Antitumor effect of resveratrol oligomers against human cancer cell lines and the molecular mechanism of apoptosis induced by vaticanol C
Tetsuro Ito1,3,
Yukihiro Akao1,4,
Hong Yi1,
Kenji Ohguchi1,
Kenji Matsumoto1,
Toshiyuki Tanaka3,
Munekazu Iinuma2,3 and
Yoshinori Nozawa1
1 Gifu International Institute of Biotechnology, Mitake, Kani-gun, Gifu 505-0116, Japan
2 Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-5858, Japan
3 Gifu Prefectural Istitute of Health and Environmental Sciences, Kakamigahara, Gifu 504-0838, Japan
4 To whom correspondence should be addressed Email: yakao{at}giib.or.jp
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Abstract
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Twenty resveratrol (3,5,4'-trihydroxystilbene) (Res) derivatives, which were isolated from stem bark of Vatica rassak (Dipterocarpaceae), were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. Among them, seven compounds displayed marked cytotoxicity. Vaticanol C (Vat C) as a major component induced a considerable cytotoxicity in all cell lines tested and exhibited growth suppression in colon cancer cell lines at low dose. Vat C caused two cell lines (SW480 and HL60) to induce cell death at four to seven times lower concentrations, compared with Res. The growth suppression by Vat C was found to be due to apoptosis, which was assessed by morphological findings (nuclear condensation and fragmentation) and DNA ladder formation in the colon cancer cell lines. The apoptosis in SW480 colon cancer cells was executed by the activation of caspase-3, which was shown by western blot and apoptosis inhibition assay. Furthermore, the mitochondrial membrane potential of apoptotic SW480 cells after 12 h treatment with Vat C was significantly lost, and concurrently the cytochrome c release and activation of caspase-9 were also detected by western blot analysis. Over-expression of Bcl-2 protein in SW480 cells significantly prevented the cell death induced by Vat C. Taken together, the findings presented here indicate that Vat C induced marked apoptosis in malignant cells mainly by affecting mitochondrial membrane potential.
Abbreviations: PBS, phosphate-buffered saline; PTP, permeability transition pore; Res, resveratrol; Vat C, vaticanol C
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Introduction
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Increasing interest has been paid to primitive medicinal plants to find new substances with potentially useful biological activities. Typically, red wine's health benefits initially received a great deal of attention following the reports indicating that a greater wine consumption was linked to a lower incidence of cardiovascular disease so-called French Paradox (13). Wine contains a broad range of polyphenols that are present in the skin and seeds of grapes. Possible mechanisms by which these phenolic compounds exert their beneficial effects include the reactive oxygen species scavenging ability. Recently, a monomeric stilbenoid, resveratrol (3,5,4'-trihydroxystilbene) (Res) (Figure 1), included in wine and some popular herbal medicines used for liver, skin and circulatory diseases, has been discussed in terms of cancer preventive or anticancer substances; it exerts an anticarcinogenic effect in a two-stage mouse skin cancer model (4), and shows tumor growth inhibition in rat (5). Res is therefore regarded as one of the important candidates for tumor suppressive agents. Stilbenoids are known to be abundantly distributed in the plants belonging to Vitaceae, Dipterocarpaceae, Leguminosae and Cyperaceae as a variety of Res oligomers ranging from dimer to octamer. Some of their derivatives have also been reported to possess various biological properties such as anti-bacterial (6), anti-HIV (7) and anti-inflammatory activity (8,9). Stilbene oligomers composed of Res are, therefore, considered to be useful resources of lead compounds for drug development. To evaluate biological activities of stilbene oligomers in nature and also to determine their exact underlying mechanisms will provide a substantial clue for the development of new drug candidates.
In the course of our phytochemical study to search for biologically active Res derivatives in Dipterocarpaceae, the structures of 43 Res oligomers including 34 novel compounds were characterized (1019) and the antibacterial activity against methicillin-resistant Staphylococcus aureus in some stilbenoids was found (20). Recently we have reported that a novel Res tetramer vaticanol C (Vat C) (Figure 1) exerts inhibitory effect on cell growth of colon cancer cell lines (21). In the present study, we have demonstrated the cytotoxicity of the Res oligomers against human cultured cancer cells and also the apoptosis-inducing effect of Vat C on colon cancer cell lines, which was mainly caused by loss of the mitochondrial membrane potential and consequent caspase activation.
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Materials and methods
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Reagents
A series of Res oligomers were isolated from the genus belonging to Vatica, Shorea and Vateria (Dipterocarpaceae), and their structures were determined in our previous studies (10,11,1318). We have examined 20 compounds against nine tumor cell lines: Res (1), piceid (2) as monomers; (-)-
-viniferin (3) as dimer; Vat A (4), vaticaside A (5), Vat E (6), Vat G (7), vaticaside D (8),
-viniferin (9) as trimers; Vat B (10), vaticaside B (11), Vat C (12), hemsleyanol D (13), (-)-hopeaphenol (14), (+)-isohopeaphenol (15) as tetramers; Vat D (16), Vat H (17), Vat I (18) as hexamers; Vat J (19) as heptamer; vateriaphenol A (20) as octamer. Compounds 18, 1012 and 1619 were isolated from the stem bark of Vatica rassak, 9 and 1315 from the stem bark of Shorea hemsleyana, and 20 from the stem bark of Vateria indica. The isolation procedure and spectroscopic data of all compounds were described in previous papers (10,11,1318). A stock solution of Vat C, Res or other stilbenoid oligomers was prepared in 10 mM DMSO, and was further diluted to the working concentration before use.
Cell culture, morphological study and cell viability
Nine human cancer cell lines(colon) SW480, DLD-1 and COLO201; (prostate) PC3 and LNCaP; (leukemia) HL60, K562, U937; (neuroblastoma) SH-SY5Ywere grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma, Tokyo) and 2 mM L-glutamine under an atmosphere of 95% air and 5% CO2 at 37°C. Human peripheral blood lymphocytes from healthy donor were stimulated with concanavalin-A (75 µg/ml) for 48 h and used for growth suppression of Vat C or other compounds. The number and viability of cells were determined by the trypan blue dye-exclusion assay. For evaluating IC50, the starting cell number was 34 x 105/ml. For evaluating apoptotic cell death, cells were seeded at a density of 5 x 105/ml in 15-mm diameter wells and allowed them for 12 h to adhere, and then DNA ladder formation was examined in the time course after the start of treatment with the compounds. For morphological examination of apoptotic changes, cells were stained with Hoechst 33342 (5 µg/ml) at 37°C for 30 min, washed twice with phosphate-buffered saline (PBS), pipetted dropwise onto a glass slide, and examined by fluorescence microscopy using an Olympus microscope (Tokyo, Japan) equipped with an epi-illuminator and appropriate filters.
Plasmid construction and DNA transfection of BCL-2 gene
For establishment of transfectants expressing Bcl-2, we used a pcDNA3 eukaryotic expression vector (Invitrogen, San Diego, CA). For construction of pcDNA3-BCL-2, the full-length human BCL-2 gene pB4 (22) was digested with EcoRI and then inserted into the EcoRI-cleaved pcDNA3 vector. SW480 cells were transfected with pcDNA3 or pcDNA3-BCL-2 by using liposomes (Lipofect-AMINE) according to the manufacturer's Lipofection protocol (Gibco BRL, Rockville, MD). Selections were started 3 days after transfection by using medium containing 0.7 mg/ml geneticin (Gibco BRL). Individual clones were isolated, and their characterization was made by RTPCR and western blot analysis. RTPCR was performed as described previously (23). In brief, total cellular RNA of transfectants and original cells was isolated by the phenolguanidium thiocyanate method with DNase I treatment. By reverse transcription of 2 µg of total RNA, cDNAs were obtained, and amplification of the respective cDNA region was conducted by PCR. PCR primers were as follows: for BCL-2, (sense) 5'-TGCACCTGACGCCCTTCAC-3'; and (antisense) 5'-AGACAGCCAGGAGAAATCAAACAG-3'. This primer can specifically amplify the 293-bp DNA fragments of BCL-2. ß-Actin cDNA was used for an internal standard. The PCR reaction consisted of 30 cycles (94°C for 30 s, 57.5°C for 1 min, 72°C for 1 min) after an initial denaturation step (95°C for 1 min). PCR products were analyzed by electrophoresis on 2% agarose gels. Two stable clones over-expressing Bcl-2 protein (SW/Bcl-2-1 and -2), which were obtained by limiting dilution, were confirmed by RTPCR and western blot analyses. SW/Bcl-2-1 cells were used for the experiments. The clone transfected with pcDNA3 alone vector (SW/Vec) was used as control.
Analysis of DNA fragmentation by agarose gel electrophoresis
Cellular DNA was extracted from whole cells by the procedure described previously (24). RNase was added to the DNA solution at the final concentration of 20 mg/ml, and the mixture was incubated at 37°C for 30 min. After electrophoresis on a 2.5% agarose gel, DNA was visualized by ethidium bromide staining.
Western blot analysis
Before and after treatment with Vat C, cells were washed twice with PBS, lysed in lysis buffer (2x PBS, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1 mM phenylmethanesulfonyl fluoride), and then homogenized. Homogenized samples were used after centrifugation at 12000 r.p.m. for 5 min. In the case of adherent cells, the cells were detached with a cell scraper, and then treated as described above. The mitochondrial and cytosolic fractions were prepared as reported ealier (25). Ten micrograms of protein of each homogenized sample was separated by SDSPAGE by using an adequate percent of polyacrylamide in the gel and electroblotted onto a PVDF membrane (Du Pont, Boston, MA). After blockage of non-specific binding sites for 1 h by 5% non-fat milk in TPBS (PBS and 0.1% Tween 20), the membrane was incubated overnight at 4°C with anti-human caspase-3 (Transduction Laboratories, Lexington, KY), anti-human caspase-8 (Transduction Laboratories), anti-human caspase-9 (Transduction Laboratories), anti-human cytochrome c (Research Diagnostic Inc., Flanders, NJ), anti-human Bcl-2 (100) (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-human Bid (R&D Systems, Minneapolis, MN). The membrane was then washed three times with TPBS, incubated further with alkaline phosphatase-conjugated goat anti-mouse antibody (Promega, Madison, WI) or anti-rabbit antibody (New England Biolabs, Beverly, MA) at room temperature, and then washed three times with TPBS. The immunoblot was visualized by use of an enhanced chemiluminescence detection kit (New England Biolabs).
Colorimetric protease assay
The activation of caspase-8 was also determined by colorimetric protease assay. Briefly, the cells treated with Vat C were harvested at the indicated times, suspended in cell lysis buffer, and then incubated on ice for 10 min. The lysate containing 150 µg protein was incubated with 200 µM IETD-pNA substrate (MBL, Nagoya) at 37°C for 2 h. Levels of released pNA were measured with a Hitachi F-3000 spectrofluorometer at 405 nm. The cell lysate of Jurkat cells treated with anti-Fas antibody (clone CH-11) (MBL) for 3 h was used as a positive control for caspase-8 activation.
Inhibition of apoptosis by pan-caspase inhibitor
For the study of inhibition of apoptosis, the tripeptide pan-caspase inhibitor Z-VAD-FMK (MBL, Nagoya) was added 12 h before treatment with Vat C. Optimal concentration of the inhibitor was determined from doseresponse curve for the extent of cell death. Inhibition of apoptosis by Z-VAD-FMK was evaluated by the blockage of the process of nucleosomal DNA fragmentation, which was observed as ladder formation.
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was measured by use of a fluorescent dye, Mito-Tracker Green (Molecular Probes, #M-7514, Eugene, OR), which estimates the mitochondrial volume, and Mito-Tracker Orange (Molecular Probes, #M-7511), which accumulates selectively in active mitochondria and becomes fluorescent when oxidized. The cells were treated with 5 µM Vat C for 12 h. After the cells were washed twice with RPMI-1640 medium, the Vat C-treated or untreated cells were incubated with Mito-Tracker fluorescent probes (100 nM each) for 30 min at 37°C. After the whole cells were collected and washed twice with PBS, the cells were resuspended in PBS. The fluorescence of Mito-Tracker Orange and Green was analyzed by flow cytometry (Becton Dickinson, San Jose, CA) (23). The whole cell population was subjected to analysis.
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Results
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We examined the effects of 20 Res oligomers at various concentrations on the cytotoxity in nine human cancer cell lines and the results of treatment with the compounds at 10 µM was shown in Table I. Among these Res oligomers tested, 11 compounds caused cytotoxicity against more than two cell lines, as judged by trypan blue-exclusion test. Especially,
-viniferin (9), Vat C (12), Vat D (16), Vat H (17), Vat I (18), Vat J (19) and vateriaphenol A (20) showed a marked cytotoxic activity against several cell lines. Res displayed significant cytotoxic effect only on HL60 cells at less than 10 µM. Of these Res oligomers, Vat C was found to induce marked cytotoxicity in all the cell lines tested (Table I). It was to be noted that the susceptibility of SW480 and HL60 cells to Vat C was four to seven times higher than that to Res (Table II). The growth of the colon cancer cell lines, SW480, DLD-1 and COLO201, was markedly suppressed after the Vat C treatment (5 µM), as compared with the control without Vat C treatment (Figure 2). Then we examined the mechanism for the growth suppression in these cell lines. In the treatment with 5 µM Vat C for 72 h, we observed the typical morphological characteristics of apoptosis, such as nuclear condensation and segmentation, in the majority of the detached SW480, DLD-1 and COLO201 cells (Figure 3A). To verify further the apoptotic response, fragmentation of DNA was examined. DNA ladder formation was observed in SW480 cells treated with 5 µM Vat C for 12, 24 or 36 h (Figure 3B). DNA ladder formation and apoptotic morphological changes were also observed in DLD-1 and COLO201 cells after the same treatment (data not shown). These findings indicated that the marked suppression of cell growth by Vat C was attributed to the apoptotic cell death. In this context, we selected SW480 cells to study the molecular mechanism of Vat C-induced apoptosis.

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Fig. 2. Effect of Vat C on cell growth of the colon cancer cell lines at 5 µM. Cell numbers of three colon cancer cell lines SW480, DLD-1 and COLO201 after treatment with Vat C were evaluated by the trypan-blue dye exclusion test. Each value represents the mean of the results obtained in two independent experiments. Closed symbols and open symbols indicate the control and Vat C-treated cells, respectively.
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Fig. 3. Vat C-induced apoptosis in SW480, DLD-1 and COLO201 colon cancer cell lines. (A) Morphological aspects of the cells. The cells were stained with Hoechst 33342 (5 µg/ml) for 30 min and then examined by fluorescence microscopy. Left, cells without treatment; right, after treatment with 5 µM Vat C for 72 h. (B) Nucleosomal DNA fragmentation of SW480 cells after exposure to 5 µM Vat C. Three micrograms of DNA was loaded onto each lane. Lane M is a DNA size marker.
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Apoptosis is known to be executed by the linkage activation of caspases such as initiators (caspase-8 and -9) and executioners (caspase-3 and -7). To know which caspase(s) is involved in Vat C-induced apoptosis, we examined formation of active forms of caspases in cell lysate by western blot analysis. As shown in Figure 4A, the processed active forms of caspase-3 were observed in SW480 cells after treatment with 5 µM Vat C. The 17 kDa active forms of caspase-3 appeared at 24 h after the treatment and the levels of their inactive proforms and active forms were inversely changed at 36 h after the treatment. Pre-treatment with the pan-caspase-like protease inhibitor Z-VAD-FMK caused a concentration-dependent inhibition of DNA ladder formation by Vat C (Figure 4B). On the other hand, western blot analysis (data not shown) and colorimetric protease assay (Figure 4C) did not show activation of caspase-8, which is known to be implicated in the extrinsic pathway triggered by cytokines and Fas ligand and the intrinsic pathway mediated by genotoxic stimuli such as anticancer drugs. Thus, these results indicated that Vat C induced the apoptotic cell death and suggest that the apoptosis was executed by the activation of caspases-3, but not via the caspase-8. Next we examined the mitochondrial pathway, which plays a crucial role in propagation and determination of cell death. The mitochondrial membrane potential and the release of cytochrome c were examined in SW480 cells after the Vat C treatment. In 12 h-treated cells, the mitochondrial membrane potential was markedly decreased when examined by FACS analysis using Mito-Tracker fluorescent probe (Figure 5A). Western blot analysis showed that the amount of released cytochrome c following the Vat C treatment was increased time-dependently (Figure 5B). Caspase-9, which is known to bind to the cytochrome cApaf-1 complex, was activated at 12 h after the treatment, concurrent with the release of cytochrome c from mitochondria (Figure 4A). Furthermore, to examine the effect of an anti-apoptotic protein, bcl-2which is a constituent of permeability transition pore (PTP) and prevents the release of cytochrome c (26)on Vat C-induced apoptosis, we established Bcl-2 over- expressants of SW480 cells (23). The expression level was determined by RTPCR (data not shown) and western blot analysis (Figure 6A). One of the clones over-expressing Bcl-2 (SW/Bcl-2-1) was selected for further experiments. It was shown that while growth suppression of bcl-2 over-expressants treated with Vat C was still observed (Figure 6B), the growth suppression by Vat C was markedly reduced by over-expression of Bcl-2 protein in the early phase of the treatment as seen in Res (Figure 6B). Such a preventive effect of Bcl-2 on apoptosis was not observed in taxol which mainly damages microtubule. As expected from the absence of caspase-8 activation (Figure 4A), the truncated Bid protein, which directly acts on mitochondria to promote apoptosis without loss of mitochondrial membrane potential (27), was not observed in the SW480 cells treated with 5 µM Vat C, as assessed by western blot analysis (data not shown).

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Fig. 4. Vat C-induced apoptosis in SW480 cells. (A) Activation of caspase-3 and -9 in SW480 cells after the treatment with 5 µM Vat C examined by western blot analysis. SW480 cells were treated with 5 µM Vat C for indicated times, and then lysed and homogenized. After centrifugation, 5 µg of protein in the supernatant was electrophoresed on a 12% SDSpolyacrylamide gel. Closed triangles indicate the proforms of caspases, and arrows their active forms. (B) Activation of caspases in SW480 cells examined by apoptosis inhibition assay. Blockage of the apoptosis by pan-caspase inhibitor in SW480 cells. Three micrograms of DNA was applied onto each lane. The inhibitor for pan-caspase, Z-VAD-FMK, was added 12 h before exposure to 5 µM Vat C. The rescue of cell death was evaluated at 72 h after Vat C exposure by the block of formation of nucleosomal DNA fragments at each concentration of the inhibitor. Lane 1, DNA from cells treated with 5 µM Vat C for 72 h; lane 2, treatment with 10 µM inhibitor; lane 3, with 20 µM inhibitor; lane 4, with 50 µM inhibitor; lane 5, with 100 µM inhibitor; lane 6, with 200 µM inhibitor; lane 7, in the presence of 0.01% DMSO and 200 µM Z-VAD-FMK (control). Lane M is a DNA size marker. (C) Colorimetric protease assay of caspase-8 in Vat C-induced apoptosis. Each sample was prepared by the method described in Materials and methods. 1, Jurkat cells without treatment; 2, Jurkat cells treated with anti-Fas antibody for 3 h; 3, Jurkat cells treated with anti-Fas antibody and caspase-8 inhibitor (Ac-IETD-CHO); 4, SW480 cells without treatment; 5, treated for 3 h; 6, treated for 6 h; 7, treated for 12 h. The values of absorbance at 405 nm are expressed. Means ± SD of three independent experiments are given.
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Fig. 5. Mitochondrial membrane potential and the release of cytochrome c in SW480 cells after treatment with 5 µM Vat C. (A) The relative fluorescence intensity of Mito-Tracker Orange was measured by flow cytometry. The whole cell population was subjected for analysis. Control was represented by bold line. Mito-Tracker Green demonstrated approximately identical peaks between control and cells treated with Vat C (data not shown). (B) The release of cytochrome c in SW480 cells after the treatment with Vat C detected by western blot analysis. The considerable amount of cytochrome c was identified after the treatment with 5 µM Vat C. Each sample was prepared by the method as described in Materials and methods.
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Fig. 6. Effect of Vat C on cell growth of human Bcl-2 over-expressants of SW480 cells. (A) Western blot analysis of Bcl-2 protein. Lane 1, SW/Vec cells; lane 2, SW/Bcl-2-1 cells; lane 3, SW/Bcl-2-2 cells. (B) Viable cell numbers of Bcl-2 over-expressants SW/Bcl-2-1 after treatment with 5 µM Vat C were measured by the trypan-blue dye exclusion test. Empty circle, SW/Vec; filled circle, SW/Bcl-2-1. The values are expressed as percentages of the viable cell number in cultures treated with each compound to those in non-treated cultures at concentrations indicated. Means ± SD of three independent experiments are given.
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Considering the possible application of Vat C for chemopreventive or chemotherapeutic agent, we examined the effect of Vat C (10 µM) on normal human blood lymphocytes and compared with those by its related compounds. As shown in Figure 7, the effects of the stilbenoid derivatives except Res (monomer) on growth suppression of mitogen-stimulated lymphocytes were similar to those observed in
-viniferin (trimer), Vat C (tetramer), Vat D (hexamer) and vateriaphenol A (octamer) on the cancer cell lines tested.

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Fig. 7. Growth suppression effect of Vat C and its related compounds on mitogen-stimulated human peripheral lymphocytes. Viable cell numbers of mitogen-stimulated human peripheral lymphocytes after treatment with 10 µM each compound were measured by the trypan-blue dye exclusion test. Empty circle, Res; filled circle, -viniferin; empty triangle, Vat C; filled triangle, Vat D; empty square, vateriaphenol A. The values are expressed as percentages of the viable cell number in cultures treated with each agent to those in non-treated cultures. Means ± SD of three independent experiments are given.
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Taken together, we concluded that the main signal transduction pathway of the Vat C-induced apoptosis in SW480 cells was mediated via the mitochondrial pathway without activation of caspase-8.
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Discussion
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This study was undertaken to explore biological activities of the stilbenoid compounds. Twenty Res oligomers, which were isolated from Asian tropical trees Dipterocarpaceae and chemically characterized in our laboratory, were examined for effects on cytotoxicity. Among them, we found that seven Res oligomers have cytotoxic activity at <10 µM against several human tumor cell lines tested. There was a tendency that the oligomers with more than four basic Res structures exhibited marked cytotoxicity. Especially, a Res tetramer Vat C showed considerable cytotoxic effect even at lower concentrations in all the cell lines tested. Res, which is the basic stilbene compound, has been demonstrated to possess cancer chemopreventive activity in a number of experimental models (4,5) and to induce apoptosis in cells through mitochondrial pathway (28,29). However, in the present study we have found that Vat C was 47-fold potent compared with Res as reflected in the IC50s of HL60 and SW480 cell lines and also that this growth suppression was due to apoptosis mediated via the loss of mitochondrial membrane potential in SW480 cells. Such apoptotic pathway via mitochondria was also observed in leukemic cell line U937 (data not shown). Many chemopreventive agents act through the induction of apoptosis, which leads to inhibition of the carcinogenesis process. During the last several years, it has become increasingly clear that mitochondria play a major rate-limiting role in apoptosis. The decision/effector phase of the apoptotic process converges on the mitochondria, where permeabilization of mitochondrial membranes is triggered as a result of the action of the permeability transition pore complex (PTPC) (30). In our study, it was shown that Vat C-induced apoptosis was executed via the mitochondrial pathway without activation of caspase-8 in SW480 cells. Thus, it can be assumed that Vat C may directly affect the mitochondrial membrane proteins consisting of PTP. Furthermore, we have shown that over-expression of Bcl-2 prevented the Vat C-induced apoptosis in SW480 cells, suggesting that Vat C may be associated with Bcl-2 and/or its closely related protein molecules such as peripheral benzodiazepine receptor and Bax on mitochondria (30). The mechanism of the growth suppressive activity of Vat C could be considered to be different from that of anticancer drugs and even the genotoxic agents such as etoposide, adriamycin, camptothecin, which induce apoptosis in various cancer cells (31,32), because they do not act directly on the mitochondrial proteins. However, the molecules interacting with these stilbenoid compounds, especially Vat C and Res in the apoptotic process should be specified by further investigation. The growth suppression of Vat C in mitogen-stimulated normal peripheral blood lymphocyts was observed at 10 µM, but this suppressive effect was considerably attenuated at the concentrations <5 µM (data not shown). Importantly, Res exhibited an unexpected potent effect on normal lymphocytes stimulated with mitogen (Tables I and II, Figure 7), suggesting that Vat C or the related compounds may also be a candidate not only for a chemopreventive agent but also for a chemotherapeutic one, because the doses of the compounds can be considerably lowered for the treatment to exhibit the same activity as seen in Res.
The apoptosis-inducing effect of Res has been explained, in part, by the antioxidant action, which inhibits both phorbol ester-mediated activation of PKC and AP-1 (33). However, our results have shown that the antioxidant activity of the stilbenoid compounds tested is unlikely to explain the cytotoxic action. It has also been reported that Res completely blocks the Cox-2 expression (34). Haung et al. have shown that the wild-type p53 is required for induction of Res-induced apoptosis in p53-deficit cells; over-expression of wild p53 induces the apoptosis (35). In contrast, Vat C induced apoptosis in colon cancer SW480 cells, which have only mutant p53, and furthermore our recent preliminary data demonstrated that Vat C reduced the amount of the mutant p53 transcripts, suggesting that Vat C may also modulate the transcription level of mutant p53 that causes abnormal transformation in colon cancer cell.
The potent ability to induce apoptosis was shown in stilbenoid compounds and it was also suggested that Vat C is a possible candidate as a chemopreventive and chemotherapeutic agent. However, further experiments should be required to assess the anticancer effect of Vat C in animal model and also to define the mechanisms of Vat C at the molecular level, which are under current progress in our laboratory.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for Scientific Research by the Ministry of Education, Science, Sports and Culture of Japan.
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Received July 8, 2002;
revised March 25, 2003;
accepted June 12, 2003.