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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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∇
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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] 18 19
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