwan, and

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Hawaii is very unique, as it does not containing CAPE nor ARC, but a variety of 3



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prenylflavonoids including nymphaeols, derived from a common origin plant, Macaranga

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tanarius.17 Taiwan Propolis (TP) contains propolin G, which induce apoptosis in brain cancer

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cells, and protect rat cortical neurons from oxidative stress.11 In the case of OP, nymphaeol-A

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was found to be the major compound responsible for the inhibition of tumor-induced

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angiogenesis in HUVEC cells.18 Other prenylflavonoids were also isolated from OP and

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showed strong anti-oxidant, anti-bacterial, and anti-angiogenic activities.17–19 However, the

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effects of OP on PAK1 activity and on longevity, melanogenesis, and cancer cell proliferation,

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remain to be clarified.

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In the present study, the effect of OP on PAK1 activity, C. elegans longevity,

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melanogenesis and cancer cell proliferation were investigated. We describe the lifespan

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extending effect in C. elegans, the anti-melanogenic effect in melanoma cells (B16F10), and

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the anti-cancer effect against A549 lung cancer cells of OP.

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MATERIALS AND METHODS

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C. elegans. The wild-type strain N2 (var. Bristol), and the transgenic strain CL2070

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(dvls) of C. elegans, together with the OP50 strain of Escherichia coli, were purchased from

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the C. elegans Genomic Center (University of Minnesota). C. elegans PAK1-deficient mutant

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(Strain RB689) was kindly donated by Dr. Sumino Yanase of Daito Bunka University (Tokyo,

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Japan).

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Chemicals and reagents. The human lung cancer cell (A549) line was purchased

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from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Murine

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B16F10 melanoma cell line was purchased from the American Type Culture Collection

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(Rockville, MD, USA). Dulbecco’s modified minimum essential medium (D-MEM),

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3-isobutyl-1-methylxanthine (IBMX), Triton X-100, and fetal bovine serum (FBS) were

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obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Anti-PAK1 antibody IgG 4


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was from Cell Signaling Technology (Danvers, Massachusetts, USA). Resveratrol,

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), protein A-agarose

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beads, and myelin basic protein (MBP) bovine were bought from Sigma-Aldrich (Saint Louis,

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Missouri, USA). Kinase Glo reagent and adenosine triphosphate (ATP) were purchased from

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Promega (Madison, Wisconsin, USA). (+)-5-Fluoro-2’-deoxyuridine (FUdR) was obtained

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from Kanto Chemicals (Tokyo, Japan).

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Extraction of Okinawa Propolis (OP). Propolis was supplied by the Okinawa Yoho

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Bee-farm (Okinawa, Japan) on June 30, 2015. Dried propolis was grounded with sterilized

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pestle and mortar. 50 mL of 100% ethanol was used for the extraction of one gram ground

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propolis. The mixture was sonicated for 3 h (AS ONE, Tokyo, Japan), and shaken for 21 h at

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25 °C (Bio-Shaker, BR-300LF, TAITEC CORPORATION, Tokyo, Japan). The suspension

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was filtered using Whatman® cellulose filter paper, and the filtrate was subjected to several

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rounds of centrifugation at 14,357 × g for 5 min. To obtain a yellow, dry, crude extract (320

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mg, yield 32%) of OP, the clear supernatant was evaporated under reduced pressure. 250 mg

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of crude extract was dissolved in 1 mL of 100% ethanol to prepare 25% tincture of OP. The

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sample was stored at -20 °C for subsequent use. The sample was diluted in dimethyl sulfoxide

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(DMSO) or ethanol to achieve appropriate final concentrations.

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Liquid chromatography-mass spectrometry (LC-MS) analysis. The major

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compounds in OP were determined as previously described.17 OP filtrate (5 µL of 100 µg/mL)

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was measured using a Shimadzu LC-MS (LC-20AD XR, Shimadzu Co., Kyoto, Japan)

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coupled with a UV-Vis detector (SPD-M20A, Shimadzu). The separation was achieved on a

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COSMOSIL 5C18-AR-II (150 mm x 2 mm I.D.; Nacalai Tesque, Kyoto, Japan). The mobile

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phase was 0.5% acetic acid (v/v) in water (solvent A) and acetonitrile (solvent B); the rate of

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flow was 0.2 mL/min; and the samples were detected at 280 nm. The gradient elution was

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done as follows: 0-30 min, isocratic conditions 80% B; 30-35 min, linear gradient 80-100% 5



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B; and 35-40 min, linear gradient 100-50% B. All MS data were acquired in negative

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ionization mode. Each compound was quantified based on the peak area and molecular weight

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according to Kumazawa et al.,17. The three major components in OP were prenylflavonoids

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called nymphaeol A, B, and C (Figure 1).

