Resveratrol analog, 3,4,5,4'-tetrahydroxystilbene, differentially induces pro-apoptotic p53/Bax gene expression and inhibits the growth of transformed cells but not their normal counterparts

Jiebo Lu, Chi-Tang Ho, Geetha Ghai and Kuang Yu Chen,,,

1 Department of Chemistry,
2 Center for Advanced Food Technology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8087, USA and
3 New Jersey Cancer Institute, New Brunswick, NJ 08901, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resveratrol, a trihydroxystilbene found in grapes and other plants, has been shown to be active in inhibiting multistage carcinogenesis. Using resveratrol as a prototype, we have synthesized a number of polyhydroxy- and polymethoxy-stilbenes and tested their anti-proliferative effect in normal and transformed human cells. Here we show that one of the resveratrol analogs, 3,4,5,4'-tetrahydroxystilbene (R-4), specifically inhibited the growth of SV40 virally transformed WI38 cells (WI38VA) at 10 µM, but had no effect on normal WI38 cells at even higher concentrations. R-4 also prominently induced apoptosis in WI38VA cells, but not in WI38 cells. RNase protection assay showed that R-4 significantly induced the expression of p53, GADD45 and Bax genes and concomitantly suppressed the expression of bcl-2 gene in WI38VA, but not in WI38 cells. A large increase in p53 DNA binding activity and the presence of p53 in the Bax promoter binding complex suggested that p53 was responsible for the Bax gene expression induced by R-4 in transformed cells. Within 4 h of treatment with R-4, the Bax to bcl-2 protein ratio in WI38 and WI38VA cells was, respectively, 0.1 and 105, a difference of three orders of magnitude. While R-4 prominently induced the p53/Bax pro-apoptotic genes, it also concomitantly suppressed the expression of Cox-2 in WI38VA cells. Taken together, our study suggests that the induction of p53 gene by R-4 in transformed cells may play a key role in the differential growth inhibition and apoptosis of transformed cells.

Abbreviations: APC, adenomatous polyposis coli; BPS, Bax promoter sequences; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDL, population doubling level; R-4, 3,4,5,4'-tetrahydroxystilbene; TUNEL assay, TdT-mediated dUTP nick end-labeling assay.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resveratrol (3,5,4'-trihydroxy-trans-stibene), a phytoalexin present in grapes, peanuts and pines, has antioxidant and anti-inflammatory activities (13) and is the active ingredient in Leguminosae that inhibits cellular events associated with tumor initiation, promotion and progression in a mouse skin cancer model (3). In vitro, resveratrol inhibits the growth of several tumor cell lines, including human oral squamous carcinoma (SCC-25) (4), human promyelocytic leukemia (5), human breast cancer cells (6) and prostate cancer cells (7). However, it is unclear whether resveratrol also exerts the same inhibitory effect on the normal cells. At molecular levels, it has been reported that resveratrol inhibits Cox-2 gene expression in 12-O-tetradecanoylphorbol 13-acetate-treated mammary epithelial cells (8), inhibits human P450 1A1 (9) and increases the p53 protein level in mouse epidermal cells (10). Because of the involvement of Cox-2 in colon cancer and other inflammatory processes (11), the effect of resveratrol on Cox-2 gene expression is of interest (8). Resveratrol, as a phytoestrogen, has also been proposed to provide cardiovascular protection (12). In view of the pharmacological potential of resveratrol, we have synthesized additional stilbene derivatives using resveratrol as a prototype. Here we report the test of eight polyhydroxy- and polymethoxy-stilbenes, including resveratrol, on the growth of a matched pair of normal and transformed fibroblasts. Among the compounds tested, 3,4,5,4'-tetrahydroxystilbene (R-4) and its methoxy-derivative (MR-4) preferentially inhibited the growth of transformed cells, but had almost no effect on the growth of the normal cells. Resveratrol and other analogs did not exhibit such a differential effect. We showed that R-4 also differentially induced p53 gene expression, caused a large increase in the Bax/bcl-2 ratio and induced apoptosis in transformed cells, but not in normal cells. In addition, R-4 preferentially suppressed the expression of Cox-2 gene in the transformed cells. These features should make R-4 an interesting compound for further evaluation as a potential chemopreventive and chemotherapeutic agent.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Gibco BRL (Gaithersburg, MD). [{gamma}-32P]ATP (end-labeling grade, >7000 Ci/mmol) and [{alpha}-32P]UTP were purchased from ICN (Costa Mesa, CA). Human Stress-1Multi-Probe Template Set was purchased from PharMingene (San Diego, CA). Anti-Cox-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI) and antibodies for p53, Bax, BclxS BclxL Bcl-2, ß-actin were from Santa Cruz Biotechnology (Santa Crutz, CA). Other chemicals were from Sigma (St Louis, MO).

