More than one million people are diagnosed with colorectal cancer (CRC) worldwide every year, and it is the third most common cause of cancer death in the United States [1
]. Studies conducted over the past two decades have clearly established chemo-preventive roles for flavonoids against CRC [3
]. Though these studies mainly focused on the therapeutic potential and activity of the parent compounds, limited reports exist on the therapeutic role for their metabolites [8
]. The backbone of flavonoids comprises of an aromatic ring A bound to a heterocyclic ring C, which is in turn attached to a third aromatic ring B through a carbon-carbon bond (Figure 1
]. They are further classified into anthocyanins, flavonols, flavan-3-ols, flavones, flavanones and isoflavones based on the individual functional groups appended or attached to this backbone. The hydroxylation pattern in the A-ring is mostly conserved whereas the B-ring can have 1 to 3 hydroxyl groups [6
]. Members of the flavonoid family are highly unstable and are known to undergo light, temperature and pH dependent degradation [10
]; the number and position of –OH groups are inversely correlated with stability [12
]. Flavonoids are reported to be more stable in acidic conditions (pH < 4); however, at neutral or alkaline pH (pH > 8, as in the intestine), they undergo auto-degradation which generates simpler phenolic acids [13
]. Additionally, owing to their low absorption (1–15%) in the small intestine, flavonoids undergo degradation in the colon due to the action of gut microbiota [9
]. The flavonoids absorbed into the systemic circulation may also revert back to the small intestine as conjugated metabolites secreted in the bile fluid, eventually reaching the colonic microflora [8
]. These metabolites may undergo de-conjugation and get re-exposed to the gastrointestinal (GI) epithelial cells. Therefore, it is likely that colonic stem/epithelial cells are exposed to these metabolites at relatively high concentrations. In fact, it is reported that phenolic acid concentrations obtained from dietary sources in human fecal matter is relatively high [16
Following degradation, different flavonoids give rise to different phenolic acids [14
]. For example, the B-ring generates 3,4,5-trihydroxybenzoic acid (3,4,5-THBA/gallic acid) from delphinidin with three –OH groups, 3,4-dihydroxybenzoic acid (3,4-DHBA/protocatechuic acid) from cyanidin with two –OH groups, and 4-hydroxybenzoic acid (4-HBA) from pelargonidin with one –OH group [11
]. The degradation of other flavonoids such as catechin and epigallocatechin gallate also produce these phenolic acids [20
]. The A-ring from most flavonoids generate an unstable and reactive 2,4,6-trihydroxybenzaldehyde (2,4,6-THBAld) [22
], which is later oxidized to 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) facilitated by host enzymes in the colon [9
]. Due to the reported low bioavailability of flavonoids and their highly unstable nature which results in the generation of phenolic acids, it is suggested that the observed anti-cancer effects of flavonoids may actually come from the degraded products rather than the parent compounds [9
]. This is further substantiated by several reports demonstrating the ability of 3,4-DHBA and 3,4,5-THBA to reduce cancer cell growth [24
]. However, limited studies exist regarding the potential of the A-ring derivative 2,4,6-THBA to act as an anti-cancer agent, which is the focus of this study.
