Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their anti-oxidant and anti-inflammatory properties. It is hypothesized that the metabolites of flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter. Consistent with this, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation. These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC.

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Published in Cancers, v. 11, issue 3, 427, p. 1-18.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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This research was funded by NIH, grant number 5RO3CA133061-02 and Research/Scholarship Support Fund 2018 from the Office of Research, South Dakota State University to G.J.B. and the National Science Foundation/EPSCoR Cooperative Agreement, grant number IIA-1355423, and the South Dakota Research and Innovation Center, BioSNTR, to S.V.s.

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The following are available online at https://www.mdpi.com/2072-6694/11/3/427/s1, Figure S1: Effect of 2,4,6-THBA on HCT-116 cell proliferation; Figure S2: Western Blot analysis showing the levels of various CDKs and cyclins in SLC5A8-pLVX cells in response to 2,4,6-THBA; Figure S3: Western Blot demonstrating the expression levels of SLC5A8 protein in HCT-116, HT-29, MDA-MB-231 and SLC5A8-pLVX cell lines; Figure S4: Effect of 2,4,6-THBA (A), 3,4,5-THBA (B), 3,4-DHBA (C) and 4-HBA (D) on colony formation in HT-29 cells; Figure S5: Effect of different concentrations of 3,4-DHBA on colony formation in SLC5A8-PLVX (A), MDA-MB-231 (B), and HCT-116 (C) cells; Figure S6: Effect of different concentrations of 3,4,5-THBA on colony formation in SLC5A8-PLVX (A), MDA-MB-231 (B), and HCT-116 (C) cells; Figure S7: Effect of different concentrations of 4-HBA on colony formation in SLC5A8-PLVX (A), MDA-MB-231 (B), and HCT-116 (C) cells; Figure S8: HPLC analysis showing the cellular uptake in SLC5A8-pLVX, MDA-MB-231 and HCT-116 cells following incubation with 3,4-DHBA (A), 3,4,5-THBA (B) and 4-HBA (C) in the cytosol.

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