Resveratrol directly targets COX-2 to inhibit carcinogenesis

Abstract

Targeted molecular cancer therapies can potentially deliver treatment directly to a specific protein or gene to optimize efficacy and reduce adverse side effects often associated with traditional chemotherapy. Key oncoprotein and oncogene targets are rapidly being identified based on their expression, pathogenesis and clinical outcome. One such protein target is cyclooxygenase-2 (COX-2), which is highly expressed in various cancers. Research findings suggest that resveratrol (RSVL; 3,5,4'-trihydroxy-trans-stilbene) demonstrates nonselective COX-2 inhibition. We report herein that RSVL directly binds with COX-2 and this binding is absolutely required for RSVL's inhibition of the ability of human colon adenocarcinoma HT-29 cells to form colonies in soft agar. Binding of COX-2 with RSVL was compared with two RSVL analogues, 3,3',4',5',5-pentahydroxy-trans-stilbene (RSVL-2) or 3,4',5-trimethoxy-trans-stilbene (RSVL-3). The results indicated that COX-2 binds with RSVL-2 more strongly than with RSVL, but does not bind with RSVL-3. RSVL or RSVL-2, but not RSVL-3, inhibited COX-2-mediated PGE(2) production in vitro and ex vivo. HT-29 human colon adenocarcinoma cells express high levels of COX-2 and either RSVL or RSVL-2, but not RSVL-3, suppressed anchorage independent growth of these cells in soft agar. RSVL or RSVL-2 (not RSVL-3) suppressed growth of COX-2(+/+) cells by 60% or 80%, respectively. Notably, cells deficient in COX-2 were unresponsive to RSVL or RSVL-2. These data suggest that the anticancer effects of RSVL or RSLV-2 might be mediated directly through COX-2.

Figures

Figure 1

Structure of resveratrol and its derivatives. Resveratrol (RSVL, A) and two of its analogues, RSVL-2 (B) and RSVL-3 (C), are polyphenolic compounds differing by the number of hydroxyl or methoxy groups, respectively.

Figure 2

In vitro and ex vivo verfication of resveratrol binding with COX-2. (A), Expression of COX-2 in HCT116 or HT-29 human colon cancer cell lines. (B), Resveratrol binding with COX-2 in vitro was confirmed by immunoblotting with an antibody against COX-2. Lane 1–input control: COX-2 recombinant protein; lane 2–negative control: Sepharose 4B was used to pull down COX-2 as described in Materials and Methods; lane 3–COX-2 was pulled down using resveratrol-Sepharose 4B affinity beads. (C), COX-2 binding with resveratrol ex vivo was confirmed by immunoblotting with an antibody against COX-2. Lane 1–input control: whole-cell lysate from HT-29 human colon cancer cells; lane 2–negative control: a lysate prepared from HT-29 human colon cancer cells was precipitated with Sepharose 4B beads as described in Materials and Methods; lane 3–whole-cell lysate prepared from HT-29 cells was precipitated by resveratrol-Sepharose 4B affinity chromatography as described in Materials and Methods. (D), Comparison of sensograms representing the interaction of RSVL, RSVL-2 or RSVL-3 (each at 300 μM) with 0.1 μg His-COX-2 as visualized by SPR (surface plasmon resonance) produced by a BIAcore X as described in Materials and Methods. The dissociation phase was started at 180 seconds. One representative graph from results of two independent experiments is shown.

Figure 3

Resveratrol directly binds with COX-2. Binding of resveratrol to COX-2 was determined by fluorescence spectroscopy. (A), Increasing concentrations of resveratrol (5−120 μM) amplify the fluorescence signal indicating an enhanced dose-dependent binding of resveratrol with COX-2. (B), Resveratrol (10 μM) was incubated with or without (negative control) 0.2 μM COX-2 recombinant protein and the emission spectra were collected after excitation at 320 nm; (a.u.) = absorbance units at 389 nm. (C), The Kd (dissociation kinetic value) of resveratrol's interaction with COX-2 was determined. COX-2 (0.2 μM) was incubated with increasing concentrations of resveratrol (0−110 μM). Fluorescence intensity at 389 nm was used to observe the change in fluorescence associated with resveratrol and protein binding. The inset shows the fluorescence unit change at 389 nm used to determine the concentration of bound and free resveratrol. The graph is representative of three independent experiments.