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Assay for the anti-cancer activity of OP. Human lung cancer cells (A549) carrying

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the oncogenic K-RAS mutant were seeded into 6-well plates (Iwaki, Tokyo, Japan) at a

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density of 2 × 105 cells/well in 10% FBS supplemented D-MEM growth medium. After 24 h

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pre-culture, various concentrations of OP (10, 20, and 40 µg/mL) were applied for another 72

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h. Later on trypan blue staining was performed as previously described.20 Both dead (stained

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in blue) and live cells were counted using a hemocytometer (OneCell, Hiroshima, Japan).

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Anti-cancer activity of OP was evaluated by comparing the percentage of viable treated cells

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in relation to the control (untreated) cells. The assay was performed in three independent

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experiment in triplicate.

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Assay for the anti-PAK1 activity of OP. “Macaroni-Western” kinase assay21 was

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used to evaluate anti-PAK1 activity of OP. Briefly, A549 cells (2 × 105 cells/well) were

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pre-cultured in D-MEM medium for 24 h, and treated with several concentrations (1, 5, and

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10 µg/mL) of OP for another 24 h. Cells were then lysed in 500 µL of lysis buffer (50 mM

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Tris-HCl, pH 7.5, 150 mM NaCl, and 1% Triton X-100), on ice for 30 min. Cell lysates were

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centrifuged at 100 × g for 5 min at 4 °C and, for immunoprecipitation experiments, the

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supernatants were incubated with 50 µL of dilution buffer alone (50 mM Tris-HCl, pH 7.5,

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150 mM NaCl, 100 µg/mL BSA) or in the presence of anti-PAK1 IgG (1:50 dilution) for 1 h

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at 4 °C. Samples were then incubated further at 4 °C for 1 h in the presence of 10 µL of

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protein A-agarose with constant shaking using a rotary mixer (Nissin, Tokyo, Japan). After

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centrifugation, the pellet (containing PAK1) was rinsed twice with 500 µL of washing buffer

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(50 mM Tris-HCl, pH 7.5 and 150 mM NaCl). The immunoprecipitated (IP)-PAK1 pellet was 6


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resuspended in 35 µL of kinase buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20

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mM MgCl2, and 0.1 mg/mL BSA. The kinase reaction was carried out by adding 10 µL of 2

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µM ATP in distilled water, and 5 µL of MBP substrate (1 mg/mL in distilled water). The

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reaction was incubated for 1 h at 37 °C with constant agitation. An equal volume of kinase

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Glo reagent (50 µL) was added to each well after incubation, and to stabilize the luminescent

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signal the plate was further incubated for 30 min. The cleared supernatant was then

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transferred to a 96-well plate for analysis. Luminescence was recorded by an MTP-880 Lab

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microplate reader (Corona, Ibaraki, Japan) with an integration time of 0.5 s per well. PAK1

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activity of OP was calculated as percentage relative to the control.

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Assessment of HSP-16.2-dependent GFP expression. The strain of C. elegans used

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in this study was CL2070, which carries the transgenic HSP-16.2-GFP fusion gene

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(HSP-16.2::GFP) as previously described.4 The nematodes were grown in nematode growth

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medium (NGM) agar plates and fed with E. coli (OP50) at 20 °C. About 30 adult worms (in

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each group) were transferred to fresh NGM plates with OP (0.5, 1 and 5 µg/mL) and

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incubated at 20 °C for 48 h. The worms were heat-shocked for 2 h at 35 °C and then

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incubated for 4 h at 20 °C and allowed to recover; each group was then fixed with a drop (1

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µL) of 0.5 M sodium azide for microscopic visualization. To measure the fluorescence,

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photographs of the GFP transgenic worms were obtained using a KEYENCE BZ-X700

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fluorescence microscope.