Preparation of resveratrol and its analogs
The synthesis of resveratrol using 4-methoxybenzyl alcohol and 3,5-dimethoxy-benzaldehyde as the starting materials has been described (13,14). Similar strategy was employed to prepare resveratrol analogs. The identity and purity of each of these compounds have been confirmed by thin layer chromatography, NMR and GC-Mass. Figure 1Go shows the chemical structures of resveratrol and its analogs. Polyhydroxystilbenes are designated as R followed by a number indicating the number of hydroxyl groups. Polymethoxystilbenes are designated as MR followed by a number indicating the number of methoxy groups. Thus, resveratrol is designated as R-3 because it has three hydroxyl groups on the stilbene ring.



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Fig. 1. Structure of resveratrol and resveratrol analogs. The number after R or MR indicates the number of hydroxyl or methoxyl groups on the stilbene ring. MR-4 and MR-4' both contain four methoxy groups but with one at a different position.

 
Cellular proliferation assay
WI38 (cell strain AG06814E, PDL = 16), a normal human diploid lung fibroblast cell strain with a finite life span, and the SV40 virally transformed WI38 cells (cell strain AG07217) were obtained from the National Institute on Aging Repository, Coriell Institute for Medical Research (Camden, NJ). Cells were cultured in DMEM containing 10% fetal bovine serum at 37°C. For proliferation assay, cells were plated at ~1x105 cells per 35 mm dish, with or without resveratrol or its analogs, and the number of viable cells was counted under a phase contrast microscope (15).

DNA fragmentation assay
Confluent cultures were treated with R-4 at 50 µM for various times. Cells were harvested and suspended in a lysis buffer (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 25 mM EDTA, 0.5% SDS and 100 µg/ml Proteinase K) for 20 h at 37°C. DNA was extracted with a mixture of phenol and chloroform, precipitated by ethanol, dried and dissolved in a TE buffer. RNA was digested with 2 µg/ml RNase Cocktail (Ambion, Austin, TX). The DNA samples were analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide (0.5 µg/ml) and visualized under UV illumination.

TUNEL assay
Apoptosis was also analyzed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay as described previously (15). Briefly, cultures were treated with R-4 (50 µM) for 20 h. Cells were fixed with 4% of formaldehyde solution, washed and incubated in a buffer containing fluorescein-12–dUTP and terminal deoxynucleotidyl transferase for 1 h. Apoptotic cells were visualized under a fluorescent microscope using an FITC filter.

Reverse transcription–polymerase chain reaction (RT–PCR)
Confluent cultures were serum-deprived for 48 h and then stimulated with 10% fresh fetal bovine serum in the presence or absence of 50 µM R-4. Cells were harvested at indicated times and the total RNA was prepared using RNeasyTM Total RNA Kit (Qiagen, Chatsworth, CA). The total RNA (1 µg) was reverse transcribed into cDNA by incubating with SuperScriptTM RNase H reverse transcriptase (Gibco BRL, Grand Island, NY) using oligo(dT)12–18 as primer. For PCR amplification, gene-specific primers, both sense and antisense, were used. The sequences of the sense primers are:

PCR conditions were chosen to ensure that the yield of the amplified product was linear with respect to the amount of input RNA. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was used as an internal control. The PCR products were analyzed by electrophoresis on a 1% agarose gel containing 0.5 µg/ml ethidium bromide.

