Induction of DNA damage and caspase-independent programmed cell death by vitamin E.

Vitamin E comprises 8 functionally unique isoforms and may be a suitable candidate for the adjuvant treatment of prostate cancer. In this study, we examined the ability of 2 vitamin E isoforms [α-tocotrienol (γ-TT) and δ-tocotrienol (δ-TT)] and 4 synthetic derivatives [γ- and δ-tocotrienol succinate (γ-TS, δ-TS), α-tocopheryl polyethylene glycol succinate (TPGS), and α-tocopheryl polyethylene glycol ether (TPGS-e)] of vitamin E to induce cell death in AR- (DU145 and PC-3) and AR+ (LNCaP) prostate cancer cell lines. Our results show that δ-TT and TPGS-e are the most effective isoform and synthetic derivative, respectively, of all compounds examined. Overall, the results of our study suggest that isoforms and synthetic derivatives of vitamin E have the potency to trigger both caspase-dependent and -independent DNA damage and dominant caspase-independent programmed cell death. The capacity of vitamin E to trigger caspase-independent programmed cell death suggests that it may be useful in the chemotherapy of prostate cancer since it may prevent the tumor resistance commonly associated with the use of classical chemotherapeutic agents that trigger caspase-dependent programmed cell death.

Commitment to erythroid differentiation in mouse erythroleukemia cells is controlled by alterations in topoisomerase II alpha phosphorylation.

To explore the program of cell differentiation in Friend murine erythroleukemia (MEL) cells, we used three clonal variants: phorbol 12-myristate 13-acetate (PMA)-hypersensitive TS-19-101, PMA-resistant TR19-9, and hexamethylene bis-acetamide (HMBA)- and PMA-resistant DS19/R1. After treating TS19-101 cells with HMBA, topoisomerase II (topo II) enzymatic activity was dramatically reduced, and cells became terminally differentiated. The initial reduction in activity was soon followed by reduced topo II alpha phosphorylation, but only later did the protein level drop significantly. PMA, which completely blocked HMBA-induced differentiation in TS19-101 cells, increased the phosphorylation of topo II alpha and restored the enzymatic activity to its original levels. Reduced topo II activity and phosphorylation were also evident in HMBA-treated TR19-9 cells. PMA failed to restore topo II activity and phosphorylation to their original levels in TR19-9 cells. Predictably, the topo II activity and phosphorylation of DS19/R1 cells showed little change in response to HMBA or PMA treatment. Structural changes in chromatin became evident in sensitive cells 24 h after HMBA treatment, suggesting that alterations in topo II alpha phosphorylation may control cell differentiation by altering nuclear architecture.

Separation of the complexes formed between the regulatory and catalytic subunits of cyclic adenosine monophosphate-dependent protein kinase and topoisomerase I activity in preovulatory follicle-enriched immature rat ovaries.

Our previous studies have shown that the regulatory subunits of the type II form of cAMP-dependent protein kinase (RII) present in soluble extract of immature rat ovaries elute from diethylaminoethyl-cellulose as three separate peaks of activity, based on their association with the catalytic subunit (C) of this enzyme, as R2IIC2, an apparent R2IIC, and R2II. Based upon the existence of ovarian RII in three different subunit arrangements, the large amount of C subunit-free R2II in this tissue, and a previous report which indicated that RII exhibited intrinsic topoisomerase I activity, we determined whether DNA topoisomerase I activity was associated with any of these molecular complexes of the ovarian RII subunits. Cyclic AMP-binding activities in soluble extracts of preovulatory follicle-enriched immature rat ovaries were separated by diethylaminoethyl-cellulose chromatography and sucrose density gradient centrifugation. Topoisomerase I activity cosedimented with the apparent R2IIC and R2II obtained from sucrose gradients but was not detected in fractions containing R2IIC2. Upon cAMP affinity purification of the RII derived from fractions containing R2IIC2, R2IIC, and R2II, respectively, no topoisomerase I activity could be detected in any fraction. Phosphorylation of the affinity purified RIIs by the C subunit of beef heart cAMP-dependent protein kinase did not alter this result. These data indicate that none of the RII subunits in soluble extracts of preovulatory follicle-enriched ovaries exhibit intrinsic topoisomerase I activity.