Two important observations previously reported by us on 2,4,6-THBA led to the present study. First, while determining the ability of salicylic acid derivatives (2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid) to inhibit Cyclin Dependent Kinase (CDK) enzyme activity, we observed that 2,4,6-THBA also inhibited its activity in vitro [28
]. Secondly, among all the hydroxybenzoic acids tested in that report, 2,4,6-THBA exhibited the highest level of CDK inhibition which prompted us to further examine its potential to inhibit cancer cell growth. CDKs are often dysregulated and overexpressed in cancers and therefore are attractive targets for cancer therapy [29
]. The present investigation was carried out to study: (1) the effect of 2,4,6-THBA on CDKs 1, 2 and 4; (2) the interactions of 2,4,6-THBA with CDKs though in silico analysis; (3) to identify a transporter for its cellular uptake; and (4) its ability to inhibit cell proliferation in colon cancer cell lines. In this study we demonstrate that 2,4,6-THBA dose-dependently inhibits activities of CDKs 1, 2 and 4, and identify the specific amino acids involved in these interactions. We show that SLC5A8, a member of the monocarboxylic acid transporter (MCT) family, is required for the cellular uptake of 2,4,6-THBA. In cell lines ectopically expressing SLC5A8, 2,4,6-THBA induced the expression of CDK inhibitors p21Cip1
(mRNA and protein) and this was associated with decreased cell proliferation. Interestingly, the other two metabolites, 3,4-DHBA and 3,4,5-THBA, which failed to inhibit CDK activity, potently inhibited cancer cell proliferation independent of a functional SLC5A8. These results suggest that the flavonoid metabolite 2,4,6-THBA may mediate its anti-cancer effects through a CDK- and SLC5A8-dependent pathway, whereas, 3,4-DHBA and 3,4,5-THBA are likely to exert their effect through a CDK- and SLC5A8-independent pathway.
In this research paper, we report several novel observations including a mechanism by which flavonoid metabolites known to be generated in the GI tract may exert their anti-cancer effects against CRC through CDK-dependent and -independent pathways. This study investigated 2,4,6-THBA generated from flavonoid A-ring and 4-HBA, 3,4-DHBA and 3,4,5-THBA from the flavonoid B-ring for their potential to inhibit cancer cell growth. Our results demonstrated that the inhibitory effect of 2,4,6-THBA on purified CDKs in vitro was dose dependent with IC50
observed between 262–580 µM, while molecular docking studies revealed the key amino acid interactions. We also identified SLC5A8 as a transporter for 2,4,6-THBA, and using SLC5A8-pLVX cells, we demonstrated that its cellular uptake for 72 h was sufficient to cause induction of p21Cip1
, leading to inhibition of cell proliferation. Although endogenous SLC5A8 was detected in HCT-116 cells, they were insensitive to 2,4,6-THBA treatment. Additionally, using clonogenic assays, we showed that 2,4,6-THBA was highly effective in inhibiting clonal formation in the cell lines expressing functional SLC5A8. The IC50
of clonal inhibition corresponded to the IC50
of CDK inhibition, suggesting that 2,4,6-THBA may mediate its effect through a CDK-dependent pathway. We believe the inhibition of proliferation can occur through at least two different mechanisms—one by direct binding of 2,4,6-THBA to CDKs leading to their inhibition and the other through upregulation of CDK inhibitory proteins p21Cip1
. Although 3,4-DHBA and 3,4,5-THBA were incapable of inhibiting CDKs, they efficiently inhibited cell proliferation in all cell lines tested including HCT-116 and HT-29 (Supplementary Figures S4–S6
). This suggests that the mechanism of inhibition by 3,4-DHBA and 3,4,5-THBA is both an SLC5A8- and CDK-independent phenomenon. Not all metabolites exhibited anti-proliferative effects as 4-HBA although taken up by all cell lines tested was ineffective in inhibiting cell proliferation which suggests that the metabolites are selective in their actions. Our results, for the first time demonstrate a role for flavonoid metabolites in the inhibition of cancer cell growth occurring through both CDK-dependent and -independent mechanisms, and also establishes a functional role for SLC5A8 in the transport of 2,4,6-THBA.
Molecular docking studies indicated that 2,4,6-THBA binds to CDKs 1, 2 and 4 at specific sites within these enzymes. The CDK structure is highly conserved among the different family members with a bi-lobe fold that harbors a conserved ATP-binding domain [37
]. The interactions of 2,4,6-THBA with CDKs 2 and 4 at the ATP-binding site may explain the enhanced inhibition of enzyme activity in comparison to the lesser inhibition of CDK1 by 2,4,6-THBA which interacts at an allosteric site. Interestingly, the structurally related compound 3,4,5-THBA failed to inhibit CDK activity although docking studies provided evidence of its binding to all CDKs (Table 1
and Figure 5
). This suggests that the presence of hydroxyl groups at specific positions on the benzene ring in these compounds confers the ability to bind and cause enzyme inhibition. The –COOH group seems to be important in increasing the potency of this inhibition as phloroglucinol which lacks the –COOH group was only marginally effective in inhibiting CDK1 and CDK2. Therefore, we suggest that both the presence of –COOH and the position of the –OH groups are important for efficient CDK inhibition. Further studies involving kinetics of inhibition are required to fully understand the nature of these interactions and its implications towards inhibition of enzyme activity.