RNase protection assay
The RNA probes corresponding to various genes were generated from the Human Stress-1Multi-Probe Template and labeled with [{alpha}-32P]UTP and T7 polymerase. The labeled probes were purified by extraction and ethanol precipitation. Total RNA (8 µg of each sample) was mixed with the labeled probe (~105 c.p.m.) in a hybridization buffer (80% formamide, 0.4 M NaCl, 40 mM PIPES, 1 mM EDTA, pH 8.0) at 90°C for 2 min, 56°C for 16 h and 37°C for 15 min. The sample was then treated with 40 µg/ml RNase Cocktail for 45 min at 30°C, and incubated with 300 µg/ml Proteinase K for 15 min at 37°C. The protected RNA fragments were analyzed on an 8 M urea–denatured polyacrylamide gel and visualized by autoradiography.

Western blot analysis
Cells were harvested, washed and sonicated in a lysis buffer (150 mM NaCl, 100 mM Tris, pH 8.0, 1% Tween-20, 1 mM EDTA, 50 mM DDT, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin). The lysates were analyzed by electrophoresis on a 10% SDS–polyacrylamide gel. The resolved proteins on the gel were transferred onto a nitrocellulose membrane for western blot analysis. The membrane was incubated with various antibodies at appropriate dilution. The affinity purified goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA) was used as the second antibody. The blots were detected with the ECL Plus western blot detection kit (Amersham Pharmacia, Piscataway, NJ).

Gel mobility shift assay
The double-stranded oligonucleotide probes for p53 binding site (sense 5'-TACAGAACATGTCTAAGCATGCTGGGG-3'), mutated p53 binding site (sense 5'-TACAGAATCGCTCTAAGCATGCTGGGG-3'), and the Bax promoter sequence (BPS, sense 5'-AAGTTAGAGACAAGCCTGGGC-3') (16) were annealed and labeled with [{gamma}-32P]ATP and T4 kinase. Approximately 200 pg of 32P-labeled probe was mixed with 20 µg of whole-cell extract in a final volume of 20 µl in a binding buffer containing 20 mM HEPES, pH 7.9, 1 mM MgCl2, 150 mM KCl, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 0.5 µg poly(dI-dC) and 5 µg bovine serum albumin. The binding reaction was carried out for 30 min at room temperature. For gel mobility supershift assay, anti-p53 antibody was added to the binding mixture and the incubation was carried out for another 20 min. At the end of incubation, the binding mixture was analyzed by electrophoresis on a 4% non-denaturing polyacrylamide gel and autoradiography.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of resveratrol and its analogs on cell proliferation
We first compared the anti-proliferative effects of resveratrol and its analogs on a pair of normal (WI38) and transformed (WI38VA) human cells. Figure 2Go shows that among the eight compounds tested, only R-4 and MR-4 exhibited differential growth inhibitory effect. R-4 at 10 µM completely blocked the growth of WI38VA cells, but had no effect on the growth of normal WI38 cells. R-4 at higher concentrations (>50 µM) appeared to be slightly cytostatic to WI38 cells as indicated by the lengthening of the doubling time (data not shown). Similarly, MR-4 preferentially inhibited the growth of WI38VA, but not WI38 cells. R-3 (resveratrol) or its methoxy derivative, MR-3, inhibited the growth of both WI38 and WI38VA cells with the same efficacy. The dihydroxy analog of resveratrol (R-2), however, blocked the growth of WI38 cells at 10 µM, but had no effect on WI38VA cells even at 50 µM. The dimethoxy derivative of R-2 (MR-2) did not inhibit the growth of either WI38 or WI38VA cells even at a concentration of 100 µM. MR-4' at 100 µM inhibited the growth of WI38 cells, but not the transformed WI38VA cells and MR-5 inhibited the growth of both WI38 and WI38VA cells only at 100 µM. These results showed that the anti-proliferative activity of polyhydroxy- and polymethoxystilbenes could be modulated by a slight modification of the chemical structure. It is possible that R-4 and MR-4 can specifically target at sites critical to the proliferation of cancer cells but not to normal cells.



