Cymopolia barbata (Linnaeus) V.Lamouroux (Dasycladaceae) is widespread in shallow waters and is seen covering rocks by the shorelines in tropical marine habitats. Known to grow to about 10-cm high, these green algae (Chlorophyta) have tufts at the end of their stems that are lightly calcified. Extracts from this plant have previously shown significant pharmacological properties such as antifungal, antitumor, antimicrobial, and antimutagenic activities 1 2 3 4 5 6 7 8 . Although the cymopols are known halogenated natural products which have been isolated from C. barbata, active ingredients responsible for the displayed biological activities remain unspecified. In this study, we investigated bioactivities of prenylated bromohydroquinones (PBQ), cymopol-related metabolites which are known to accumulate in C. barbata, and report, for the first time, biological activities from single ingredients isolated from this marine algae. Two of these compounds namely, 7-Hydroxycymopochromanone (PBQ1) and 7-hydroxycymopolone (PBQ2), shown in Figure 1 were investigated for cytotoxicity against three cancerous cell lines, one normal cell line, in addition to their potential for chemoprevention via inhibition of cytochrome P450 (CYP) 1 enzymes.

The CYP1 family of enzymes and in particular CYP1B1 appears to be a universal molecular cancer marker and a target for drug discovery. Findings of the over-expression of CYP1B1 in many tumor tissues compared with normal surrounding cells have led to the search for prodrugs reliant on CYP1B1 metabolism for the conversion into cytotoxic therapeutics 9 . The modification in the expression levels of CYP1B1 has been shown to modulate tumor progression 10 and thus specific inhibitors are expected to be of therapeutic/preventive benefit. Further, the involvement of CYP1 enzymes in the bioactivation of procarcinogens such as polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines, aromatic amines, and nitro polycyclic hydrocarbons 11 , in addition to the biotransformation of anticancer drugs, has stimulated research into inhibitors of CYP1 enzyme activity 12 13 . Such inhibitors are thought to be potential anti-carcinogens if they could inhibit the activities of CYP1B1 and CYP1A1 to metabolize PAHs to toxic intermediates and/or decrease their ability to detoxify cancer drugs. A number of natural products have been found to be direct inhibitors of CYP1 enzymes, as well as generate metabolites that are CYP inhibitors with cytotoxic properties. In the study described in this article, PBQ2 demonstrated potent inhibition against CYP1B1 activity, together with promising and specific activity against the colon cancer cell line HT29. The examination of a close structural relative, PBQ1, also allows identification of structural motifs critical for biological activity.

Two PBQs (Figure 1) were isolated from the marine alga C. barbata and investigated for biological activity. The ability of these compounds to interfere with the reduction of the tetrazolium salt in the MTS assay was examined as a measure of impact on cell viability (Figure 2) using normal and cancer colon cells (CCD18 Co and HT29, respectively) along with liver and breast cancer cells (HepG2 and MCF-7, respectively). IC₅₀ values were calculated for test compounds and positive control known drug entities, doxorubicin, fluorouracil, and tamoxifen (Table 1). PBQ2 selectively impacted the viability of colon cells, HT29 with comparable potency to fluorouracil (for HT29 cancer cells: IC₅₀, 19.82 ± 0.46 μM compared to 23.50 ± 1.12 μM and normal colon cells, CCD18 Co IC₅₀, 55.65 ± 3.28 compared to 55.51 ± 3.71 μM, respectively). PBQ1 had no significant impact (<10% at 60 μM) on any of the cell lines tested.

Key NI, no impact (<10% inhibition at 60 μM); NA, not applicable; ND, not determined.

To verify the accuracy of experimental techniques employed to detect CYP inhibition, assays with known inhibitors were carried out with furafylline (against CYP1A2 activity), ketoconazole (against activities of CYP1A1, CYP1B1, and CYP3A4), (−)-N-3-Benzyl-phenobarbital (against 2 C19) and quinidine (against CYP2D6 activity) and the obtained IC₅₀ values (0.8 ± 0.2, 0.04 ± 0.01, 6.3 ± 1.7, 0.06 ± 0.01, 0.3 ± 0.01, 0.03 ± 0.01 μM, respectively) compared well with published values (0.99, <10, <10, 0.06, 0.25, and 0.04 μM, respectively; 17 18 19 20 ). Michaelis constant, K _M, was determined for each marker substrate under the specified experimental conditions, in order to determine suitable substrate concentrations for assessing inhibitory potential of test compounds 21 .