Detection of 2,4,6-THBA in SLC5A8-pLVX cells through HPLC suggests that SLC5A8 is a natural transporter for this monocarboxylic acid. The attributed physiological functions of SLC5A8 includes transport of short chain fatty acids (SCFAs), salicylates, propionate, butyrate, lactate, pyruvate, and benzoates to name a few [33
]. Numerous studies have shown that SLC5A8 is likely to be a tumor suppressor protein [34
] and the mechanism is predicted to involve inhibition of histone deacetylases (HDACs) through the uptake of SCFAs by these transporters [33
]. It is a sodium-coupled MCT and is expressed in the apical membrane of the colonic and intestinal epithelium [32
]. It is silenced in many cancers including colon, thyroid, kidney, stomach, brain, breast, pancreas and prostate. Studies suggest that silencing of SLC5A8 contributes to tumorigenesis [39
]. We speculated that the lack of response observed following 2,4,6-THBA treatment of HCT-116 and other CRC cell lines is due to the absence of a wild-type functional SLC5A8. In support of this view, no cellular uptake of 2,4,6-THBA was observed in these cells. Importantly, in SLC5A8-pLVX cells expressing the functional SLC5A8, 2,4,6-THBA not only induced p21Cip1
RNA and protein expression, but also reduced cell proliferation. Our demonstration that 2,4,6-THBA inhibits cell growth in cancer cells expressing functional SLC5A8 suggests that such a phenomenon may indeed exist in vivo and may contribute to its anti-cancer effects against CRC.
The mechanism by which 2,4,6-THBA inhibits CDK activity requires additional investigations. Unlike the conventional drugs Palbociclib and Ribociclib that target CDK4/6 and cause G0/G1 cell cycle arrest [40
], 2,4,6-THBA appears to target CDKs 1, 2, and 4. The lack of inhibition at any particular stage of the cell-cycle observed in our study (Figure 8
A) may be attributed to the effect of 2,4,6-THBA on multiple CDKs (Figure 2
A–C), leading to a decrease in the overall growth rate without causing accumulation of cells at any given stage. Exposure of SLC5A8-pLVX cells to 2,4,6-THBA at concentrations from 125–1000µM slowed the proliferation (Figure 6
C) without causing significant apoptosis (Figure 8
B) suggesting that it is more likely to be a cytostatic rather than a cytotoxic agent.
The exposure of SLC5A8-pLVX cells as well as MDA-MB-231 cells to 2,4,6-THBA for 14–21 days resulted in a decrease in colony formation. The ability of 2,4,6-THBA to inhibit cell proliferation in MDA-MB-231 cells was surprising considering the low expression levels of SLC5A8 and the fact that acute treatment (72 h) with 2,4,6-THBA did not cause any significant decrease in cell proliferation. This discrepancy between acute and chronic treatment in MDA-MB-231 cells can be explained by the limited uptake of 2,4,6-THBA during acute exposure, whereas chronic exposure of these cells to 2,4,6-THBA could result in significant accumulation sufficient to cause inhibition of cell proliferation. This was consistent with the HPLC data that showed lower uptake of 2,4,6-THBA in MDA-MB-231 cells when compared to SLC5A8-pLVX cells (Figure 7
A). Consistent with the previously reported lack of functional SLC5A8 in HCT-116 cells [36
], 2,4,6-THBA had no effect on the colony formation (Figure 7
D), and HPLC also showed no uptake of 2,4,6-THBA in these cells (Figure 7
A). These results suggest that the transporter activity of SLC5A8 is required for 2,4,6-THBA-mediated inhibition of cell proliferation.