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Brood size assay. Approximately 20 worms of wild-type (N2) and RB689 C. elegans

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strains were synchronous cultured, and the L1 animals were then transferred at 20 °C onto

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NGM agar plates, where E. coli (OP50) and OP (0.5-1 µg/mL) were applied. The number of

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eggs were counted 10 h after the adults worms started laying eggs. The number of eggs per

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mother was used to calculate the brood size, as previously described.4

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Lifespan assay. The synchronized C. elegans eggs (60-100) were placed on fresh 7



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NGM agar plates containing E. coli (OP50) and supplemented with OP (1 µg/mL) or

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resveratrol (10 µg/mL). 400 µL of FUdR (0.5 mg/mL) was added to the agar plate after the

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worms reached adulthood (Day 3) for the prevention of progeny production. Worms were fed

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with OP50 every 2 days, and the number of the surviving nematodes was counted every day,

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as previously described.4,22

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Measurement of B16F10 cell viability. B16F10 melanoma cells were cultured in

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D-MEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin

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(10,000 U/mL and 100 µg/mL, respectively), at 37 °C in a humidified atmosphere containing

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5% CO2. Cell viability (or growth rate) was determined using trypan blue assay, as previously

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described.21 Briefly, B16F10 cells (2 × 104 cells/well) were seeded onto a 24-well plate. After

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24 h of preculture, various concentrations of OP (10, 20, and 40 µg/mL) were applied for 72 h

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at 37 °C. Cells were then washed twice with phosphate buffer and harvested with Accutase®,

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and the viable (not-stained) cell number were calculated by performing trypan blue exclusion

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test.

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Measurement of melanin content. Melanin content was assayed as previously

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described.23 In short, B16F10 cells were plated in a 24-well plate at a density of 2 × 104

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cells/well. After 24 h of prelature, the culture medium was changed with normal

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(supplemented with 10% FBS) or serum-free medium, and cells were treated with several

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concentration (5-40 µg/mL) of OP. After 1 h, IBMX (a melanogenic hormone; 100 µM),

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which activates PAK1, was added to the medium and cells were incubated for another 72 h at

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37 °C. Cells were dissolved in 500 µL NaOH (1 N) containing 10% DMSO after washing

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with phosphate buffer. To promote melanin solubilization samples were incubated at 80 °C for

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1 h. The optical density of the mixed homogenate was measured at 490 nm.

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Measurement of intracellular tyrosinase activity. Tyrosinase activity was

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determined as described,24 with slight modifications. B16F10 cells were seeded in a 24-well 8


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plate at a density of 2 × 104 cells/well. After 24 h of pre-culture, the culture medium was

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replaced by normal (containing 10% FBS) and serum-free medium, and supplemented with

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varying OP concentrations (10-40 µg/mL). After 1 h, IBMX (100 µM) was added and

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incubated at 37 °C for another 72 h. Cells were then washed with ice cold phosphate buffer

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and lysed with phosphate buffer (pH 6.8) containing 1% Triton X-100 (500 µL/well). The

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plates were frozen for 30 min at -80 °C. 100 µL of 1% L-DOPA was added to each well after

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thawing and mixing. Following incubation for 2 h at 37 °C, the absorbance was measured at

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490 nm.

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Statistical analysis. Data are expressed as mean ± standard errors (SE). Statistical

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comparisons were done by one-way ANOVA followed by Duncan’s multiple-range test. For C.

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elegans lifespan, the Median Survival Time (MST) was analyzed using Kaplan-Meier survival

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curves and log-rank test. Statistical analysis was conducted using SPSS (version 16.0,

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Chicago, Illinois), and p < 0.05 was considered significant.

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RESULTS AND DISCUSSION

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Anti-cancer and anti-PAK1 activity of OP. Although both ARC-containing

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Brazilian green propolis (GP) and CAPE-based propolis such as Bio30, block PAK1 and have

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been used for the treatment of cancer and some other PAK1-dependent diseases, the

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anti-cancer and anti-PAK1 activities of OP still remain to be clarified. Thus, we used A549

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cell line (the human lung cancer cell) for testing anti-cancer and anti-PAK1 activities and

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found that OP inhibited cell growth concentration-dependently with an IC50 of 12 µg/mL

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(Table 1). This cancer cell line is important as PAK1 has been indicated as the Achilles’s heel

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for over 70% human cancer cells, and for around 64% of squamous non-small cell lung

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cancers (NSCLC).8,25,26 A549 cell growth is strongly depended on PAK1 and carry an

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oncogenic Ki-RAS mutant.1 Thus, we tested the effect of OP against PAK1 in this NSCLC 9



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cell line, using the Macaroni-Western kinase assay, which is an effective system for screening

