Elucidation of molecular targets of mammary cancer chemoprevention in the rat by organoselenium compounds using cDNA microarray*

Karam El-Bayoumy1, Bhagavathi A. Narayanan, Dhimant H. Desai, Narayanan K. Narayanan, Brian Pittman, Shantu G. Amin, Joel Schwartz and Daniel W. Nixon

American Health Foundation Cancer Center, Institute for Cancer Prevention, 1 Dana Road, Valhalla, NY 10595, USA

1 To whom correspondence should be addressed Email: kelbayou{at}ifcp.us


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We employed cDNA microarray analysis to identify, in mammary adenocarcinomas induced by 7,12-dimethylbenz[a] anthracene (DMBA) in the rat, target genes as potential biomarkers for cancer chemoprevention by 1,4-phenylenebis(methylene)selenocyanate (p-XSC). Confirmation of selected genes was conducted by reverse transcription polymerase chain reactions (RT–PCR). The glutathione conjugate, p-XSeSG, a putative metabolite of p-XSC was also employed to test our hypothesis that p-XSeSG is a more effective cancer chemopreventive agent in the mammary cancer model than p-XSC. Mammary adenocarcinomas were induced by a single oral administration of 5 mg DMBA in 0.2 ml olive oil per rat at 50–55 days of age. Consistent with our previous reports, dietary p-XSC at a non-toxic dose (10 p.p.m. as selenium) significantly inhibited adenocarcinoma development, independent of feeding duration. Moreover, p-XSeSG appears to be just as effective as p-XSC when fed after DMBA administration, but was significantly less effective than p-XSC in inhibiting the induction of mammary adenocarcinomas when it was fed before DMBA and continued until termination. To delineate the molecular basis for cancer chemoprevention by organoselenium compounds, we focused our analysis on differential expression of genes known to be involved in DMBA metabolism, as well as those related to cell cycle, cell proliferation and apoptosis. p-XSC and p-XSeSG were significantly and equally effective in inhibiting levels of expression of genes associated with cytochrome P450 isoforms, but the former was more active than the latter in up-regulating the expression of those related to certain phase II enzymes. p-XSC and p-XSeSG were significantly more effective in the up-regulation of pro-apoptotic genes, such as p21CIP1/WAF1, p27KIP1, APO-1 and Caspase-3, while down-regulating cell growth regulatory genes, such as c-myc, cyclin D1, cyclin D2 and proliferating cell nuclear antigen (PCNA). To our knowledge, this is the first report that provides insights into the effects of p-XSC and p-XSeSG at the molecular level that may account for mammary cancer chemoprevention in vivo in the rat.

Abbreviations: Cy3, cyanine 3-dUTP; Cy5, cyanine 5-dUTP; DMBA, 7,12-dimethylbenz[a]anthracene; p-XSC, 1,4-phenylenebis(methylene)-selenocyanate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Knowledge gained from epidemiological studies, although sometimes ambiguous, suggests that an increased risk for certain human diseases, including cancer, is related to insufficient intake of selenium (13). A key achievement is the double-blind, randomized trial of selenium-enriched yeast in patients with non-melanoma skin cancer that led to the unexpected discovery that selenium protects against colon, lung and prostate cancers; insufficient breast cancer cases in this study prohibited any firm conclusion regarding the effect of selenium on breast cancer (46). The outcome of this trial stimulated the initiation of two new clinical intervention trials in the US (Selenium and Vitamin E Cancer Prevention Trial, SELECT) and in Europe (Prevention of Cancers by Intervention with Selenium, PRECISE) (79).

Pre-clinical investigations have led to a systematic study of selenium compounds as one group of cancer chemopreventive agents that merits further research (10). Yet the toxicity of certain selenium compounds, including the inorganic form of selenium, which have been used in the past inhibited further research until we introduced novel synthetic organoselenium compounds (11); these organoselenium compounds were examined as chemopreventive agents in several animal tumor models and have been shown to be superior to those employed in the past. Developing organoselenium chemopreventive agents with optimal chemopreventive potency and low toxicity continues to be a primary goal in our laboratories to develop organoselenium chemopreventive agents. Multi-organ sensitivity to anticarcinogenesis by the organoselenium 1,4-phenylenebis(methylene)selenocyanate (p-XSC) has been documented by our group in pre-clinical investigations (12).