Tumor progression involves distinct stages—initiation, promotion, progression and metastasis. Of these stages, it is suggested that chemo-preventive agents preferentially act within the initiation and promotion stages to reverse the process of carcinogenesis [41
]. They are further classified into blocking agents and suppressing agents depending on their ability to prevent carcinogenesis and suppress neoplastic cell growth respectively [42
]. Based on the properties reported for phenolic compounds [43
] and the results presented in this study, we propose that 2,4,6-THBA can act as both—a blocking and a suppressing agent. Since it is a phenolic compound with multiple hydroxyl groups, it can act as a blocking agent and exert its effect through its anti-oxidant properties [45
]. It can also be viewed as a suppressing agent through its direct inhibitory effect on CDKs 1, 2 and 4 leading to reduced rate of cell proliferation. Its anti-oxidant and anti-proliferative actions could collectively contribute to cancer prevention by slowing down the rate of cell proliferation and providing an opportunity to repair the DNA damage or for immune surveillance by Natural Killer- and cytotoxic T-cells for cancer cell destruction [46
Although 3,4-DHBA and 3,4,5-THBA failed to inhibit CDK activity in vitro, their ability to inhibit colony formation in all cell lines tested irrespective of the SLC5A8 expression is also an important observation. It suggests that these compounds exert their anti-proliferative effect through an SLC5A8- and CDK-independent mechanism. These flavonoid metabolites have previously been shown to inhibit cancer cell growth through multiple signaling pathways [24
]; however, no direct primary target(s) have been identified. It appears that depending upon the cell line tested, 3,4-DHBA and 3,4,5-THBA can inhibit colony formation within a range of 7.3–250 μM and 7.3–31.25 μM respectively. These concentrations are significantly lower compared to those of 2,4,6-THBA. The lack of detection of these compounds in HPLC studies suggests that they are not taken up by the cells, and hence we suggest that their primary target is likely to be extracellular.
The discovery of the flavonoid metabolite 2,4,6-THBA as an inhibitor of CDKs and the demonstration of its ability to inhibit cell proliferation is a significant finding. Although the IC50
of 2,4,6-THBA and other compounds tested in this study are in micromolar concentrations, it could be argued that it is physiologically relevant in view of their abundance in the diet [8
]. CDKs and other potential cellular targets probably have evolved to be less sensitive to these compounds to avoid complete inhibition or perturbance of cell cycle at lower concentrations. We suggest that the reported chemo-preventive actions of flavonoids could be explained at least in part through the ability of its metabolite 2,4,6-THBA to target CDKs by direct inhibition and through upregulation of CDK inhibitory proteins; this would tip the balance strongly towards cell-cycle arrest, leading to reduced cell proliferation. A model depicting how flavonoid metabolites may prevent the occurrences of CRC is shown in Figure 9
. Since the metabolites are also generated through microbial degradation, it highlights the importance of GI microflora in the prevention of CRC.
Effective cancer prevention may therefore require both the chemo-preventive agents and the responsible partners for their degradation. Identification of the microbial species contributing to this process as well as the mechanism of action of the metabolites on GI tissue is an important area for future study. Additionally, in vivo studies would be required to further confirm the chemo-preventive role of 2,4,6-THBA against CRC.
4. Materials and Methods
4.1. Cell Lines
HCT-116, HT-29, Caco-2 and MDA-MB-231 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231 cells expressing functional SLC5A8 (SLC5A8-pLVX cell line) was kindly provided by Dr. Puttur Prasad, Medical College of Georgia. The cells were cultured in their respective medium (RPMI media for both the MDA-MB-231 cells and McCoy’s 5A media for all other cell lines) containing 10% Fetal Bovine Serum (FBS) with antibiotics for 24 h before treatment with specified compounds for indicated times. SLC5A8-pLVX cells were grown in the presence of doxycycline (5 μg/mL) for the induction of SLC5A8. Authentication of cell lines was done by ATCC through their DNA-STR profile.