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potent and safe PAK1 blockers.21 As shown in Table 1, OP strongly inhibited PAK1 with an

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IC50 of 6 µg/mL. Since the apparent anti-PAK1 activity of OP is stronger than the anti-cancer

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activity in the same cell line, it is most likely that OP inhibits PAK1 directly, as reported in

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New Zealand propolis and GP.8,9 However, further research is necessary to validate this

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hypothesis. It has been reported that, New Zealand propolis and GP inhibit PAK1 through its

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direct effect on PAK1 signaling by suppressing its autophosphorylation.8,9 In agreement with

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this, OP might affect PAK1 signaling as we found stronger effect of OP on PAK1 using

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Macaroni-Western kinase assay. As summarized in Table 1, OP appears to be the strongest

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anti-PAK1 blocker compared to the other two propolis (IC50 of Bio30 = 60 µg/mL, and IC50

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of GP > 150 µg/mL).8,21 Moreover, the OP anti-cancer activity was several fold more potent

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than that of the ARC-based GP (IC50 = 100 µg/mL), but slightly less potent than that of the

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CAPE-based Bio30 (IC50 = 8 µg/mL).21 The above quantitative data based on the standard

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cancer cell line (A549) could be useful for the quality control of various propolis and other

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PAK1-blocking herbal products present on the market.

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Effect of OP on heat-shock (HSP-16.2) gene expression, and C. elegans

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reproduction and lifespan. HSP-16.2 gene has been found to be inactivated by PAK1 in C.

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elegans and it is among the key regulators of lifespan and stress/heat shock resistance, as it

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serves as a stress-sensitive indicator to predict lifespan.4,27 Hence, in this study, we tested the

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effect of OP on HSP-16.2::GFP expression. At 0.5-1 µg/mL, OP significantly upregulated

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HSP-16.2::GFP expression by more than 31 ± 0.7%, compared to that of control (Figure 2).

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The effect of OP was clearly stronger than that of the positive control resveratrol.

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Both CAPE from New Zealand propolis (Bio30) and ARC from GP upregulated the

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expression of the heat-shock protein HSP-16.2 in transgenic C. elegans CL2070, and

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suppressed the PAK1-dependent reproduction in wild-type C. elegans.4 Thus, a further 10


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investigation was carried out to test the influence of OP treatment on C. elegans brood size.

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The brood size decreased by 19% and 52% at 0.5 and 1 µg/mL of OP, respectively, compared

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with the control (Figure 3), while resveratrol (10 µg/mL), on the other hand, reduced the

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brood size by only 33%. The brood size of PAK1-deficient strain (RM689) was smaller than

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the wild-type (N2), confirming that PAK1 promotes reproduction, as previously described.4

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The longevity of PAK1-deficient C. elegans mutant (RB689) was 60% higher than

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the wild type,4 and PAK1-blockers such as CAPE and apigenin from Bio30, as well as a

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PAK1-blocking leaf extract from the Okinawa plant called Alpinia zerumbet showed positive

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effects on the lifespan of C. elegans.22,28–31 Thus, to determine the potential lifespan-extending

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effect of OP, worms were treated with either OP (1 µg/mL) or resveratrol (10 µg/mL) (Figure

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4). OP significantly increased the nematodes’ lifespan by 33% (24 ± 0.2 days) compared with

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the control (18 ± 1.1 days), as measured by MST. Moreover, the lifespan extension observed

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with OP was clearly better than that with resveratrol (22 ± 1 days). Taken together, our results

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suggest that OP has a positive effect on the lifespan of C. elegans and this is achieved through

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PAK1 inhibition. To the best of our knowledge, this is the first example in which the propolis

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extract extends C. elegans lifespan at such a low dose.

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Anti-melanogenic effects of OP. The inhibition of melanogenesis can be useful not

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only for skin-whitening purposes, but also for the treatment of hyperpigmentation. In our

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previous study, we have demonstrated that propolis Bio30 inhibited PAK1-dependent

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melanogenesis.21 Tyrosinase is the key enzyme for melanin production and very recently we

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have found that melanogenesis depends on PAK1 by more than 50%. As the OP inhibit PAK1

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(Table 1), we hypothesized that it would also inhibit intracellular tyrosinase activity and

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decrease melanin content of B16F10 cells. In the present study, we found that OP also has a

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potent anti-melanogenic effect. As shown in Figures 5 and 6, OP had a strong

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anti-melanogenic activity in B16F10 melanoma cells (IC50 of 37 µg/mL). 11



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Furthermore as shown in Figure 7, OP downregulated the intracellular tyrosinase

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with an IC50 of 30 µg/mL. All of these results demonstrate that OP efficiently inhibited

244

melanogenesis in B16F10 melanoma cells.

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In conclusion, this is the first report showing that OP significantly prolongs the

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lifespan of C. elegans, and inhibits melanogenesis and cancer cell growth by blocking the

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oncogenic/ageing/melanogenic kinase PAK1. So far there are two major anti-cancerous

248

propolis extracts available on the market: CAPE-based propolis (Bio30), and ARC-based

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Brazilian green propolis (GP).8 Our results clearly indicate that, like Bio30, OP would be

250

among the best healthcare products to promote longevity, and for the treatment or prevention

251

of different PAK1-dependent disorders such as cancer and hyperpigmentation.

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Finally, it would be worth noting that further identification of the major

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PAK1-blocking nymphaeols in OP could be useful for developing a new potent

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PAK1-blocker(s) for both clinical and cosmetic uses.

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ACKNOWLEDGEMENT

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We are grateful to Drs. Hiroshi Maruta (PAK Research Center, Melbourne) and

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Sumino Yanase (Daito-Bunka University, Saitama, Japan) for their critical reading of this

258

manuscript and technical expertise. Nematode strains (RB689 and CL2070) used in this work

259

were provided by the Caenorhabditis Genetics Center (University of Minnesota). We are also

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grateful to the International Research Center, University of the Ryukyus, Okinawa, Japan for

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providing the facilities for LC-MS and the fluorescence microscope.

13



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Figure captions Figure 1. HPLC profile and chemical structures of the main constituents of OP. (A) nymphaeol-B; (B) isonymphaeol-B; (C) nymphaeol-A; (D) 3’-geranyl-naringenin; (E) nymphaeol-C.

Figure 2. HSP-16.2 gene activation by OP of in C. elegans (CL2070). (A) Image of HSP-16.2::GFP expression in the control worms and worms treated with OP (1 µg/mL). (B) GFP intensities from for three experiments were quantified in each CL2070 group. Each value represents the mean ± SE of three independent determinations. Statistical differences between treatment and control are indicated by asterisks (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***).

Figure 3. Reduction in Brood size by OP. OP significantly reduced the number of eggs laid by wild-type nematodes. Resveratrol was used as positive control; RB689 is the PAK1-deficient strain. Each value represents the mean ± SE of three independent determinations. Statistical differences between the treatment and the control are indicated by asterisks (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***).

Figure 4. Effects of OP on C. elegans lifespan. (A) Survival curves of C.elegans, (B) Median survival time (MST) in worms. OP extended both the survival % and median survival time of C.elegans. Statistical differences between the treatment and the control was demonstrated by log-rank test using the Kaplan-Meier survival analysis (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, n = 156).

Figure 5. OP has no effect on the viability of melanoma cells. The results are displayed as percentage of control samples (mean ± SE, of three independent experiment in triplicate). 20


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Statistical differences between the treatment and the control are indicated by asterisks (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***).

Figure 6. Anti-melanogenic activity of OP in melanoma cells. The results are displayed as percentage of control samples (mean ± SE, of three independent experiment in triplicate). Statistical differences between the treatment and the control are indicated by asterisks (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***).

Figure 7. Downregulation of intracellular tyrosinase activity in melanoma cells by OP. The results are displayed as percentage of control samples (mean ± SE, of three independent experiment in triplicate). Statistical differences between the treatment and the control are indicated by asterisks (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***).

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Table 1. Anti-cancer and anti-PAK1 activity of different propolis products. IC50 value (µg/mL)

Propolis type

Anti-cancer activity

Okinawa propolis Green propolis

a

Anti-PAK1 activity

12

5

100

>150

a

a

Bio 30 8 60 These propolis have been reported to inhibit A549 cancer cell growth by inhibiting PAK1 directly (References 8 and 19 for Green propolis and Bio 30, respectively).

Figure 1.

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Figure 2.

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Figure 3.

24


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Figure 4.

25



Figure 5.

26


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Figure 6.

27



Figure 7.

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84x47mm (96 x 96 DPI)


Effect of Okinawa Propolis on PAK1 Activity, Caenorhabditis elegans

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