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Fig. 2. Effect of resveratrol (R-3) and its analogs on the growth rate of normal WI38 and SV40 transformed WI38VA cells. Cells were plated at ~1.5x105 cells/ml for WI38VA cells and ~1.0x105 cells/ml for WI38 cells in a 35 mm dish on day 1 in the absence (open circles) or in the presence of 1 µM (open triangles), 10 µM (closed squares), 50 µM (open diamonds) or 100 µM (closed circles) of resveratrol analogs. The viable cells were counted at indicated times. Each point represents an average of three separate dishes with standard errors <10%.

 
R-4-induced apoptosis in WI38VA cells
After the treatment of R-4, WI38VA cells exhibited clear cytoplasmic and nuclear condensation (data not shown), suggesting that R-4 may cause apoptosis in WI38VA cells. The characteristic cleavage of DNA into oligonucleosomal fragments has been regarded as a hallmark of apoptosis (17), we therefore used both DNA fragmentation analysis and TUNEL assay to determine whether R-4 indeed may induce apoptosis differently in normal and transformed cells. Figure 3AGo shows that R-4 induced DNA ladder formation, indicative of internucleosomal DNA damage, in WI38VA, but not in WI38 cells. Similarly, TUNEL assay as shown in Figure 3BGo indicates that while almost all WI38VA cells underwent apoptosis, as revealed by labeling with fluorescein-12–dUTP, only a few WI38 cells appeared to be labeled in response to R-4 treatment. These results suggest that apoptosis could be the cause for the differential inhibitory effect of R-4 on the growth of normal and transformed cells.



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Fig. 3. (A) DNA fragmentation analysis. Confluent cultures of WI38 and WI38VA cells were treated without (lanes 1 and 5) or with R-4 at a final concentration of 50 µM (lanes 2–4 and 6–8). The cells were harvested at the indicated times and the formation of DNA ladder was monitored by electrophoresis on a 1% agarose gel. (B) TUNEL assay of the effects of R-4 on apoptosis. WI38 and WI38VA cells at 90% confluence were treated with 50 µM R-4 for 20 h. Apoptotic cells were detected by labeling with fluorescein-12–dUTP using terminal deoxynucleotidyl transferase. The labeled cells were detected by fluorescent microscope using FITC filter (1) and (3). All intact cells in the culture, including the apoptotic ones, were shown by phase contrast microscope (2) and (4).

 
Analysis of apoptotic pathway activated by R-4
Bax and bcl-2 genes are considered to be key players in the apoptosis pathways (18,19). To further understand the molecular basis for the differential effect of R-4 on the growth and apoptosis of normal and transformed cells, we examined the effect of R-4 on the expression of Bax, bcl-2 and other related genes in WI38 and WI38VA cells. RNase protection assay was employed to determine the effect of R-4 on the expression of bcl-x, p53, GADD45, c-fos, p21, Bax, bcl-2 and mcl-1 genes. Figure 4AGo shows that R-4 had little or no effect on the expression of all these genes in normal WI38 cells over a 24 h time period. In contrast, R-4 rapidly and markedly induced the increase in the expression of p53, GADD45 and Bax genes in WI38VA cells. Concomitantly, R-4 suppressed the mRNA levels of bcl-x, bcl-2 and mcl-1 in WI38VA cells. Thus, R-4 was potent in inducing the expression of pro-apoptotic genes (p53 and Bax) and in suppressing the expression of anti-apoptotic genes (bcl-2, bcl-x and mcl-1) in transformed cells, but not in normal cells. Figure 4BGo shows that the changes of mRNA levels of these apoptosis-related genes were also reflected at the protein levels. Thus, R-4 induced an increase in p53 and Bax protein in WI38VA cells by 10- and 20-fold, respectively, after 12 h of treatment. The Bax/bcl-2 protein ratio has been considered as a rheostat for measuring the susceptibility of cells to apoptosis (19). Figure 5Go shows the dramatic effect of R-4 on the Bax/bcl-2 ratio in WI38VA cells. Bax/bcl-2 ratio in WI38VA cells increased from 1.5 to 105 after 4 h of R-4 treatment and remained at this high level for at least 24 h. In contrast, the Bax/bcl-2 ratio in WI38 cells treated with R-4 was consistently lower than that of the control, and remained at a level below 2.