2,4,6-THBA, 3,4-DHBA, 3,4,5-THBA, 4-HBA and trypsin-EDTA were obtained from Sigma Aldrich (St. Louis, MO, USA); H1 Histones and Immobilon membranes from EMD Millipore (Billerica, MA, USA); 32P γ-ATP from MP Biochemicals (Solon, OH, USA); RT-PCR reagents from New England Biolabs (NEB, Ipswich, MA, USA); qPCR reagents from Applied Biosystems (Foster City, CA, USA); Annexin V/7-AAD kit from Beckman Coulter (Miami, FL, USA); Super Signal™ West Pico Chemiluminescent Substrate, protease inhibitor tablets and all other chemicals were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).
4.3. Recombinant Proteins and Antibodies
anti-p21Cip1, anti-p27Kip1, anti-CDK1, anti-CDK2, anti-CDK4, anti-cyclinA2, anti-cyclinB1, anti-cyclinD1, and anti-β tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-SLC5A8 from Invitrogen (Carlsbad, CA, USA); goat anti-rabbit and goat anti-mouse antibodies were obtained from Bio-Rad (Hercules, CA, USA). CDK1/Cyclin B1, CDK2/Cyclin A2, CDK4/Cyclin D1, Retinoblastoma (C-term) and kinase buffer were purchased from SignalChem (Richmond, BC, Canada).
4.4. Cell Lysate Preparation and Western Blotting
Following treatment with compounds at the specified concentrations, cells were washed with 1X phosphate buffered saline (PBS) and lysates were prepared as previously described [48
]. Fifty micrograms of total protein was separated on an 8 or 12% polyacrylamide gel, transferred to immobilon membrane and immunoblotted with indicated antibodies.
4.5. In Vitro CDK Assay
In vitro CDK assays were performed as described by the manufacturer. For this, purified enzyme was aliquoted into the reaction buffer and incubated with indicated compounds at various concentrations for 10 min at room temperature. The reaction mixture was incubated with kinase buffer containing 15 µM ATP, 2 µCi of [γ 32P] ATP, 5 µg of H1 Histone or retinoblastoma, at 30 °C for 20 min in a final volume of 50 μL. The reactions were halted by adding EDTA to a final concentration of 20 mM and addition of 4× loading buffer. The samples were boiled for 10 min, analyzed by 8 or 10% SDS-PAGE, stained using coomassie brilliant blue (R250), dried and exposed to X-ray film. NIH ImageJ software was used to quantify the intensities of the bands.
4.6. Molecular Docking Studies
The crystallographic three dimensional structures of CDK1 (4Y72 A chain), CDK2 (1FIN A chain) and CDK4 (3G33 A chain) were retrieved from the Protein Data Bank (PDB). Energy minimization for these proteins was performed using Gromacs 3.3.1 package utilizing GROMOS96 force field [49
]. The energy-minimized molecules were used as the receptors for virtual small molecule docking with 2,4,6-THBA, 3,4-DHBA, 3,4,5-THBA, 4-HBA and phloroglucinol using AutoDockVina. The results were visualized by PYMOL molecular graphics system version 1.3.
4.7. HPLC Analysis
High Performance Liquid Chromatography (HPLC) was utilized to determine the uptake of flavonoid metabolites. Cells were exposed to different compounds at indicated concentrations and lysates were prepared as described previously [28
]. Protein concentration was determined; 300 μL of the lysates were taken and proteins were precipitated using 15 μL of trifluoracetic acid (TFA). Samples were centrifuged at 14,000 rpm for 5 min, the supernatant was transferred to fresh tubes and the pH was adjusted to about 4–5. Finally, 300 μL of acidified methanol was added to the samples. HPLC analysis was conducted using a Waters HPLC system (Milford, MA, USA) equipped with a 1525 binary pump and a W2998 PDA detector. An isocratic method was used to elute the compound in reverse phase using a ZORBAX SB-CN, 5 µm, 4.6 × 250 mm column (Agilent, Santa Clara, CA, USA). The mobile phase contained 20 mM ammonium acetate (adjusted to pH 4.0 with acetic acid) and methanol (90:10 ratio), with a flow rate of 0.7 mL/min. The injection volume was 40 μL. Compounds were detected at a wavelength of 260 nm. Quantification was done by generating a standard graph using known amounts of each compound.
4.8. RNA Isolation and qRT-PCR
Total RNA was isolated from treated and untreated cells as previously described using TRIzol reagent [50
]. RT-PCR was performed according to the manufacturer’s instructions (NEB). Briefly, total RNA from 2,4,6-THBA treated cells were isolated and reverse transcribed for 1 h at 42 °C using M-MuLV reverse transcriptase followed by amplification of the cDNA product. The qPCR experiment was set by adding equal volumes of each template to the SYBR Fast Master mix along with ROX dye (Applied Biosystems) and 5 pmols of each primer (p21Cip1
Forward: AGCTCAATGGACTGGAAGG Reverse: TGGATGAGGAAGGTCGCT; p27Kip1
Forward: GCTGAG GAACTGACGTGG Reverse: AGGGCAGTGAGGATAGGT). The final volume was adjusted to 20 µL using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 min; and one melt cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse: ACCCTGTTGCTGT AGCCA).
4.9. Flow Cytometric Analysis
SLC5A8-pLVX cells were treated for 72 h with varying concentrations of 2,4,6-THBA. Adherent cells were collected by trypsinization and pooled with floating cells, washed with 1X PBS. Cell cycle analysis was performed by adding Vybrant®
DyeCycle™ Green Stain (Invitrogen, Carlsbad, CA, USA) at a final concentration of 250 nM to 1 mL cell suspension. The samples were analyzed by flow cytometry following incubation for 30 min at 37 °C. To detect apoptosis, cells were stained with an Annexin V/7-AAD kit as previously described [7
]. All experiments were carried out by the CytoFLEX flow cytometer (Beckman Coulter, Miami, Indianapolis, IN, USA) using CytExpert 2.0 software (Beckman Coulter, Indianapolis, IN, USA).
4.10. Clonogenic Assay
Clonogenic assays were performed as previously described [51
]. Cells were seeded at a density of 500 cells/100 mm plate and grown for 48 h following which specified compounds were added at the concentrations indicated. The spent media was replaced with fresh media containing the respective compounds every 5–6 days. Cells were incubated for 14–21 days, fixed with 100% methanol for 20 min, and stained with 0.5% crystal violet prepared in 25% methanol. The colonies were then photographed and quantified using the ImageJ software (NIH, Bethesda, MD, USA).
4.11. Statistical Analysis
All experiments were repeated 3–6 times independently of each other. One-way ANOVA followed by Tukey’s post-hoc analysis was used to analyze group differences to the control, and significance was defined at p < 0.05.
In this research paper, using a variety of biochemical, molecular biology and computational approaches, we report that the flavonoid metabolite 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) inhibits CDK enzyme activity and exhibits potent anti-proliferative effects. We showed that cellular uptake of 2,4,6-THBA required the expression of SLC5A8, a monocarboxylic acid transporter. Investigations were also carried out to determine the effectiveness of three other metabolites—4-hydroxybenzoic acid (4-HBA), 3,4-dihydroxybenzoic acid (3,4-DHBA) and 3,4,5-THBA. Of these only 3,4-DHBA and 3,4,5-THBA inhibited cancer cell proliferation and this was independent of both SLC5A8 transport and CDK inhibition. These findings for the first time, suggests that the flavonoid metabolite 2,4,6-THBA along with 3,4-DHBA and 3,4,5-THBA may contribute to the chemo-preventive effects of flavonoids against CRC. In addition, our studies also highlight the need of further investigations directed towards the role(s) played by flavonoid metabolites in the prevention of cancer. Our finding that 2,4,6-THBA is an effective inhibitor of CDKs that acts as an anti-proliferative agent suggests that it has the potential to be developed into a novel class of CDK inhibitors.