Genistein induces maturation of cultured human breast cancer cells and prevents tumor growth in nude mice.

Results of recent studies in animal models of mammary carcinogenesis showed that the soybean isoflavone genistein is a chemopreventive agent. The objective of the present study was to determine whether soybean isoflavones can be used for the prevention of human breast carcinogenesis. Human adenocarcinoma cells that are either estrogen-receptor positive (such as MCF-7) or estrogen-receptor negative (such as MDA-MB-468) were used as our model system. Treatment of these cells with genistein concentrations of 15, 30, and 45 micromol/L resulted in cell growth inhibition, which was accompanied by the expression of maturation markers. Maturation was monitored by the induction of intracytoplasmic casein and lipids and the membrane protein intercellular adhesion molecule-1. These maturation markers were optimally expressed after 9 d of treatment with 30 mmol genistein/L. Both estrogen receptor-positive and -negative cells became differentiated in response to genistein treatments, suggesting that the antiestrogenic function of genistein is unrelated to the mechanism of cell differentiation. Daidzein, the other major isoflavone component of soybeans, did not induce differentiation in either MCF-7 or MDA-MB-468 cells. To explore the potential applications of this result, we used the nude mouse xenograft model of carcinogenesis. Treatment of either cell line with genistein before implantation into nude mice diminished the cells' tumorigenic potential. These data suggest that initiation of the differentiation program provides a protective effect against tumor growth in mouse xenografts.

Phenoxodiol, a novel isoflavone derivative, inhibits dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in female Sprague-Dawley rats.

The present study was undertaken to evaluate the potential cancer chemopreventive effects of novel synthetic derivatives of isoflavones. Initially these agents were tested in a mouse mammary organ culture (MMOC) model. Phenoxodiol (2H-1-benzopyran-7-O1,3-(4-hydroxyphenyl)), the most effective in this assay, was selected for further testing in female Sprague-Dawley rats. The agent was tested at 0 (basal diet), 50 and 75 mg/kg diet. Mammary carcinomas in these three groups were induced by dimethylbenz[a]anthracene (DMBA) injected 1 week after the animals started eating the experimental diets. Phenoxodiol significantly reduced tumour incidence rate at both doses (P

Genistein inactivates bcl-2, delays the G2/M phase of the cell cycle, and induces apoptosis of human breast adenocarcinoma MCF-7 cells.

The aim of this study was to identify the molecular mechanism of action of the isoflavone, genistein. Genistein at 0.15 mM caused MCF-7 apoptotic cell death, which was accompanied by cell cycle delay in the G2/M phase. Twenty-four hours post-treatment, 47.3% of the MCF-7 cells accumulated at G2/M, compared with 19.9% in the untreated controls. At 0.15 mM, genistein caused an increase in the steady-state levels of the wild-type tumour suppressor p53, which was attributed to stabilising the tumour suppressor protein, since p53 mRNA levels did not increase. Prior to the upregulation of p53, which became evident within 6 h of genistein treatment, there was increased bcl-2 phosphorylation at 30 min post-treatment. Although early changes (30-120 min) in the phosphotyrosine peptide patterns were not detected, after 24h, genistein inhibited phosphorylation of several peptides. These results suggest that genistein's dual roles of protein tyrosine kinase inhibitor and topoisomerase II inhibitor are essential for the initiation of apoptosis.

Genistein induces apoptosis and topoisomerase II-mediated DNA breakage in colon cancer cells.

The present study was undertaken to determine if (a) genistein induces topo II-mediated DNA damage in HT-29 colon cancer cells; and (b) if this damage is required to induce apoptosis. DNA damage was evaluated using the comet assay. Apoptosis was determined by the ethidium bromide/acridine orange staining technique. DNA breakage was noted within 1 h of treatment. Apoptosis was only induced with high concentrations (>/=60 microM) of genistein. Marked inhibition of HT-29 cell growth was evident at concentrations ranging from 60 to 150 microM. This was associated with a cell cycle arrest at G(2)/M. Similar findings were obtained in SW-620 and SW-1116 colon cancer cell lines. Aclarubicin, a topo II antagonist, reduced genistein-induced DNA breaks but did not reduce apoptosis. These data suggest that, in colon cancer cells, topo II serves as the enzymatic target of genistein. Furthermore, topo II-mediated DNA cleavage is not required for the induction of apoptosis.

Regulation of lactate dehydrogenase gene expression by cAMP-dependent protein kinase subunits.

The studies described in this report suggest a rather complex, albeit incomplete, sequence of molecular events that we believe form part of the cascade of reactions through which a series of hormones, via cAMP, regulates the expression of specific gene products. The majority of our own studies relate to cAMP-mediated induction of LDH. Some, if not all, of the molecular steps discussed in this paper may ultimately be recognized as part of a universal mechanism by which cAMP controls gene expression in higher eukaryotes. The idea of a functional role for cAMP-dependent protein kinase subunits in cAMP-mediated gene control has already had experimental support, but our identification of the regulatory subunit RII as a topoisomerase now more firmly points to a complex function for the kinase in regulating gene function at the DNA level. We look forward to the elucidation of the function of those nuclear proteins that serve as substrate for the catalytic subunit of cAMP-dependent protein kinase. Further studies related to the molecular interaction of RII with chromosomal DNA should be a fruitful area for future research.

Betulinic acid reduces ultraviolet-C-induced DNA breakage in congenital melanocytic naeval cells: evidence for a potential role as a chemopreventive agent.

Melanoma transformation progresses in a multistep fashion from precursor lesions such as congenital naevi. Exposure to ultraviolet (UV) light promotes this process. Betulinic acid (BA) was identified by our group as a selective inhibitor of melanoma that functions by inducing apoptosis. The present study was designed to investigate the effect of BA and UV-C (254 nm) on cultured congenital melanocytic naevi (CMN) cells, using the single-cell gel electrophoresis (comet) assay to detect DNA damage. Exposure to UV light induced a 1.7-fold increase in CMN cells (P = 0.008) when compared with controls. When a p53 genetic suppressor element that encodes a dominant negative polypeptide (termed GSE56) was introduced into the CMN cells, the transfected cells were more sensitive to UV-induced DNA breakage. This suggests that p53 can protect against UV-induced DNA damage and subsequent melanoma transformation. Pretreatment with BA (3 microm) for 48 h resulted in a 25.5% reduction in UV-induced DNA breakage in the CMN cells (P = 0.023), but no changes were observed in the transfected cells. However, Western blot analysis revealed no changes in the p53 or p21 levels in BA-treated cells, suggesting that BA might mediate its action via a non-p53 pathway. These data indicate that BA may have an application as a chemopreventive agent in patients with congenital naevi.

Inhibition of N-methyl-N-nitrosourea-induced mammary tumors in rats by the soybean isoflavones.

Soy-based diets, rich in the isoflavones genistein and daidzein, are thought to protect against breast and prostate cancer. We used the N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis animal model to test the effectiveness of these two isoflavones as chemopreventive agents. Each isoflavone was injected daily into 35-day-old rats for six months while we monitored the animals' body weight and mammary tumor appearance. Genistein was effective in reducing tumor multiplicity, but it reduced tumor incidence only marginally. Daidzein was less effective in reducing both tumor incidence and multiplicity. To investigate genistein's mechanism of action, we determined the topoisomerase II (topo II) activity and detected the phosphotyrosine-containing peptides in the extracts of mammary tissues isolated from control and isoflavone-treated animals. Mammary tumors contained over 60-fold higher topo II enzymatic activity than the mammary glands. Similarly, more tyrosine phosphopeptides were detectable in mammary tumors than in mammary glands. Tissue samples from genistein treated animals contained similar topo II and protein tyrosine kinase (PTK) activities as the control group. These data suggest that mammary tumorigenesis is accompanied by an extensive increase in topo II and PTK activities. The mechanism of chemoprevention by genistein, however, is independent of topo II or PTK inhibition.

A novel use for the comet assay: detection of topoisomerase II inhibitors.

BACKGROUND: The simple and quick comet assay can quantitatively detect DNA cleavage in cells. This study aimed to determine whether the comet assay could be used to detect topoisomerase (topo) II inhibitors. MATERIALS AND METHODS: HT-29 colon cancer cells were pre-incubated with aclarubicin, a topo II antagonist, then treated with topo II poisons: etoposide (VP-16), teniposide (VM-26), 4'-(acridinylamino) methansulfon-m-anisidide (m-AMSA) and adriamycin (doxorubicin). We also tested a topo I poison (camptothecin) and a microtubule depolymerization inhibitor (taxol). RESULTS: Aclarubicin significantly reduced DNA cleavage induced by topo II poisons, but not that induced by camptothecin. In HL-60/MX2 cells (containing no topo II beta and reduced topo II alpha), DNA breakage induced by topo II poisons was lower. Also, aclarubicin antagonized topo I-mediated camptothecin-induced DNA cleavage in these resistant cells. CONCLUSIONS: The comet assay can be used to detect topo II poisons in cultured cells. Also, aclarubicin has a dual topo I and topo II antagonism, with "preferential antagonism" of topo II when topo II beta catalytic activity is normally expressed.

Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.

Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

Solubilization and stabilization of carotenoids using micelles: delivery of lycopene to cells in culture.

The use of the organic cosolvents tetrahydrofuran and dimethylsulfoxide was found to be unsuitable for prostate tumor cell cultures because of solvent cytotoxicity and the poor solubility and instability of lycopene. For example, the half-life of lycopene in organic/aqueous solution was found to be less than 2 h. Therefore, a micellar preparation of lycopene was developed for the solubilization and stabilization of lycopene in cell culture media. Neither the micelles themselves nor lycopene solubilized in micelles at concentrations up to 10 microg/mL in the cell culture media produced cytotoxicity or inhibition of cell proliferation in either LNCaP human prostate cells or Hs888Lu human lung cells. Lycopene solubilized in micelles was stable for at least 96 h under standard cell culture conditions so that a constant lycopene supply could be provided to the cells. During the culture process, lycopene was taken up by LNCaP cells and reached a plateau at approximately 12 h. Micelles provide a convenient, inexpensive, and nontoxic vehicle for dissolving and stabilizing carotenes such as lycopene in tissue culture media and then delivering them to cells growing in culture.

Caspase-independent pathways of programmed cell death: the unraveling of new targets of cancer therapy?

In the past few years, accumulating evidence in the literature supports the existence of pathways of caspase-independent programmed cell death (CI-PCD). These pathways are likely to be acting as 'death backup systems' that ensure effective removal of defective cells from the organism. Similar to classical apoptosis i.e. caspase-dependent programmed cell death (CD-PCD), the mitochondrion is the main organelle orchestrating the series of events which are required for the induction of CI-PCD. In addition, the pro-apoptotic proteins Bax and Bid are also key participants in CI-PCD. However, contrary to CD-PCD, CI-PCD involves executioners other than the caspases which include the cathepsins, the calpains and serine proteases. The protein AIF may also play an important role in the induction of CI-PCD. In this review we report current knowledge on CI-PCD and provide evidence for its regulation by chemotherapeutic agents currently used in the clinic and under investigation in clinical trials. Lastly, we discuss how the study of natural and synthetic agents triggering CI-PCD may help in the pharmacological design of a new generation of more effective chemotherapeutic drugs. The use of such drugs activating both CD-PCD and CI-PCD pathways should achieve a more successful eradication of carcinogenic cells and the attainment of lower levels of tumor resistance.

Dietary retinoids and carotenoids in rodent models of mammary tumorigenesis.

In this review of the scientific literature the relationship between retinoids, carotenoids, and mammary carcinogenesis is examined. Several retinoids have shown promise as chemopreventive agents against chemically induced mammary carcinogenesis in mice and especially in rats. The most promising retinoids are retinyl acetate (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide). In rats, dietary administration of these retinoids reduced tumor incidence and multiplicity, and increased the latency of DMBA or MNU-induced mammary cancers. In mice, 4-HPR reduced the number of hyperplastic alveolar nodules and the number of tumors in MTV- and MTV+ mice, respectively. Among retinoids, 4-HPR is at present the most promising analogue, due to its ability to concentrate in the mammary gland. The combination of 4-HPR with tamoxifen not only is more effective in suppressing breast cancer than either agent alone, but also inhibits the appearance of subsequent cancers following the surgical removal of the first tumor. These studies suggest that retinoids, like tamoxifen, may be applicable to the prevention of contralateral breast cancer in women who underwent breast cancer surgery. It is also becoming evident that differentiation therapy and chemoprevention can become attractive alternative approaches to intensive cytotoxic chemotherapy. The role of carotenoids in the prevention of mammary carcinogenesis, however, is ambiguous. Poor absorption and low levels of carotenoids that reach the target tissues complicate interpretation of data in rodent models of mammary carcinogenesis. Very few animal studies are presently available in which purified carotenoids were found effective against mammary carcinogenesis. These results do not justify undertaking clinical evaluation of individual carotenoids against breast cancer at this time.

The phosphoform of the regulatory subunit RII of cyclic AMP-dependent protein kinase possesses intrinsic topoisomerase activity.

The phosphoform of the type II regulatory subunit (phospho-RII-cAMP) of cAMP-dependent protein kinase from rat liver was found to possess intrinsic topoisomerase activity towards several DNA substrates such as phi X174, pBR322, SV40, and M13. Like the type I topoisomerases from several eukaryotic cells, phospho-RII X cAMP can relax both positive and negative superhelical turns of phi X174 DNA. Topological isomers with a decreasing number of superhelical turns can be identified as transient products. Conditions under which phospho-RII X cAMP relaxes superhelical phi X174 DNA lead to transient formation of a DNA-phospho-RII X cAMP complex via DNA strand breakage and covalent attachment of the DNA to a tyrosine residue of phospho-RII X cAMP via a phospho-RII X cAMP depends on the presence of cAMP and is altered by changes in the degree of phosphorylation of RII. Both dephosphorylation and removal of cAMP from phospho-RII X cAMP abolish its topoisomerase activity.

Genistein-induced cell differentiation and protein-linked DNA strand breakage in human melanoma cells.

Genistein, an in vitro inhibitor of topoisomerase II and tyrosine kinases, elicited an inhibition of growth and increased melanin content in five human melanoma cell lines, after six days of treatment at a concentration of 45 microM. In two lines examined more thoroughly, HO and SK-MEL-131, treatment with genistein also increased other markers of differentiation, including tyrosinase activity, reactivity with CF21 monoclonal antibody, and dendrite-like structure formation. The genistein-evoked increases in melanin content and tyrosinase activity were concentration- and time-dependent. Treatment of HO and SK-MEL-131 cells with 45 microM genistein for 24 hr or 60-600 microM genistein for only 1 hr resulted in an increase in protein-linked DNA strand breaks. Our results suggest an association between the genistein-evoked, protein-linked, DNA strand breaks and the genistein-induced differentiation of human melanoma cells.

Evidence that a metabolite of equine estrogens, 4-hydroxyequilenin, induces cellular transformation in vitro.

Estrogen replacement therapy has been correlated with an increased risk of developing hormone-dependent cancers. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin and equilin which are components of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to potent cytotoxic quinoids which can consume reducing equivalents and molecular oxygen, and cause a variety of DNA lesions, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could contribute to the cytotoxic/genotoxic effects of equine estrogens in vivo. In the study presented here, we studied the oxidative and carcinogenic potential of 4-OHEN and the catechol metabolite of the endogenous estrogen, 4-hydroxyestrone (4-OHE), in the JB6 clone 41 5a and C3H 10T(1/2) murine fibroblast cells. The relative ability of 4-OHEN and 4-OHE to induce oxidative stress was measured in these cells by oxidative cleavage of 2',7'-dichlorodiacylfluorosceindiacetate to dichlorofluoroscein. 4-OHEN (1 microM) displayed an increase in the level of reactive oxygen species comparable to that observed with 100 microM H(2)O(2). In contrast, 4-OHE demonstrated antioxidant capabilities in the 5-50 microM range. With both cell lines, we assessed single-strand DNA cleavage using the comet assay and the formation of oxidized DNA bases, such as 8-oxodeoxyguanosine, utilizing the Trevigen Fpg comet assay. 4-OHEN caused single-strand breaks and oxidized bases in a dose-dependent manner in both cell lines, whereas 4-OHE did not induce DNA damage. Since oxidative stress has been implicated in cellular transformation, we used the JB6 clone 41 5a anchorage independence assay to ascertain the relative ability of 4-OHEN and 4-OHE to act as tumor promoters. 4-OHEN caused a slight but significant increase in the extent of cellular transformation at the 100 nM dose; however, in the presence of NADH, which catalyzes redox cycling of 4-OHEN, the transformation ability of 4-OHEN was dramatically increased. 4-OHE did not induce transformation of the JB6 clone 41 5a in the 0.1-10 microM range. The initiation, promotion, and complete carcinogenic transformation potentials of both metabolites were measured in the C3H 10T(1/2) cells. 4-OHEN demonstrated activity in all stages of transformation at doses of 10 nM to 1 microM, whereas 4-OHE only demonstrated promotional capabilities at the 10 microM dose. These data suggest that oxidative stress could be partially responsible for the carcinogenic effects caused by 4-OHEN and that 4-OHEN is a more potent transforming agent than 4-OHE in vitro.

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea.

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a beta-glucosidase produced by the organism. Genistein was co-extracted with erythromycin from the fermentation broth, then separated from erythromycin during the second step of the purification process for the production of erythromycin.

Chemopreventive effects of soy protein and purified soy isoflavones on DMBA-induced mammary tumors in female Sprague-Dawley rats.

There are conflicting reports on the effect of soy and its components on mammary carcinogenesis in adult female rats, mainly because of different rodent models that are used in chemoprevention studies. The present study was undertaken to compare the tumor-preventative effects of soy protein isolate (SPI) and two of its isoflavones in a "standard" model that had been used for the identification of many chemopreventive agents. Six groups of female Sprague-Dawley rats were provided with modified cornstarch AIN-76A diets supplemented as follows: no additional agents (control), purified genistein (200 mg/kg diet), purified daidzein (200 mg/kg diet), genistein + daidzein (100 mg/kg diet each), SPI containing normal levels of isoflavones (SPI-n), or SPI depleted of isoflavones (SPI-d). Mammary carcinomas were induced by 7,12-dimethylbenz[a]anthracene (DMBA) introduced 1 wk after the animals began consuming the experimental diets. At the end of the study (120 days after DMBA treatment), no significant differences were found among the six groups with respect to tumor incidence or survival, nor was there a significant reduction in tumor multiplicity in the genistein or genistein + daidzein group. However, there was a 32% reduction in tumor multiplicity in the daidzein and SPI-n groups relative to the control group (P < 0.05). The most effective diet was SPI-d, which produced a 50% reduction in tumor multiplicity relative to the control (P < 0.01). The difference between the SPI-d group and the daidzein or SPI-n group was not significant. Median tumor latency was increased from 53 days in the control group to 68 days in the daidzein group and to 72 days in the SPI-d group, but these differences were not statistically significant. These results show that daidzein and SPI (with normal or low levels of isoflavones) are effective inhibitors of DMBA-induced mammary tumors in adult rats.