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Fig. 4. (A) RNase protection assay. WI38 and WI38VA cells at 90% confluence were serum-deprived for 36 h and then replenished with complete growth medium containing 10% fetal bovine serum without (–) or with (+) R-4 at 50 µM. The cells were harvested at indicated times for total RNA preparation. RNA samples were analyzed by RNase protection analysis as described in Materials and methods. (B) Western blot analysis. WI38VA cells at 90% confluence were serum-deprived for 48 h and then serum stimulated with 10% fetal bovine serum in the presence of R-4. Cells were harvested at indicated time and the whole cell extracts were prepared for western blot analysis using specific antiserum as described in Materials and methods. Each lane contained 30 µg of proteins. The ß-actin level was used as an internal standard.

 


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Fig. 5. The effect of R-4 on the Bax/bcl-2 protein ratio in WI38 and WI38VA cells. Western blot analysis was performed as described in Materials and methods for WI38 and WI38VA cells with or without R-4 treatment (50 µM) for 4, 16 and 24 h. The relative protein levels were estimated by densitometric tracing of the protein bands. The ratio was an average calculated based on the results from two separate experiments.

 
Activation of Bax gene promoter involves p53
Figure 6AGo shows that the increase in p53 gene expression was accompanied by a large increase in p53 DNA binding activity in WI38VA cells. This increase in p53 DNA binding activity is likely to be the cause for the increase in Bax gene expression because Bax promoter contains p53 binding sites (20). Indeed, using Bax gene promoter fragment as a probe, R-4 induced a striking increase in DNA binding activity with a time course similar to that of p53 DNA binding (Figure 6BGo, lanes 3–5 versus Figure 6AGo, lanes 2–4). Moreover, when the specific antiserum against p53 was added into the binding mixture, the position of the DNA binding complex was further retarded (Figure 6BGo, lanes 6 and 7 versus lanes 4 and 5), indicating that p53 protein was likely to be present in the binding complex. Taken together, these data strongly suggest that p53 was responsible for the large increase in Bax/bcl-2 ratio in transformed cells.



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Fig. 6. Gel mobility shift assay (A) and gel mobility supershift assay (B). WI38VA cells at 90% confluence were serum-deprived for 36 h and then stimulated with 10% fetal bovine serum in the presence of 50 µM of R-4. The cells were harvested at the indicated times for the preparation of whole-cell extract. The binding and the electrophoretic analysis on polyacrylamide gel were performed as described in Materials and methods. The probes used in binding reaction were consensus p53 binding site (CS), mutated p53 binding site (mCS) and BPS. (A) Lane 1, free CS; lanes 2–4, CS with cell extracts; lane 5, competitor (100x unlabeled CS); lanes 6 and 7, mCS with cell extracts. (B) Lane 1, free BPS; lane 2, BPS with cell extracts and competitor (100x unlabeled BPS); lanes 3–5, BPS with cell extracts; lanes 6 and 7, BPS with cell extracts and anti-p53 antibody (0.5 µg).

 
Effect of R-4 on the expression of Cox and BRCA genes
Overexpression of p53 has been shown to suppress the expression of Cox-2 gene expression, presumably due to a direct competition of p53 with TBP for the TATA binding sites on the Cox-2 promoter (8,21). In light of the striking effect of R-4 on p53 gene expression in the transformed cells, it is of interest to examine whether R-4 may differentially suppress the expression of Cox-2 gene. We also included BRCA1 and BRCA2 in this study, because of the previous finding that BRCA1 may induce an accumulation of p53 protein (22). Figure 7Go shows that R-4 was more potent in suppressing the serum-induced expression of Cox-2 gene in WI38VA (Figure 7Go, lanes 12–14 versus lanes 9–11) than in WI38 cells (Figure 7Go, lanes 5–7 versus lanes 2–4). Colorectal carcinoma tissues from both human and rodent contain elevated levels of Cox-2 (23,24). It is therefore of interest to examine whether R-4 also blocks the induction of Cox-2 gene in Caco-2 cells. Figure 7Go shows that indeed R-4 prominently suppressed the serum-induced Cox-2 gene expression in Caco-2 colon cancer cells. As expected, Cox-1 was constitutively expressed in all cell types and its expression was not affected by R-4. R-4 did not significantly affect the expression of BRAC2 gene, but appeared to downregulate BRAC1 gene expression in all three cell lines.



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Fig. 7. Effect of R-4 on the expression of growth and apoptosis-related genes. WI38, WI38VA and Caco-2 cells at 90% confluence were serum-deprived for 48 h and then stimulated with 10% fetal bovine serum in the absence (–) or presence (+) of 50 µM of R-4 for various times. The cells were harvested at indicated times for total RNA preparation. The relative level of mRNA of each indicated gene was analyzed by RT–PCR as described in Materials and methods. The mRNA level of housekeeping gene, GAPDH, was used as an internal standard.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resveratrol is a naturally occurring trihydroxystilbene, which has been demonstrated to have cancer chemopreventive activity in a number of experimental systems (3,25). In the present study we have tested the effect of resveratrol and seven resveratrol analogs (Figure 1Go) on the growth of normal and transformed human cells. Among the compounds tested, R-4 and MR-4 exhibited a clear differential growth inhibitory effect toward transformed WI38VA cells (Figure 2Go). The transformed cells, but not the normal cells, underwent apoptosis after the treatment with R-4 (Figure 3Go), suggesting that apoptosis is the major cause for the differential growth inhibitory effect of R-4 on the transformed cells.

p53 is considered to be a guardian of the genome and a gatekeeper for growth and division because of its role in controlling critical checkpoints in response to DNA damage, hypoxia, viral infection and various oncogene activations (26,27). The p53 target genes can be functionally grouped into either growth inhibitory or pro-apoptotic ones. Both p21 and GADD45 are downstream p53-dependent growth inhibitory genes whereas Bax is the p53-dependent pro-apoptotic gene (27). R-4 significantly induced the expression of p53 in transformed WI38VA, but not in normal WI38 cells (Figure 4Go). The increase in p53 gene expression was accompanied by a prominent increase in p53 DNA binding activity (Figure 6AGo) and Bax promoter binding activity (Figure 6BGo) in R-4-treated WI38VA cells. In contrast, R-4 almost had no effect on the expression of p53 and p53-dependent genes in normal cells (Figure 4AGo). The striking difference in the Bax/bcl-2 protein ratio between normal and transformed cells after R-4 treatment (Figure 5Go) is consistent with the finding that R-4 induced apoptosis only in transformed cells, and not in normal cells (Figure 3Go).

Since p53 may be involved in Cox-2 gene regulation via competition at the TATA binding site (8), the suppression of Cox-2 gene expression by R-4 (Figure 7Go) could be causally related to p53. Direct genetic evidence linking Cox-2 to tumorigenesis has been shown by using the APC gene knockout mice (28). Inhibition of Cox-2 enzyme by specific inhibitors can reduce the risk of colon cancer (29). Thus, the prominent inhibitory effect of R-4 on Cox-2 gene expression in Caco-2 cells could enhance the merit of R-4 as a potentially useful chemopreventive agent for cancers of epithelial origin.

In summary, our study shows that R-4, a resveratrol analog, preferentially inhibited the growth of transformed human cells, most likely via the induction of p53 gene expression and subsequent activation of the p53-dependent pro-apoptotic genes. Our findings also suggest that further modification of the chemical structure of polyhydroxystilbene may produce more specific and potent chemotherapeutic or chemopreventive reagents.


    Notes
 
3 To whom correspondence should be addressed at: Department of Chemistry, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854-8087, USA Email: kychen{at}rutchem.rutgers.edu Back


    Acknowledgments
 
The authors thank Dr Zong Ping Chen and Monika Linowaski for performing the initial studies. We also acknowledge the helpful discussions with Drs M.T.Huang, R.Rosen and D.Evans. The work was supported by grant SNJ-CST 3403 from the Commission on Science and Technology, State of New Jersey.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received August 5, 2000; revised October 19, 2000; accepted November 3, 2000.