Our results and those described in the literature indicate that the chemopreventive efficacy of selenium as an anticarcinogen depends on the chemical form in which it is administered, indicating that metabolism is a prerequisite for cancer prevention (13). Understanding the metabolism of organoselenium compounds is essential to determine whether the parent compound and/or its metabolites are responsible for chemoprevention. The identification of TSC as an in vivo metabolite of p-XSC led us to postulate the following metabolic pathway: p-XSC->glutathione conjugate (p-XSeSG)->aromatic selenol moiety (p-XSeH)->TSC (14). In this pathway, the formation of p-XSeH may be a critical step, as the selenol moiety is considered an important entity in cancer chemoprevention by selenium compounds (13). To test our hypothesis p-XSeSG was synthesized, and compared with p-XSC in azoxymethane-induced rat colon tumors; the outcome of this comparative study appears to support our hypothesis (15). In the present study we tested the same hypothesis by comparing the efficacy of p-XSC and its glutathione conjugate in the rat mammary tumor model using 7,12-dimethylbenz[a]anthracene (DMBA) as the carcinogen. To provide basic knowledge at the molecular level about the contrasting difference in the efficacy of the two selenium compounds, a follow-up study using cDNA microarray analysis followed by quantitative RT–PCR using rat oligoarrays and gene sequence-specific primers were applied to mammary adenocarcinomas isolated from rats treated with DMBA and fed control diet, or diet supplemented with p-XSC or p-XSeSG. Specifically, we focused on the effects of selenium compounds on genes that are associated with DMBA activation/detoxification as well as those related to cell cycle, cell proliferation and apoptosis; modulation of such genes is considered critical in cancer chemoprevention especially by selenium compounds (10,16).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
DMBA was obtained from Aldrich Chemical Co. (Milwaukee, WI). p-XSC and p-XSeSG were synthesized as described earlier (15,17).

Animal and diet preparation
Pathogen-free, female Sprague–Dawley rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). A semi-purified diet (AIN-76A) was used throughout the study; this diet contains 0.1 p.p.m. of selenium as sodium selenite. Levels of selenium (10 p.p.m.) for incorporation into the diet were based on numerous bioassays conducted previously in our laboratories (10,15,17). The incorporation of p-XSC and p-XSeSG into the semi-purified diet was done as described (17). The levels and stability of the selenium compounds in the diet were confirmed by HPLC analysis as reported previously (17).

Chemoprevention studies
Rats were divided into five groups and all groups were maintained on the control diet (Table I). At 50–55 days of age, all groups were given 5 mg DMBA in 0.2 ml olive oil by gastric intubation. One week after DMBA administration, groups 2 and 3 began to receive the experimental diet containing p-XSC or p-XSeSG, respectively (post-initiation); rats were kept on these diets until termination. Rats in groups 4 and 5 were fed p-XSC and p-XSeSG, respectively, 1 week before DMBA administration and remained on these diets until termination (initiation-post-initiation).


View this table:
[in this window]
[in a new window]
 
Table I. Mammary cancer chemoprevention by p-XSC and p-XSeSG

 
At weekly intervals, beginning 4 weeks after DMBA administration, the appearance and position of palpable mammary tumors in each rat were recorded. Twenty-two weeks after DMBA treatment, all rats were killed by CO2. At necropsy, the mammary gland was exposed to detect palpable and non-palpable tumors. All tumors were excised and portions were fixed in buffered formalin, blocked in paraffin, sectioned and stained with hematoxylin and eosin; the other portions were saved at -80°C for cDNA microarray analysis. Histological diagnosis of mammary tumors was based on criteria outlined by Russo et al. (18).

The percentage of rats with palpable tumors determined throughout the bioassay was compared among the groups using life table analysis. Kaplan–Meier curves (19) were generated for each group and then compared with the control group using the log-rank test (20) and adjusted for multiple comparison by means of the Bonferroni correction (21). The percentage of rats that developed mammary adenocarcinomas as determined by histopathology was compared among groups using the {chi}2 test and adjusted for multiple comparison using the Bonferroni correction (21). Tumor multiplicity was compared among the groups using ANOVA, followed by Duncan's multiple comparison test (22).

Oligonucleotide microarray analysis
Gene expression profiling was performed using oligoarrays from Clontech (Rat 1.0 Atlas Glass Arrays containing 1081 genes). Total RNA from mammary adenocarcinomas was isolated from rats treated with DMBA and fed control diet (group 1), or diet supplemented with p-XSC (group 4) and p-XSeSG (group 5) (Table I). Based on the histology, the total RNA used in this study was isolated from adenocarcinoma, the major type of cancer induced by DMBA in this animal model. Tumors from each rat (n = 4 rats/group) were used to obtain a minimum of 50 µg RNA, which was isolated using Trizol reagent, and purified on Qiagen columns (Life Technologies, Rockville, MD and Qiagen, Valencia, CA); this was converted to cDNA using superscript reverse transcriptase. Universal RNA (BD Biosciences Clontech, Palo Alto, CA) extracted from rat mammary glands was labeled with cyanine 3-dUTP (Cy3) and was used as control probes. RNA from different treatment groups was labeled with cyanine 5-dUTP (Cy5). Hybridization of the microarray (overnight, 65°C) to the probe was carried out as per the manufacturer's instructions (BD Biosciences Clontech, Palo Alto, CA) and with appropriate modifications as described in our earlier reports (23,24).

Scanning and data analysis
Scanning of the hybridized microarray slides were done at the Microarray Unit, AHF Cancer Center, using the ScanArray Express-HT Microarray Scanner, a confocal-scanning instrument that contains two lasers which excite cyanine dyes at appropriate wavelengths, 635 (Cy5) and 532 nm (Cy3), respectively, with high-resolution (10 µ pixel size) photo multiplier tubes that detect fluorochrome emission ScanArray Express Pro software (PE/Packard Biosciences, Boston, MA) was used to analyze the images and to extract the data sets into a Microsoft Excel spreadsheet. The data sets consist of signal and background intensity, standard deviation of signal and background intensity, and ratio of media and/or mean or total intensity including flags. A Microsoft Excel macro program that corrects the intensity of each spot for variations in the overall intensity of the image with respect to a control image was used to perform normalization. Data analysis was carried out using a Genespring bioinformatics software package (Silicon Genetics, Redwood City, CA) as we described recently (23).

Confirmation of microarray results
To verify the gene expression pattern, the experiments were repeated with three similar sets of rat Atlas arrays containing similar sets of genes and prosite motifs. The level of expression pattern was compared with similar spots present in the respective quadrants in all the three slides simultaneously; 87% of the spots showed similarity in their expression in all three arrays that are hybridized with similar samples. The expression of highly expressed genes was confirmed with RT–PCR using sequence-specific primers. Primers (Table II) for the target genes were designed with the assistance of the Oligo 6.0 Primer Design and Analysis Software (Molecular Biology Insights, Cascade, CO). The RT–PCR product signals were compared with the signal intensity of Cy5/Cy3 ratio as described in our earlier publications (23,24).


View this table:
[in this window]
[in a new window]
 
Table II. Oligonucleotide primer sequences used to perform RT–PCR analysis

 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of p-XSC and p-XSeSG on DMBA-induced mammary adenocarcinomas
Figure 1 shows the time course of palpable mammary tumor development. The percent incidence of palpable mammary tumors in the p-XSC- and p-XSeSG supplemented groups was lower through the course of the bioassay. As shown, the growth curve for group 4 is significantly different from group 1 (control) at P < 0.05 after adjustment for multiple comparisons. Group 5 is also significant at P < 0.05 when adjustment for multiple comparisons was ignored. The remaining two treatment groups were not significantly different from group 1.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. Incidence (%) of palpable mammary tumors in rats treated with DMBA and fed either control diet or diets supplemented with selenium compounds. G1, DMBA-treated control; G2, post-initiation; G3, post-initiation; G4, pre-initiation to end; G5, pre-initiation to end.

 
As shown in Table I (only adenocarcinomas are reported), dietary p-XSC significantly inhibited mammary tumor formation (tumor multiplicity ± SD; incidence) from 4.0 ± 2.77 (88%) to 1.28 ± 2.51 (P<0.01); 48% (P<0.05) when fed 1 week before DMBA and continued until termination. When p-XSC was fed 1 week after DMBA and continued until termination, tumor multiplicity was reduced to 2.04 ± 2.26 (P < 0.05) and tumor incidence was reduced to 72% but this was not significant. Depending on the feeding regimen, on the basis of tumor multiplicity, p-XSeSG appears to be equally effective as p-XSC when fed after DMBA administration (group 2 versus group 3); percent tumor incidence in group 2 and group 3 was comparable and was not significantly different from group 1. When fed continuously, p-XSC was significantly more effective than the conjugate (group 4 versus group 5, P < 0.05).

Differential gene expression pattern
Genes whose expression was altered by >2-fold by organoselenium compounds were selected for further cluster analysis of functional genes as described by Kaminski et al. and Eisen et al. (25,26). The results are shown in Figure 2, as scatter graphs that represent the gene expression patterns based on the magnitude of change in the intensity of color (Cy5/Cy3 ratio). An in-depth analysis of microarray data was performed in order to obtain an overall gene expression pattern on specific cluster of genes that were modulated by selenium compounds. Biochemical functions of the genes in the expression profiles are diverse and include oncogenes, transcription factors, genes involved in the carcinogen metabolism, pro- and anti-apoptotic genes, growth factors and genes involved in map kinase signaling pathways. Table III provides a partial list of differentially expressed genes and the results indicate that selenium compounds had a profound effect on various functional groups of genes, such as those related to Phase I and Phase II enzymes especially those involved in the metabolism of DMBA as well as pro-inflammatory, cell cycle regulatory and apoptosis-related genes. The changes in the levels of expression observed with the microarray analysis were confirmed with quantitative RT–PCR for a few selected genes using sequence-specific primers (Figure 3).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2. Scatter plot view of expressed genes using DNA microarray analysis. Comparative analysis on the distribution of differentially expressed genes in mammary adenocarcinomas induced by DMBA in rats fed with control diet (A), mammary adenocarcinomas induced by DMBA in rats fed p-XSC (B) or p-XSeSG (C). Signal intensities between two fluorescent images were normalized (Cy5/Cy3) to arrive at the ratio of expression. Differentially expressed genes, upregulated (>2-fold) genes are shown above the median diagonal line and down-regulated (<2-fold) genes are shown below the median diagonal line.

 

View this table:
[in this window]
[in a new window]
 
Table III. Effect of p-XSC and p-XSeSG on the regulation of Phase I and Phase II enzymes, and cell cycle regulatory genes in DMBA-induced rat mammary tumor

 


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 3. Gene expression analysis: RT–PCR. Agarose gel (2%) showing the RT–PCR products amplified for transcripts of selected genes that were differentially expressed in microarray analysis. PCR amplifications using gene sequence-specific primers were carried our for total RNA isolated from different treatment groups, as described in ‘Materials and Methods’.

 
Differential expression of cytochrome P450 isoforms
Carcinogen-activating cytochrome P450 (Phase I) and Phase IIdetoxification enzymes are potential targets for chemopreventive agents. Therefore, in this study we analysed the expression of genes associated with Phase I enzymes, including those known to be involved in the metabolism of DMBA in the rat (2730). As shown in Figure 4, both selenium compounds significantly inhibited various P450 isozymes (P < 0.05).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 4. Comparative analysis on the levels of differential expression of cytochrome 450 isoforms. Mammary adenocarcinomas induced by DMBA in rats fed a control diet (shaded), a diet supplemented with p-XSC (black) or supplemented with p-XSeSG (white). The data expressed as mean ± SD are from four data sets of gene expression levels (Cy5/Cy3 ratio). The difference in the expression of all the CYP isoforms shown in the bar graph is statistically significant, particularly between groups 1 versus 4 and 1 versus 5 (P < 0.05), except for the expression of CYP4F1. The difference in the expression between groups 4 versus 5 for all the CYP isoforms was not significant.

 
Differential expression of GSTs and GPXs
The impact of p-XSC and p-XSeSG on the differential expression of GST isoforms that are known to be involved in carcinogen detoxification (31) was examined. As shown in Figure 5, both selenium compounds significantly induced the expression of several genes related to Phase II enzymes (P < 0.05); specifically p-XSC was significantly more effective than p-XSeSG in the up-regulation of GSHPx-P (selenoP), GST mu type2, GST1, GST kappa and GST pi 2.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5. Comparative analysis of the levels of differential expression of selected GST and GSPHX isoforms. Mammary adenocarcinomas induced by DMBA in rats fed a control diet (shaded), a diet supplemented with p-XSC (black) or with p-XSeSG (white). The data expressed as mean ± SD are from four data sets of gene expression levels (Cy5/Cy3 ratio). *Group 1 is significantly different from group 4, P < 0.05. #Group 1 is significantly different from group 5, P < 0.05 with the exception of GST 1 (theta), and GST 1. **Group 4 is significantly different from group 5, P < 0.05.

 
Differential expression of genes involved in cell cycle, cell proliferation and apoptosis
Both selenium compounds significantly altered the expression of several genes involved in cell proliferation, pro- and anti-apoptotic functions (Figure 6). Specifically, p-XSC and its conjugate significantly and equally up-regulate p27, p21 and BAD (P < 0.05). However, p-XSC was significantly more effective than its conjugate in the up-regulation of pro-apoptotic genes APO-1, and caspase-3 (P < 0.05). Furthermore, p-XSC was equally effective to that of p-XSeSG in down-regulating cyclin D1, cyclin D2, c-myc and PCNA: the extent of down-regulation was significant (P < 0.05).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. Comparative analysis of the levels of differential expression of selected genes involved in cell cycle and apoptosis. Mammary adenocarcinomas induced by DMBA in rats fed a control diet (shaded), or a diet supplemented with p-XSC (black) or with p-XSeSG (white). The data expressed as mean ± SD are from four data sets of gene expression levels (Cy5/Cy3 ratio). *Group 1 is significantly different from group 4, and group 5, P < 0.05. **Group 4 is significantly different from group 5, P < 0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To our knowledge, this is the first report describing how genes that are relevant in the development of breast cancer can be altered by chemopreventive p-XSC and p-XSeSG. In this study we have explicitly focused on only a small fraction of the genes that are known to play a critical role in the development of breast cancers both in humans and in laboratory animals. Based on the histology, the total RNA isolated in this study was from adenocarcinomas, the major type of cancer induced by DMBA in this animal model. Many genes were differentially expressed in this study, but it was decided to focus on those genes, which would test the hypothesis that selenium compounds inhibit carcinogen activation, enhance carcinogen detoxification and apoptosis while reducing cell proliferation.

Carcinogenesis is a multi-step process and selenium's effect can be on genes related to early events (i.e. initiation phase); genes related to Phase I and Phase II enzymes involved in the metabolic activation of the carcinogen DMBA employed in this study as well as on genes that are critical in the post-initiation phase of carcinogenesis (genes related to cell cycle, proliferation and apoptosis). Clearly, using mammary adenocarcinomas, the results of this study showed that selenium has an impact on genes that are involved in the multi-step carcinogenesis process. However, one could argue that molecular analysis of adenocarcinomas, which represent the ‘clones of cells’ that escaped chemoprevention by selenium compounds may not reflect the effect that can account for cancer chemoprevention by selenium. Thus, future molecular analysis should be performed on mammary epithelial cells at several stages of disease during the multi-step carcinogenesis process. Furthermore, identification of proteins and biochemical pathways involved in tumorigenesis using a ‘Proteomics’ approach should provide better insights as levels of transcripts, using cDNA microarray analysis, do not always equate to differences in levels of proteins or their activities.

Most carcinogens, including DMBA, have to be metabolized into an electrophilic species that can react irreversibly with nucleophilic sites on DNA, thereby altering gene expression. The human, as well as the rodent, cytochrome P450 family of enzymes are involved in the metabolic activation of chemical carcinogens (27). Multiple P450 isozymes in rat liver are known to activate DMBA to the corresponding proximate and ultimate carcinogenic forms; the cytochrome P450 2C subfamily appears to play a significant role (2830). Although there is evidence that the ultimate carcinogenic diol epoxide metabolites are stable, and can be transported to extrahepatic tissue where binding to DNA can occur (3234), the metabolic capacity of the target organ (mammary gland) should not be dismissed. Several investigators have shown that extra-hepatic tissue, such as mammary epithelial cells, contains a number of P450 enzyme systems that are capable of converting chemical carcinogens or their proximate metabolites to reactive electrophilic species (35, and references therein). Independent of the relative contribution of rat liver and mammary gland in the metabolic activation of DMBA, our previous studies indicate clearly that dietary p-XSC is a powerful inhibitor of DMBA–DNA adducts in the mammary gland but not in the liver (17). The balance between activation and detoxification determines the levels of active electrophiles in the target organ. In our previous study, Sohn et al. (36) showed that p-XSC had no effect on cytochrome P450 1A1, 1A2 and 2B1 in rat liver; a similar analysis in the rat mammary gland was not possible due to extremely low levels of P450 in this organ. Both selenium compounds were equally effective in inhibiting various cytochrome P450 s that can catalyze the activation of DMBA. The inductive effect of p-XSC on Phase II enzymes, such as GST alpha, GST mu, GST pi and GST Px was clearly evident in the rat mammary gland (36). A similar study that focuses on the effects of p-XSeSG on Phase I and Phase II enzymes has not yet been carried out. The results reported here show that both selenium compounds significantly induced the expression of several genes related to Phase II enzymes including those described in our previous report (36). In addition, the results of this study show that p-XSC is significantly more active than its conjugate in the induction of GST alpha, GST pi and GST mu that catalyze the detoxification of DMBA (31). Collectively these results can account for the inhibitory effect of p-XSC on DMBA–DNA adducts in rat mammary gland and suggest that p-XSeSG can exert a similar but perhaps a weaker effect on adduct formation. The effect of both selenium compounds on Phase I and Phase II enzymes may account for their relative chemopreventive efficacies in mammary carcinogenesis.

Our goal to determine the effect of selenium compounds during the post-initiation phase of mammary carcinogenesis was based on our previous knowledge that p-XSC, among other selenium compounds, can inhibit carcinogenesis by inhibiting cell proliferation and inducing apoptosis (10,12). Studies with human and rodent breast tumors suggest that we are far from having a complete picture about the etiology and the molecular changes that can account for the development of this disease (3741). Therefore, we focused on genes that are highly expressed in breast cancer and are related to cell proliferation and apoptosis. Amplification of the c-myc gene has been reported in DCIS and in early breast cancers (4244). Over-expression of c-myc can elicit genomic instability and tumorigenesis in the rat mammary gland and in the human breast (45,46). Over-expression of cyclin D1 has also been observed in human breast cancers as well as in experimental mammary carcinogenesis (4750). Furthermore, over-expression of cyclin D1 can lead to abnormal mammary cell proliferation (50,51). Our data demonstrate that both selenium compounds down-regulate the levels of c-myc, cyclin D1, cyclin D2 and PCNA and these results could account for the observed chemopreventive activity. It is of particular significance that other forms of selenium, such as methylseleninic acid, have been shown to inhibit cyclin D1 in experimentally induced mammary cancer (52).

The p21WAF1/CIP1 and p27KIP1 genes have been identified as inducers of cell cycle arrest at the G1-checkpoint. Alterations of both genes have been suggested to contribute to the development and progression of a variety of human malignancies due to a loss of critical anti-proliferative mechanisms (53). Decreased expression of both p21WAF1/KIP1 and p27KIP1 protein has been identified as predictors of a poor clinical prognosis in patients with malignancies including breast cancer (54,55). Although low levels of expression of p21 and p27 genes were observed in mammary adenocarcinomas, in our study, both selenium compounds significantly enhanced the expression of both genes.

Bcl-2, a cytoplasmic oncoprotein, has been shown to play an important role as a suppressive regulator of apoptosis (5658). Numerous reports have indicated the expression of Bcl-2 in human breast cancers (5961). On the other hand, BAD is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with BCLX; phosphorylation of BAD at Ser-170 is a critical event in blocking the pro-apoptotic activity of BAD (62). Both selenium compounds in our study showed a significantly higher expression of BAD and caspase-3 as well as APO-1, indicative of their inductive role in apoptosis. Our results further support the relative chemopreventive efficacy as p-XSC was significantly more effective than its conjugate in the induction of caspase-3 and APO-1 which are pro-apoptotic indicators. Clearly, our results on the effect of selenium compounds on genetic alterations appear to be consistent with their effect on apoptosis and cell proliferation. In fact, p-XSC has been shown to inhibit cell proliferation and enhance apoptosis in breast cancer cell lines (63).

In summary, consistent with our previous study, p-XSC is a highly effective chemopreventive agent in DMBA-induced rat mammary tumors. Depending on the feeding regimen, its conjugate was equally or less active than p-XSC itself. Thus, the parent compound (p-XSC) and not its conjugates (p-XSeSG) should be the choice in future mammary cancer chemoprevention studies. On the basis of our previous biochemical investigations (12,17), combined with the altered gene expression profile found, we formulated a scheme (Figure 7) showing molecular and cellular alterations that may account for mammary cancer prevention by p-XSC and its glutathione conjugate. Briefly, selenium compounds down-regulate genes related to CYP450 and induce those related to Phase II enzymes leading to low levels of DMBA–DNA adducts in the rat mammary gland (17). Modulation of genes involved in cell proliferation and apoptosis provide a mechanistic basis for cancer prevention in this animal model system. This study suggests that the molecular targets modulated by organoselenium compounds may be highly useful indicators of success in clinical cancer chemoprevention intervention trials.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 7. Proposed mechanistic pathways of mammary cancer chemoprevention by p-XSC and p-XSeSG.

 

    Notes
 
* This study was presented at the American Association for Cancer Research 93rd Annual Meeting, 2002. Back


    Acknowledgments
 
The authors wish to thank Ms Elizabeth Appel for editorial assistance and for preparing the document for submission. This work was supported by grants from NCI: P01 CA-46589 and P30 CA-17613, the AHF Cancer Center Support Grant.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Shamberger,R.J. and Frost,D.V. (1969) Possible protective effect of selenium against human cancer. Can. Med. Assoc. J., 100, 682.[Medline]
  2. Schrauzer,G.N., White,D.A. and Schneider,C.J. (1977) Cancer mortality correlation studies. III. Statistical association with dietary selenium intakes. Bioinorg. Chem., 7, 35–56.[CrossRef][ISI][Medline]
  3. Clark,L.C., Cantor,K.P. and Allaway,W.H. (1991) Selenium in forage crops and cancer mortality in US counties. Arch. Environ. Health, 46, 37–42.[ISI][Medline]
  4. Clark,L.C., Combs,G.F. Jr, Turnbull,B.W. et al. (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. J. Am. Med. Assoc., 276, 1957–1963.[Abstract]
  5. Clark,L.C., Dalkin,B., Krongrad,A. et al. (1998) Decreased incidence of prostate cancer with selenium supplementation: results of a double-blind cancer prevention trial. Br. J. Urol., 81, 730–734.[CrossRef][ISI][Medline]
  6. Duffield-Lillico,A.J., Reid,M.E., Turnbull,B.W., Combs,G.F. Jr, Slate,E.H., Fischbach,L.A., Marshall,J.R. and Clark,L.C. (2002) