Homodimers of Vanillin and Apocynin Decrease the Metastatic

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Homo-Dimers of Vanillin and Apocynin Decrease Metastatic Potential of Human Cancer Cells by Inhibiting the FAK/PI3K/Akt Signaling Pathway Phatcharida Jantaree, Kriengsak Lirdprapamongkol, Wilailak Kaewsri, Charnsak Thongsornkleeb, Kiattawee Choowongkomon, Korakot Atjanasuppat, Somsak Ruchirawat, and Jisnuson Svasti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05697 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Homo-Dimers of Vanillin and Apocynin Decrease Metastatic Potential of Human

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Cancer Cells by Inhibiting the FAK/PI3K/Akt Signaling Pathway

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Phatcharida Jantaree,†,‡,§ Kriengsak Lirdprapamongkol,*,‡,§ Wilailak Kaewsri,†,‡ Charnsak

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Thongsornkleeb,†,⊥ Kiattawee Choowongkomon,∇ Korakot Atjanasuppat,§ Somsak

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Ruchirawat,†,‡,ǁ Jisnuson Svasti†,§

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Chulabhorn Graduate Institute, Bangkok 10210, Thailand

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Center of Excellence on Environmental Health and Toxicology (EHT), Ministry of

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Education, Bangkok 10400, Thailand

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§

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Medicinal Chemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand

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Thailand

Laboratory of Biochemistry, ⊥Laboratory of Organic Synthesis, and ǁLaboratory of

Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10903,

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*Corresponding author: Tel: +66 2553 8555 ext 8356. Fax: +66 2553 8572. E-mail:

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[email protected]

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Short title: Anti-Metastatic Potential of Vanillin and Apocynin Homo-Dimers

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ABSTRACT

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The spread of cancer cells to distant organs, in a process called metastasis, is the main

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factor that contributes to most death in cancer patients. Vanillin, the vanilla flavoring agent,

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has been shown to suppress metastasis in a mouse model. Here, we evaluated the anti-

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metastatic potential of the food additive divanillin, the homo-dimer of vanillin, and their

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structurally related compounds, apocynin and diapocynin, in hepatocellular carcinoma cells.

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The Transwell invasion assay showed that the dimeric forms exhibited higher potency than

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vanillin and apocynin in inhibiting invasion, with IC50 values of 23.3±7.4 to 41.3±4.2 µM for

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the dimers, which are 26-34 fold lower than IC50 values of vanillin and apocynin (p divanillin > apocynin

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> vanillin, indicating that the vanilloid dimers were more potent than their monomeric forms

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in binding to the FAK FERM domain. This ranking also corresponds to the anti-invasion

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potential of the test compounds (Table 1), as well as their inhibitory effects on FAK and Akt

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phosphorylation (Figure 4). The predicted ligand-protein interactions of divanillin, vanillin,

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and apocynin show several interactions with amino acid side chains at the Y397 pocket site

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of FERM domain, including hydrogen bonds with Y397 (Figure S4 in supporting

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information). Diapocynin showed the highest numbers of interactions with the target site,

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although diapocynin did not form a hydrogen bond with Y397, it could form five hydrogen

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bonds with R35 and H58, making its binding affinity stronger than other test compounds

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(Figure S4 in supporting information). Taken together, these results imply that a mechanism

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underlying the anti-metastatic potential of divanillin and diapocynin involves inhibition of

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FAK activation by interfering with Y397 phosphorylation.

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Discussion The role of vanillin and divanillin in food flavoring is recognized, but these compounds

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also have potential benefit for improving cancer treatment. This study reports the anti-

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metastatic potential of divanillin and diapocynin, which are the homo-dimers of vanillin and

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apocynin. Thus, the vanilloid dimers possess higher potency than their monomeric forms in

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terms of inhibiting cancer cell invasion and migration, as well as the regulatory pathways

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involved. To our knowledge, this is the first report to show bioactivity of divanillin and

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diapocynin in cancer cells.

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Several dimeric compounds possess higher potency compared to their monomeric

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forms. Thus, 3-hydroxytyrosol dimer is more potent than the monomer in protecting

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keratinocytes against UV-induced cell death 34. Additionally, dimeric compounds of

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oleanolic acid are more efficient in inhibiting growth of several cancer cell lines, compared to

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monomeric compounds 35, while the dimer of p-coumaric acid is more effective than the

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monomer in inhibition of tyrosinase 36. Interestingly, diapocynin also showed greater efficacy

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than apocynin in inhibition of NADPH oxidase enzymatic activity 24, and in downregulating

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expression of an NADPH oxidase subunit gene (gp91phox) 37, as well as in decreasing the

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release of TNF-α and IL-10 pro-inflammatory cytokines, and CCL2 chemokine from

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stimulated cells 37-38. All of these reports are in agreement with our results using an in vitro

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anti-metastatic model, where the vanilloid dimers (divanillin and diapocynin) were found to

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be more potent than their monomeric forms (vanillin and apocynin) in suppressing the

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metastatic potential of HepG2 human liver cancer cells.

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During cell invasion, the migrating cells grip the ECM components at the leading edge

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to establish focal adhesion points by using integrins, then pull the cell body forward followed

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by detachment of focal adhesion at the rear of the cells 39. Integrin-ECM interaction induces

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autophosphorylation of FAK which subsequently activates PI3K/Akt signaling, as well as

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Rho GTPases, via the PI3K product - PIP3. Activation of Rho GTPases, such as Rac and

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Cdc42, promotes actin polymerization at the leading edge, resulting in formation of filopodia

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and lamellipodia 30. In the present study, we used a model of cell-ECM attachment which

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occurs during cell invasion and migration. The results showed that PI3K/Akt signaling is

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downstream of FAK activation, since treatment with FAK inhibitor inhibits Akt

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phosphorylation, while PI3K inhibitor does not affect FAK phosphorylation (Figure 5). Our

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results also showed that divanillin and diapocynin, as well as their monomeric forms, could

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inhibit the FAK/PI3K/Akt signaling pathway. In the case of the FAK inhibitor I, FAK

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activation is blocked when the phosphorylation of Y397 is interfered with by the binding of

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the inhibitor to the Y397 pocket of the FERM domain of FAK 33. Molecular docking was

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used to study the binding of divanillin, diapocynin, vanillin and apocynin to FAK. These

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vanilloid compounds could bind to the same binding site as the FAK inhibitor I. Therefore,

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the higher anti-invasion and anti-migration effects of the vanilloid dimers compared to the

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monomers presumably results from the increased interactions with amino acids in the Y397

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pocket of the FERM domain of FAK. Consistent with our report, inhibition of

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FAK/PI3K/Akt signaling has been shown to mediate anti-invasion and anti-migration effects

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of several compounds in foods, such as hispolon from Phellinus linteus mushroom 40, antcin

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K form Antrodia cinnamomea mushroom 41, and nobiletin from citrus fruits 42. Recently,

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apocynin was reported to show inhibitory effect on FAK phosphorylation, as well as on

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migration and invasion of U251 glioma cells under hypoxia 43. Thus, we suggest that

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inhibition of the FAK/PI3K/Akt signaling pathway is the mechanism underlying the anti-

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metastatic potential of divanillin and diapocynin in our cellular model.

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Other vanilloid compounds in food have also been reported to decrease the metastatic

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potential of cancer cells. For example, capsaicin suppressed invasion and migration of HT-

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1080 fibrosarcoma cells 44. 6-Gingerol and 6-shogaol reduced invasiveness of MDA-MB-231

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breast cancer cells and Hep3B hepatocellular carcinoma cells 45-46. Ferulic acid attenuated

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invasion and migration of H1299 lung cancer cells 47. Although the mechanism of action of

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these vanilloid compounds are diverse, the anti-metastatic potential shared by the vanilloid

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compounds, such as divanillin and diapocynin, make these compounds of interest for further

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study, as modulators derived from foods for controlling cancer metastasis.

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In conclusion, our results demonstrate that divanillin and diapocynin could decrease the

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metastatic potential of HepG2 liver cancer cells, by using a similar target to vanillin and

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apocynin, but with greater potency. Since vanillin and divanillin are generally regarded as

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safe, therefore, the health benefit of these food additives may be of interest for further study,

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in addition to their role in food flavoring.

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Conflicts of interest

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None

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Acknowledgments This work was supported by grants from the Chulabhorn Research Institiute, the

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Chulabhorn Graduate Institute and the Center of Excellence on Environmental Health and

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Toxicology (EHT), Ministry of Education. We would like to thank Professor Dr. Gerd

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Dannhardt and Dr. Jan-Peter Kramb, Department of Pharmacy and Biochemistry, Johannes

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Gutenberg University of Mainz, for supporting preliminary synthesis of diapocynin. We are

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grateful to Assoc. Prof. Dr. Montip Tiensuwan, Department of Mathematics, Faculty of

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Science, Mahidol University, for assistance with the statistical analysis.

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Supporting Information Dimeric compound synthesis and NMR spectra, binding interfaces between the Y397

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pocket on FERM domain of FAK and the FAK inhibitor I, 2D diagrams of molecular

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interactions between the Y397 pocket on FERM domain of FAK and test compound. This

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material is available free of charge via the Internet at http://pubs.acs.org.

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Figure captions

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Figure 1. Conversion of vanillin and apocynin to divanillin and diapocynin.

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Figure 2. Inhibitory effect of divanillin, diapocynin, and their monomeric forms on HepG2

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cell invasion. (A) The cells were treated with various concentrations of test compounds for 30

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min and then allowed to invade in Matrigel-coated Transwell chambers for another 24 h. The

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invaded cells were stained with crystal violet dye and counted. Cell viability after 24 h

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treatment with test compounds was determined by the MTT assay. Data are presented as

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mean ± SEM from three independent experiments. * (p < 0.05), ** (p < 0.01) and *** (p