Cancer chemopreventive activity of resveratrol, a natural product derived from grapes.

Resveratrol, a phytoalexin found in grapes and other food products, was purified and shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis. Resveratrol was found to act as an antioxidant and antimutagen and to induce phase II drug-metabolizing enzymes (anti-initiation activity); it mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity); and it induced human promyelocytic leukemia cell differentiation (antiprogression activity). In addition, it inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin cancer model. These data suggest that resveratrol, a common constituent of the human diet, merits investigation as a potential cancer chemopreventive agent in humans.

A ligand of peroxisome proliferator-activated receptor gamma, retinoids, and prevention of preneoplastic mammary lesions.

BACKGROUND: Chemoprevention of breast cancer is an active area of investigation. Recent in vivo and in vitro studies have shown that thiazolidinediones (e.g., troglitazone) and retinoids are able to inhibit the growth of breast cancer cells. Troglitazone mediates its action via peroxisome proliferator-activated receptor gamma (PPARgamma). We evaluated the ability of troglitazone, alone or in combination with retinoids, to prevent the induction of preneoplastic lesions by 7,12-dimethylbenz[a]anthracene (DMBA) in a mouse mammary gland organ culture model. METHODS: Mammary glands of BALB/c mice were treated with DMBA (2 microg/mL) to induce preneoplastic lesions in organ culture. Effects of troglitazone, all-trans-retinoic acid (retinoic acid; ligand for retinoic acid receptor [RAR] alpha), and LG10068 (ligand for retinoid X receptors [RXRs]), singly or in combination, on the development of lesions were evaluated. Expression of retinoid receptors (RARalpha and RXRalpha) and PPARgamma was determined by western blot analysis. Statistical significance was determined by generalized chi-squared analysis using the GENCAT software program and Bonferroni correction. All P values are two-sided. RESULTS: Troglitazone (at 10(-5) M) or retinoic acid (at 10(-6) M) markedly inhibited the development of mammary lesions (both P values <.05); however, together they did not enhance the effectiveness of the other. In contrast, LG10068 (at 10(-7) M or 10(-8) M) alone had very little ability to inhibit development of these lesions, but a combination of LG10068 (at 10(-8) M) and troglitazone (at 10(-5) M or 10(-6) M) almost completely inhibited (by 85% and 100%, respectively; both P values <. 05) the development of mammary lesions. The expression of PPARgamma and RXRalpha remained unchanged with the various treatments, whereas the expression of RARalpha was substantially reduced after treatment with the combination of retinoic acid and troglitazone. CONCLUSIONS: To our knowledge, this is the first report showing the possibility of a PPARgamma ligand having chemopreventive activity. Furthermore, an RXR-selective retinoid, LG10068, appears to enhance this activity.

Prevention of preneoplastic mammary lesion development by a novel vitamin D analogue, 1alpha-hydroxyvitamin D5.

BACKGROUND: The form of vitamin D (vitamin D3) in fortified milk and the provitamin D produced by the body undergo metabolic activation to a biologically active form, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. This compound can induce cell differentiation and can prevent proliferation of cancer cells. However, because 1alpha,25(OH)2D3 is hypercalcemic (effective in increasing serum calcium level), it is not suitable for use in cancer prevention or cancer therapy trials. PURPOSE: We synthesized a vitamin D5 series analogue, 1alpha-hydroxy, 24-ethyl-cholecalciferol, or 1alpha-hydroxyvitamin D5 [1alpha(OH)D5], and evaluated its chemopreventive activity in carcinogen-treated mammary glands in organ culture experiments. METHODS: The analogue 1alpha(OH)D5 was synthesized from sitosterol acetate and was characterized by nuclear magnetic resonance. Its purity was evaluated by high-pressure liquid chromatography. The calcemic activities of vitamin D3 and D5 analogues were determined in vitamin D-deficient Sprague-Dawley rats. Mammary glands of BALB/c mice were placed in organ culture and treated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to induce preneoplastic lesions. Vitamin D analogues were added to the culture medium at four different concentrations, and formation of mammary lesions was evaluated. The effects of 1alpha(OH)D5 and 1alpha,25(OH)2D3 on the expression of vitamin D receptors (VDRs) and transforming growth factor-beta1 (TGF-beta1) were studied by immunohistochemistry. Statistical significance was determined by the chi-squared test. All reported P values were two-sided. RESULTS: 1alpha,25(OH)2D3 was fourfold more calcemic than 1alpha(OH)D5 at a dose of 0.042 microg/kg per day in rats. Both 1alpha,25(OH)2D3 and 1alpha(OH)D5 inhibited the development of DMBA-induced preneoplastic lesions in mouse mammary glands compared with untreated glands. The effect of the vitamin D3 analogue was observed at a much lower concentration (0.01 microM). Treatment with 1alpha(OH)D5 resulted in a dose-related (0.01-10.0 microM) inhibition without any toxicity, whereas the vitamin D3 analogue was highly potent but toxic at concentrations of 1.0 microM or higher. Normal mouse mammary glands poorly express VDR and TGF-beta1; incubation with 1alpha(OH)D5 or 1alpha,25(OH)2D3 dramatically induced their expression. CONCLUSIONS: This is the first report showing the possibility of chemoprevention by a vitamin D5 series compound. We conclude that 1alpha(OH)D5 is less calcemic than 1alpha,25(OH)2D3. It is nontoxic at a wide range of concentrations, but it is potent in inhibiting the development of preneoplastic lesions in mammary glands in organ culture. In addition, we show for the first time the induction of TGF-beta1 in normal mammary tissues by a chemopreventive agent. IMPLICATIONS: 1alpha(OH)D5 is a good candidate for in vivo chemoprevention studies. It may mediate its action by inducing expression of VDR and of TGF-beta1, as is seen in other systems.

Inhibition of DNA synthesis by retinyl acetate during chemically induced mammary carcinogenesis.

The effect of retinoids on DNA synthesis was studied in the female Sprague-Dawley rat. Animals were treated with either solvent, 7,12-dimethylbenz(a)anthracene, or 1-methyl-1-nitro-sourea at 50 days of age and were placed on either placebo or retinyl acetate diet at 57 days of age. [3H]Thymidine incorporation into purified DNA isolated from mammary parenchymal cells was determined. Retinyl acetate effectively inhibited mammary cell DNA synthesis in both 1-methyl-1-nitrosourea- and 7,12-dimethylbenz(a)anthracene-treated animals; however, DNA synthesis in solvent-treated animals was unaffected.

Effects of 12-O-tetradecanoylphorbol-13-acetate on carcinogen-induced mouse mammary lesions in organ culture.

Mammary glands of young female BALB/c mice develop hyperplastic nodule-like alveolar lesions (NLAL) in response to a 24-h exposure to 7,12-dimethylbenz(a)anthracene (DMBA; 2 micrograms/ml) on Day 3 during a 24-day organ culture. Experiments were conducted to determine if 12-O-tetradecanoylphorbol-13-acetate (TPA), a known tumor promoter, can influence the development of DMBA-induced NLAL in mammary gland organ culture. Mammary glands were incubated with TPA (25 ng/ml) for various periods of time in the presence of appropriate hormone combinations. Results indicated that the presence of TPA in the medium between Days 9 to 14 of the culture period enhanced both the incidence and multiplicity of DMBA-induced NLAL; however, it was ineffective when included in the medium between Days 4 to 9 or 19 to 24 of the organ culture. Toxicity of TPA was evident when it was present during the entire culture period subsequent to DMBA treatment. Computer-assisted image analysis of NLAL in the glands determined the area covered by these lesions within the gland. It was observed that 7.8% of the area was covered by NLAL in DMBA plus TPA-treated glands as compared to 2.5% by DMBA treatment alone. These results provide a model for initiation-promotion studies of mammary carcinogenesis in vitro, as well as a modified approach for quantitative analysis of structural alterations.

Inhibition of carcinogenesis by retinoids.

Retinoids are effective inhibitors of chemical carcinogenesis in the mammary gland and urinary bladder of experimental animals. Modification of the basic retinoid structure has produced retinoids with increased target organ specificity, resulting in increased anticancer activity with reduced systemic toxicity. Combining retinoid treatment with hormonal manipulation results in a synergistic inhibition of mammary carcinogenesis; this combination approach also inhibits development of additional mammary cancers following surgical removal of the first mammary cancer. Retinoids are most effective when administered shortly after the carcinogenic insult. However, even when retinoid treatment is delayed, the compounds are still effective cancer chemopreventive agents for the mammary gland and urinary bladder. The length of time that retinoid exposure can be delayed and retain an anticancer effect is directly related to tumor latency, with a longer delay permissible against tumors with long latent periods.

Distribution of retinoic acid-binding proteins in normal and neoplastic mammary tissues.

Cellular retinoic acid-binding proteins were detected in chemically induced mammary tumors using sucrose density gradient analysis. Unlabeled retinoic acid did not displace nonspecific binding in the 5S region but was, however, a competitive inhibitor for the specifically binding 2S component. Mammary gland cytosol fractions from both 1-methyl-1-nitrosourea-treated and untreated as well as from lactating rats contained low levels of retinoic acid-binding proteins. 1-Methyl-1-nitrosourea treatment did not result in the increased number of binding sites. Thus, the increase in the levels of binding proteins in tumors most probably occurred during tumor development and probably was not a result of the carcinogen per se. Retinoids which have been shown to be effective in the chemoprevention of mammary carcinogenesis only partially competed for the binding sites, indicating that they may be metabolized prior to their action as an active chemopreventive agent.

Inhibition of rat mammary gland chemical carcinogenesis by dietary dehydroepiandrosterone or a fluorinated analogue of dehydroepiandrosterone.

The chemopreventive efficacy of p.o. administered dehydroepiandrosterone (DHEA), DHEA plus N-(4-hydroxyphenyl)retinamide (4-HPR), or 16 alpha-fluoro-5-androsten-17-one (DHEA analogue 8354) was examined in rats treated with N-methyl-N-nitrosourea (MNU; 50 mg/kg body weight, i.v.) at 50 days of age. Semipurified diet (AIN-76A) containing each steroid alone, or DHEA plus 4-HPR, was administered during initiation (-1 week to +1 week post-MNU), promotion/progression (+1 week post-MNU to termination), or both phases (-1 week post-MNU to termination) of the carcinogenic process. Neither DHEA nor DHEA analogue 8354 (0.2%, w/w) significantly affected the initiation of mammary cancer when administered alone; however, DHEA (0.2%, w/w) plus 4-HPR (1 mmol/kg diet) significantly reduced cancer multiplicity (26%) when given during initiation. All three treatments were strongly effective when given during promotion/progression, significantly reducing mammary cancer multiplicity by 77% (DHEA), 84% (DHEA/4-HPR), and 66% (DHEA analogue 8354), relative to carcinogen controls. Cancer incidence was significantly inhibited by DHEA (33% inhibition) and DHEA/4-HPR (24% reduction) during promotion/progression. However, the most effective chemopreventive treatment encompassed both phases of carcinogenesis. Thus, under these conditions, DHEA (0.2% or 0.1%, w/w) reduced cancer incidence (52% and 32% reductions, respectively) and multiplicity (91% and 86% reductions, respectively). Further reduction in mammary cancer incidence was observed in animals that received DHEA (both doses) plus 4-HPR (1 and 0.5 mmol/kg diet, respectively). DHEA analogue 8354 (0.2% or 0.1%, w/w) given for the duration of the study reduced only cancer multiplicity (61% and 56% reductions, respectively). Tumor-related mortality was significantly lower in rats that received long-term treatment with DHEA or DHEA/4-HPR, when compared with carcinogen controls. Except for a slight, but significant, postcarcinogen decrease in the mean body weights of rats treated concomitantly with DHEA (plus or minus 4-HPR) and MNU, additional gross manifestations of steroid-induced toxicity were not observed.

Estrogenic and antiestrogenic properties of resveratrol in mammary tumor models.

Trans-3,4',5-trihydroxystilbene (resveratrol), a phytoalexin present in grapes and grape products such as wine, has been identified as a chemopreventive agent. Recent studies performed with MCF-7 human breast cancer cells have demonstrated superestrogenic effects with resveratrol. In contrast, studies performed using estrogen receptor-transfected cell lines have shown that resveratrol acts as a mixed agonist/antagonist. The major objective of this study was to characterize the estrogen-modulatory effects of resveratrol in a variety of in vitro and in vivo mammary models. Thus, the effect of resveratrol alone and in combination with 17beta-estradiol (E2) was assessed with MCF-7, T47D, LY2, and S30 mammary cancer cell lines. With cells transfected with reporter gene systems, the activation of estrogen response element-luciferase was studied, and using Western blot analysis, the expression of E2-responsive progesterone receptor (PR) and presnelin 2 protein was monitored. Furthermore, the effect of resveratrol on formation of preneoplastic lesions (induced by 7,12-dimethylbenz(a)anthracene) and PR expression (with or without E2) was evaluated with mammary glands of BALB/c mice placed in organ culture. Finally, the effect of p.o. administered resveratrol on N-methyl-N-nitrosourea-induced mammary tumors was studied in female Sprague Dawley rats. As a result, in transient transfection studies with MCF-7 cells, resveratrol showed a weak estrogenic response, but when resveratrol was combined with E2 (1 nM), a clear dose-dependent antagonism was observed. Similar mixed estrogenic/antiestrogenic effects were noted with S30 cells, whereas resveratrol functioned as a pure estrogen antagonist with T47D and LY2 cells. Furthermore, in MCF-7 cells, resveratrol induced PR protein expression, but when resveratrol was combined with E2, expression of PR was suppressed. With T47D cells, resveratrol significantly down-regulated steady-state and E2-induced protein levels of PR. With LY2 and S30 cells, resveratrol down-regulated presnelin 2 protein expression. Using the mouse mammary organ culture model, resveratrol induced PR when administered alone, but expression was suppressed in the presence of E2 (1 nM). Furthermore, resveratrol inhibited the formation of estrogen-dependent preneoplastic ductal lesions induced by 7,12-dimethylbenz(a)anthracene in these mammary glands (IC50 = 3.2 microM) and reduced N-methyl-N-nitrosourea-induced mammary tumorigenesis when administered to female Sprague Dawley rats by gavage. Therefore, in the absence of E2, resveratrol exerts mixed estrogen agonist/antagonist activities in some mammary cancer cell lines, but in the presence of E2, resveratrol functions as an antiestrogen. In rodent models, carcinogen-induced preneoplastic lesions and mammary tumors are inhibited. These data suggest that resveratrol may have beneficial effects if used as a chemopreventive agent for breast cancer.

Enhanced inhibition of mammary carcinogenesis by combined treatment with N-(4-hydroxyphenyl)retinamide and ovariectomy.

Bilateral ovariectomy and dietary administration of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) are both effective inhibitors of chemical carcinogenesis in the rat mammary gland. The present study was designed to determine whether an enhanced inhibitory effect is obtained with combined ovariectomy and 4-HPR administration, compared to either treatment alone. In separate experiments, 50-day-old virgin female Sprague-Dawley rats received either a single i.v. injection of 50 mg N-methyl-N-nitrosourea per kg body weight or a single intragastric dose of 20 mg 7,12-dimethylbenz(a)anthracene. The experimental design was the same in both the N-methyl-N-nitrosourea and 7,12-dimethylbenz(a)anthracene experiments: Group 1, 25 intact rats, placebo diet; Group 2, 25 intact rats, supplement of 782 mg 4-HPR per kg diet; Group 3, 50 ovariectomized rats, placebo diet; Group 4, 50 ovariectomized rats, supplement of 782 mg 4-HPR per kg diet. Feeding of the 4-HPR supplement was begun 7 days after carcinogen administration; ovariectomy was performed 7 days post-7,12-dimethylbenz(a)anthracene or 14 days post-N-methyl-N-nitrosourea. In both experiments, combined ovariectomy plus 4-HPR was significantly more active in suppressing mammary cancer induction than was either manipulation alone. 4-HPR was a more effective inhibitor of carcinogenesis in ovariectomized rats than in intact animals. These data indicate that 4-HPR is highly effective in inhibiting ovarian hormone-independent cancers and suggest that retinoid inhibition of mammary carcinogenesis does not involve an influence on ovarian hormone action.

Sulfone metabolite of sulindac inhibits mammary carcinogenesis.

Sulindac sulfoxide, a commonly prescribed anti-inflammatory drug, has cancer chemopreventive activity. During its metabolism, the inactive prodrug sulindac sulfoxide undergoes either reduction to the active anti-inflammatory metabolite sulindac sulfide or irreversible oxidation to sulindac sulfone, which lacks prostaglandin synthetase inhibitory activity. Interestingly, sulindac sulfone has been reported to have cancer chemopreventive activity. The objective of the experiments reported here was to investigate the chemopreventive activity of sulindac sulfone against mammary carcinogenesis and to study its mechanism. Rats were injected with either 12.5 or 37.5 mg of 1-methyl-1-nitrosourea (MNU)/kg body weight at 50 days of age. Sulindac sulfone was incorporated into a purified diet at a concentration of either 0.03 or 0.06% (w/w) and fed to rats beginning 7 days after the injection of MNU. Sulindac sulfoxide at a level of 0.06% (w/w) was fed as a reference for comparison. Thirty rats were assigned to each dietary group treated with the high dose of MNU, and 44 rats were assigned to each dietary group treated with the low dose of MNU. The sulfone reduced cancer incidence and the number of cancers per rat irrespective of the dose of MNU injected, and its chemopreventive activity was comparable to that of sulindac sulfoxide. Cancer latency was also prolonged significantly by sulindac sulfone; the effect was particularly notable at the low dose of carcinogen, at which the prolongation of latency was >8 weeks. The sulfone inhibited the occurrence of mammary carcinomas that were classified as having either a wild-type or a mutant codon 12 in the Ha-ras gene; however, the inhibitory effect was greater against carcinomas with a mutant Ha-ras genotype. Using a mammary gland organ culture transformation assay, it was observed that sulindac sulfone also inhibited the formation of 7,12-dimethylbenz(a)anthracene-induced hyperplastic alveolar nodules and that the inhibitory activity of the sulfone was comparable to that of the sulfoxide. These data indicate that the observed effect of the sulfone on mammary carcinogenesis in vivo is likely to be due to a tissue-specific effect rather than to other systemic effects. The findings that both the prodrug and the sulfone inhibited carcinogenesis in vivo and nodule formation in organ culture and that the sulfone lacks inhibitory activity on prostaglandin synthesis suggest a mechanism(s) of chemoprevention that is independent of the prostaglandin pathway. A candidate mechanism for the apparent clonal selection pressure exerted by the sulfone against mammary carcinogenesis is apoptosis. To test this hypothesis, MCF-7 cells were exposed to a range of concentrations of sulindac sulfone and sulfoxide. Both compounds inhibited cell growth and induced apoptosis in the absence of necrosis. Collectively, these data support a specific chemopreventive effect of sulindac sulfone against mammary carcinogenesis and indicate that this compound may have a selective effect against carcinogenesis involving alterations in the signal transduction cascade of which Ha-ras is a component. Evidence is consistent with the involvement of apoptosis in the cancer-inhibitory activity observed.

Cancer chemopreventive potential of sulforamate, a novel analogue of sulforaphane that induces phase 2 drug-metabolizing enzymes.

Chemoprevention involves the use of natural or synthetic substances to reduce the risk of developing cancer. Two dietary components capable of mediating chemopreventive activity in animal models by modulation of drug-metabolizing enzymes are sulforaphane, an aliphatic isothiocyanate, and brassinin, an indole-based dithiocarbamate, both found in cruciferous vegetables. We currently report the synthesis and activity of a novel cancer chemopreventive agent, (+/-)-4-methylsulfinyl-1-(S-methyldithiocarbamyl)-butane (trivial name, sulforamate), an aliphatic analogue of brassinin with structural similarities to sulforaphane. This compound was shown to be a monofunctional inducer of NAD(P)H:quinone oxidoreductase [quinone reductase (QR)], a Phase II enzyme, in murine Hepa 1c1c7 cell culture and two mutants thereof. Induction potential was comparable to that observed with sulforaphane (concentration required to double the specific activity of QR, approximately 0.2 microM), but cytotoxicity was reduced by about 3-fold (IC50 approximately 30 microm). In addition, sulforaphane, as well as the analogue, increased glutathione levels about 2-fold in cultured Hepa 1c1c7 cells. Induction of QR was regulated at the transcriptional level. Using Northern blotting techniques, time- and dose-dependent induction of QR mRNA levels were demonstrated in Hepa 1c1c7 cell culture. To further investigate the mechanism of induction, HepG2 human hepatoma cells were transiently transfected with QR-chloramphenicol acetyltransferase plasmid constructs containing various portions of the 5'-region of the QR gene. Sulforaphane and the analogue significantly induced (P < 0.0001) CAT activity at a concentration of 12.5 microM by interaction with the antioxidant responsive element (5-14-fold induction) without interacting with the xenobiotic responsive element. Moreover, both compounds significantly induced mouse mammary QR and glutathione S-transferase activity (feeding of 3 mg/mouse intragastric for 4 days), whereas the elevation of hepatic enzyme activities was less pronounced. Both sulforaphane and the analogue were identified as potent inhibitors of preneoplastic lesion formation in carcinogen-treated mouse mammary glands in organ culture (84 and 78% inhibition at 1 microm, respectively). On the basis of these results, the sulforaphane analogue can be regarded as a readily available promising new cancer chemopreventive agent.

Regulation of ornithine decarboxylase induction by deguelin, a natural product cancer chemopreventive agent.

Deguelin, a plant-derived rotenoid, mediates potent chemopreventive responses through transcriptional regulation of phorbol ester-induced ornithine decarboxylase (ODC) activity. To explore the mechanism of this effect, the activity of this compound was evaluated with a number of model systems. Using cultured mouse epidermal 308 cells, the steady-state levels of both 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC mRNA and c-fos were decreased by treatment with deguelin. ODC activity was also inhibited by bullatacin and various antimitotic agents (podophyllotoxin, vinblastine, and colchicine), but only deguelin and bullatacin were active as inhibitors of ODC levels in a TPA-independent c-Myc-mediated induction system using cultured BALB/c c-MycER cells. These results suggest that antimicrotubule effects, as mediated by rotenone, for example, are not responsible for inhibitory activity facilitated by deguelin. This was confirmed by use of an in vitro model of tubulin polymerization in which deguelin and a variety of other rotenoids were investigated and found to be inactive. As anticipated, however, NADH dehydrogenase was inhibited by these rotenoids. Moreover, inhibition of this enzyme correlated with a rapid depletion of ATP levels and potential to inhibit either TPA- or c-Myc-induced ODC activity. It therefore seems that deguelin-mediated interference with transient requirements for elevated energy can inhibit the induction of ODC activity and thereby yield a cancer chemopreventive response.

Phase I/II trial of tamoxifen with or without fenretinide, an analog of vitamin A, in women with metastatic breast cancer.

PURPOSE: Considerable attention has been focused on the chemopreventive properties of fenretinide against carcinogen-induced rodent mammary cancer. Less is known about its direct antitumor effects. The combination of tamoxifen and fenretinide is more effective than tamoxifen or fenretinide alone in prevention of rat mammary cancer. However, the combined toxicity of tamoxifen plus fenretinide in humans is unknown. Therefore, we performed a phase I/II trial in women with estrogen receptor (ER)-positive or progesterone receptor (PR)-positive, previously untreated metastatic breast cancer. PATIENTS AND METHODS: Groups of three patients received tamoxifen 20 mg/d, or tamoxifen plus fenretinide 100, 200, 300, or 400 mg/d. Patients who received fenretinide enjoyed a 3-day "drug holiday" every 4 weeks. Serum levels of fenretinide and its major metabolites were monitored. Patients were monitored for known toxicities of tamoxifen and vitamin A analogs, as well as for response. RESULTS: There were no significant adverse effects on renal, hepatic, hematologic, or lipid values. Nyctalopia, photophobia, cheilitis, and pruritus were not observed. Improvement or stabilization of disease occurred in 12 of 15 patients. CONCLUSION: We conclude that tamoxifen administered with fenretinide is nontoxic. Phase III trials of tamoxifen versus tamoxifen plus fenretinide are warranted.

Resveratrol inhibits rhabdomyosarcoma cell proliferation.

Rhabdomysarcoma is the most common soft tissue tumour in children under the age of 15. Although the introduction of multimodal treatment programmes, including chemotherapy, radiation therapy and excision have increased the overall survival, the chemotherapeutic agents currently used for the treatment of rhabdomyosarcoma exhibit considerable toxicity. The aim of this study was to investigate the effects and possible mechanism(s) of action of resveratrol on human embryonal rhabdomyosarcoma (RD) cells. Resveratrol is a natural polyphenolic compound produced in a number of edible plants and has received considerable attention as a potential chemopreventive and/or chemotherapeutic agent against various types of cancers. In the present study, resveratrol was shown to inhibit cell proliferation of RD cells in a dose-dependent manner with an IC50 of 48.1 micromol/l and induce an arrest in the S/G2 phase of the cell cycle. As evident from immunocytochemical data, resveratrol treatment increased the size of the RD cells. Furthermore, resveratrol treatment resulted in a significant downregulation of cyclin B expression as demonstrated by western blot analyses. In conclusion, the present study shows that resveratrol exerts a strong inhibition of rhabdomyosarcoma cell proliferation in part by arresting cells in S/G2 phase of the cell cycle. These findings warrant further investigation to establish potential use of resveratrol as a relatively non-toxic chemotherapeutic agent for the treatment of rhabdomyosarcoma.

Experimental basis for the prevention of breast cancer.

Cancer chemoprevention involves intervention in the carcinogenic process by a natural or synthetic chemical that either blocks neoplasia development or arrests malignant phenotype progression. The chemopreventive test agent must experimentally be established as safe before a clinical trial. In our laboratory, inhibition of carcinogen-induced development of precancerous lesions in the mouse mammary gland organ culture model is used as a primary screen to select chemopreventive agents for in vivo efficacy evaluation. A nearly 75% correlation apparently exists between the efficacy observed in vitro and in vivo carcinogenesis. For in vivo experiments, MNU- and DMBA-induced mammary tumours in rats are the models of choice. Numerous agents have been identified and progressed to preclinical toxicity and clinical trials. More recently, combination chemoprevention has received considerable attention, since no known chemopreventive agent sufficiently reduces tumour incidence in rats. The sequence of events for establishing the experimental basis for chemoprevention of breast cancer is described.

Distribution of fenretinide in the mammary gland of breast cancer patients.

Fenretinide has been used orally as a chemopreventive retinoid in a trial for women at risk of developing contralateral breast cancer. The levels of fenretinide and its metabolites were measured in the breast tissue obtained at surgery from women in the trial. Fenretinide was concentrated by breast tissue. Two major metabolites were detected in the tissue extract, one co-eluting with N-(4-methoxyphenyl)retinamide (4-MPR), and the other, more polar, eluting at 17 min under the conditions used. This metabolite remains unidentified. Division of the breast tissue into epithelial cells and fat fractions revealed that fenretinide and the metabolite at the 17 min peak were concentrated in the epithelial cells, whereas 4-MPR was principally localised in the fat compartment. Thus fat may serve as a storage compartment for the retinoid.

Deguelin inhibits the growth of colon cancer cells through the induction of apoptosis and cell cycle arrest.

As previously demonstrated, deguelin [(7aS, BaS)-13, 13a-dihydro-9,10-dimethoxy-3,3-dimethyl-3H-bis[1]benzo-pyrano[3,4-b:6',5'-e]pyran-7(7aH)-one mediates anti-proliferative properties in a variety of cell types. In the present study, deguelin was found to suppress the growth of HT-29 colon cancer cells with an IC(50) of 4.32 x 10(-8) M. The cells were arrested in the G1-S-phase of the cycle. Investigations of G1/S regulatory proteins by Western blot analyses showed an upregulation of p27, and decreased expression levels of cyclin E and CDK4. Furthermore, by 24 h, exposure to deguelin resulted in an increase in the hypophosphorylated form of Rb. Since hypophosphorylated pRb binds to and inactivates E2F1, additional studies were performed and downregulation of E2F1 was observed after 24 h of treatment with deguelin. These results are consistent with the observation that deguelin arrested cells in the G1-S- phase. In addition, based on ethidium bromide/acridine orange staining, detection of digoxigenin-labelled genomic 3'-OH DNA ends, and DNA laddering, it was found that deguelin exerts its growth inhibitory effects via the induction of apoptosis. Based on these data, the potential of deguelin to serve as a cancer chemotherapeutic agent for colon cancer may be suggested.

Metabolism of N-[4-hydroxyphenyl]retinamide (4-HPR) to N-[4-methoxyphenyl]retinamide (4-MPR) may serve as a biomarker for its efficacy against human breast cancer and melanoma cells.

A clinical trial of N-[4-hydroxyphenyl]retinamide (4-HPR) has been in progress for the past 4 years to evaluate its role in chemoprevention of breast cancer. However, it is currently not known whether the effect of 4-HPR in breast cells is mediated by 4-HPR directly or through one of its metabolites. In this report, we investigated in vivo and in vitro effects of 4-HPR on three different breast carcinoma cells and two different melanoma cell lines. In vitro, the growth of all three breast carcinoma cell lines was inhibited by 4-HPR. Only one of two melanoma cell lines (UISO-Mel-1) showed growth inhibition to 4-HPR. The cell lines sensitive to 4-HPR in vitro also showed inhibition to 4-HPR in a xenograft model. Dietary 4-HPR (0.5 mmol/kg diet) reduced the growth of UISO-BCA-1 xenografts in female athymic mice, but had no effect on UISO-Mel-6 xenografts. Metabolism investigations of the 4-HPR-sensitive and insensitive cell lines indicated that N-[4-methoxyphenyl]retinamide (4-MPR), the major metabolite of 4-HPR, was detected only in cells sensitive to 4-HPR. Further in vitro studies with 4-MPR suggested that it is not an active metabolite of 4-HPR as it failed to inhibit growth of 4-HPR-resistant UISO-Mel-6 cells, and showed no dose-dependent inhibition of 4-HPR-sensitive breast carcinoma and melanoma cell lines. Our results in the present study indicate that, although 4-MPR is not an active metabolite of 4-HPR, detection of this metabolite in the malignant cells may serve as an indirect biomarker to predict response of cells to 4-HPR.

Induction of differentiation by 1alpha-hydroxyvitamin D(5) in T47D human breast cancer cells and its interaction with vitamin D receptors.

The role of the active metabolite of vitamin D, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in cell differentiation is well established. However, its use as a differentiating agent in a clinical setting is precluded due to its hypercalcaemic activity. Recently, we synthesised a relatively non-calcaemic analogue of vitamin D(5), 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)), which inhibited the development of carcinogen-induced mammary lesions in culture and suppressed the incidence of chemically induced mammary carcinogmas in rats. In the present study, we determined the differentiating effects of 1alpha-(OH)D(5) in T47D human breast cancer cells and compared its effects with 1,25(OH)(2)D(3). Cells incubated with either 10 or 100 nM of the analogues inhibited cell proliferation in a dose-dependent manner, as measured by the dimethylthiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Similar growth-inhibitory effects were also observed for MCF10(neo) cells. Both vitamin D analogues induced cell differentiation, as determined by induction of casein expression and lipid production. However, MCF10(neo) cells failed to respond to either vitamin D analogue and did not undergo cell differentiation. Since the cell differentiating effect of vitamin D is considered to be mediated via the vitamin D receptor (VDR), we examined the induction of VDR using reverse transcriptase-polymerase chain reaction (RT-PCR) in both cells. The results showed that, in T47D cells, both 1,25(OH)(2)D(3) and 1alpha(OH)D(5) induced VDR in a dose-dependent manner. Moreover, both analogues of vitamin D upregulated the expression of vitamin D response element-chloramphenicol acetyl transferase (VDRE-CAT). These results collectively indicate that 1alpha-(OH)D(5) may mediate its cell-differentiating action via VDR in a manner similar to that of 1,25(OH)(2)D(3).