Both PBQs 1 and 2 potently (IC₅₀ < 1 μM) inhibited the activity of CYP1A1 (IC₅₀s of 0.39 ± 0.05 and 0.93 ± 0.26 μM, respectively). PBQ2 also potently inhibited the activity of CYP1B1 (IC₅₀, 0.14 ± 0.04 μM) as shown in Figure 3. For those interactions yielding an IC₅₀ < 10 μM against the activities of CYP1 family, further kinetic characterization was carried out to determine the nature of the inhibition, and Eadie-Hofstee plots are illustrated in Figure 4. Reversible enzyme kinetics was observed for PBQ1 with partial noncompetitive inhibition of CYP1A1 activity, noncompetitive inhibition of CYP1A2 activity (K _i s of 3.07 ± 0.69 and 9.16 ± 4.68 μM, respectively) and uncompetitive inhibition of CYP1B1 activity (K _i of 0.26 ± 0.03 μM). PBQ2 displayed uncompetitive inhibition of the activity of CYP1A1 (K _i of 0.84 ± 0.07 μM) and partial noncompetitive inhibition of the activity of CYP1B1 (K _i of 4.7 × 10^–3 ± 5.1 × 10^–4 μM).

Further characterization of the isolated test compounds against other major drug metabolizing P450 enzymes (CYP2C19, CYP2D6, and CYP3A4) was carried out. A summary table with all IC₅₀ data is presented in Table 2. As seen therein, while both PBQs 1 and 2 moderately (IC₅₀ > 1 μM) inhibited the activities of CYP2D6 (IC₅₀s, 1.03 ± 0.40 and 2.75 ± 0.96 μM, respectively) and CYP3A4 (IC₅₀s, of 5.07 ± 3.54 and 8.31 ± 4.67 μM, respectively). PBQ1 and PBQ2 potently inhibited the activity of CYP2C19 (0.08 ± 0.03 and 0.12 ± 0.06 μM, respectively).

From the panel of cell lines tested, the impact on the viability of malignant colon (HT29) and normal colon (CCD18 Co) cells by PBQ2 was similar to that imparted by the chemotherapeutic drug, fluorouracil, with comparable IC₅₀ values. PBQ2 also displayed selective cytotoxicity toward HT29 cells with no impact on cancerous liver (HepG2) and breast (MCF-7) cells. PBQ1, its structural isomer on the other hand, had no impact on any of the cell lines investigated and thus the presence of a tertiary hydroxyl group on PBQ2 appears to be critical for the observed bioactivity. Be it the formation of hydrogen bonds with key residues within the cell or during receptor-mediated cell permeability, the conjugated, open ring, and hydroxyl group presence in PBQ2 plays a crucial role in impacting cell viability compared with epoxy moiety of PBQ1.

Inhibitors of CYPs1A1 and 1B1 enzymes have received particular interest due to their role in reducing the activation of carcinogens and thus as chemoprotectors and chemotherapeutics. Several classes of natural compounds 22 , including flavonoids 23 24 and organosulfur compounds 13 25 26 , have demonstrated great potential in chemoprevention and thus provide the impetus for the search for others. Both PBQs potently (IC₅₀ < 1 μM) inhibited the activity of CYP1A1while PBQ2 also displayed potency toward CYP1B1. Studies using knock-out mice models have linked CYP1B1 with the activation of several carcinogens, such as BaP and DMBA 11 and have also been shown to play an important role in modulating tumor progression 10 27 . Thus, the potency toward both CYP1B1 and CYP1A1 activities by PBQ2 suggests potential chemopreventive bioactivity in vivo. Conversely, PBQ1 with demonstrated CYP1A1 inhibition, devoid of cytotoxicity on all cells examined in this study, also highlights it as an attractive candidate with potential for development as a chemoprotector.