Figure 3

Resveratrol directly binds with COX-2. Binding of resveratrol to COX-2 was determined by fluorescence spectroscopy. (A), Increasing concentrations of resveratrol (5−120 μM) amplify the fluorescence signal indicating an enhanced dose-dependent binding of resveratrol with COX-2. (B), Resveratrol (10 μM) was incubated with or without (negative control) 0.2 μM COX-2 recombinant protein and the emission spectra were collected after excitation at 320 nm; (a.u.) = absorbance units at 389 nm. (C), The Kd (dissociation kinetic value) of resveratrol's interaction with COX-2 was determined. COX-2 (0.2 μM) was incubated with increasing concentrations of resveratrol (0−110 μM). Fluorescence intensity at 389 nm was used to observe the change in fluorescence associated with resveratrol and protein binding. The inset shows the fluorescence unit change at 389 nm used to determine the concentration of bound and free resveratrol. The graph is representative of three independent experiments.

Figure 4

Inhibitory effect of RSVL, RSVL-2, RSVL-3 or CEL on COX-2-mediated PGE2 production. The effects of different concentrations of RSVL, its analogues, or CEL on COX-2-mediated PGE2 production in vitro, using a recombinant COX-2 protein (Sigma; A), and ex vivo, using COX-2+/+ cells (B), were determined by measuring prostaglandin E2 production as described in the protocol of the COX Inhibitory Screening Kit (Cayman Chemical). Data are presented as means ± S.D. of three samples from two independent experiments. For A and B, the asterisk (*) indicates a significant decrease (p < 0.001) in COX-2-mediated PGE2 production compared to untreated control.

Figure 5

Inhibitory effect of RSVL and its analogues on growth and colony formation of HT-29 colon cancer cells in soft agar. (A), HT-29 cells (1×104) were treated or not treated with RSVL, RSVL-2, or RSVL-3 (each at 10 μM) and cell number was calculated each day over 5 days. Data are presented as means ± S.D. from 3 samples of 2 independent experiments. The asterisk (*) indicates a significant decrease (p < 0.001) in cell number of treated cells compared to respective untreated control cells. (B), Final average number of colonies formed in soft agar. HT-29 colon cancer cells (1 × 104) were subjected to a soft agar assay with or without resveratrol or its analogues (10 or 20 μM). The numbers of colonies were counted after 10 days. Results are expressed as means ± S.D. of three independent experiments. The asterisk (*) indicates a significant decrease in colony number of treated cells compared to untreated control cells (* p < 0.03, 20 μM RSVL; ** p < 0.006, 20 μM RSVL-2).

Figure 5

Inhibitory effect of RSVL and its analogues on growth and colony formation of HT-29 colon cancer cells in soft agar. (A), HT-29 cells (1×104) were treated or not treated with RSVL, RSVL-2, or RSVL-3 (each at 10 μM) and cell number was calculated each day over 5 days. Data are presented as means ± S.D. from 3 samples of 2 independent experiments. The asterisk (*) indicates a significant decrease (p < 0.001) in cell number of treated cells compared to respective untreated control cells. (B), Final average number of colonies formed in soft agar. HT-29 colon cancer cells (1 × 104) were subjected to a soft agar assay with or without resveratrol or its analogues (10 or 20 μM). The numbers of colonies were counted after 10 days. Results are expressed as means ± S.D. of three independent experiments. The asterisk (*) indicates a significant decrease in colony number of treated cells compared to untreated control cells (* p < 0.03, 20 μM RSVL; ** p < 0.006, 20 μM RSVL-2).

Figure 6

Effect of RSVL, its analogues, or CEL on COX-2+/+ and COX-2−/− cell growth after 6 days. COX2+/+ and COX2−/− cells (inset shows COX02 expression in respective cell lines) were treated with RSVL, analogues, or CEL (10 μM) and cell number was determined each day over a 6-day period using a Coulter Z cell counter. Control represents untreated cells. Data are presented as means ± S.D. of three samples from two independent experiments. The asterisk (*) indicates a significant difference (p < 0.001) in number of treated COX-2+/+ or COX-2−/− cells compared to respective untreated cells.