The development of novel classes of therapeutics that can target both drug metabolizing enzymes and disease pathways is a multi-targeted approach that may well suit the multi-factorial origins of a disease such as cancer. Examples of such compounds include isothiocyanate which impact both Nrf-2 transcription factors, and inhibit the nuclear factor ĸß pathway to exhibit potent anti-inflammatory properties 28 . Such attractive dual qualities are displayed by PBQ2 in this study, with potent and selective targeting of HT29 colon cancer cells, as well as the inhibition of CYP1A1 and CYP1B1enzyme activities.

The potency of PBQ2 can be put into perspective with the reported inhibitory effect of eight flavonoids tested against recombinant human CYP1B1 and CYP1A1 enzyme activities which showed a range in IC₅₀s between 0.3 and 27 μM 29 . They made ideal chemoprotectants against prostate cancer 30 . PBQ2 had an IC₅₀ of 0.14 μM, which appears to be more potent at inhibiting CYP1B1 activity than all the eight flavonoids tested by Chaudhary and Willet 29 , making this isolate an ideal candidate for further research.

PBQs examined in this study displayed reversible, non or uncompetitive (partial or full) kinetics on CYP1 enzyme activities. In noncompetitive inhibition, typically the IC₅₀ value is equal to the K _i , while in uncompetitive inhibition IC₅₀ will equal twice the value of the K _i for experiments where the substrate concentration is close to the K _m value 31 , as designed in our experiments. Such approximations are observed in the kinetics of PBQ1 with CYPs1A2, 1B1 and of PBQ2 with CYP1A1, although deviations from these approximations were seen for interactions of PBQ1 with CYP1A1 and PBQ2 with CYP1B1 where the partial noncompetitive kinetics were observed and such mixed type binding may complicate relations between IC₅₀ and K _i s. Due to the non and uncompetitive nature of PBQ binding with CYP1A1, previous active site models developed for CYP1A1 using natural product quassinoids 20 32 , were therefore not useful in shedding light on structure–activity relationships within the active site. The dietary flavonoid, galangin, shown to display inhibition of DMBA-induced CYP1A1 in MCF-7 breast cancer cells, was of a noncompetitive, dose-dependent manner 33 similar to that of PBQ1.

Investigating the impact of these PBQs against the other major drug metabolising enzymes (CYP2C19, CYP2D6, and CYP3A4) allowed for the predictions of drug interaction potential. The impacts on CYP3A4 and CYP2D6, the enzymes responsible for metabolism of over 90% of drugs on the market, were only moderate by the two PBQs, suggesting unlikely metabolism-based drug interactions via these important enzymes. However, both compounds potently inhibited CYP2C19 activity. CYP2C19 is also involved in the process of carcinogenesis albeit with lower impact than the CYP1 family, and the CYP2C19 inhibition by the PBQs may prove useful in chemopreventive value, although drug interactions possibility via this enzyme that metabolises important therapeutics such as omeprazole and theophylline will remain a concern compounded by likely variations in inhibition reliant on the expression levels of this polymorphic enzyme.

The polyisoprenylated bromohydroquinone, PBQ2, was found to affect cell viability of colon cells (HT29) comparable to the chemotherapeutic drug fluorouracil, with selectivity, making this compound an ideal lead candidate suitable for further experimentation in chemotherapy of colon cancer cells. In addition, it showed potent inhibition against CYP1B1 enzyme activities, a marker for cancer and target for drug discovery. Compounds such as PBQ2 that can target both drug metabolizing enzymes and disease state cells are of high value. Such chemotherapeutic and chemopreventive potential implied by the displayed bioactivity validate the on-going search for treatment leads among natural products from endemic tropical biodiversity including marine habitats.

The authors declare that they have no competing interests.

SB carried out all CYP inhibition and cell culture assays, WG carried out extraction and purification of natural products, GH designed and assisted all cytotoxicity assays, TJT participated in coordination of cytotoxicity assays, RD conceived of the study and participated in its design and coordination. All authors read and approved final manuscript.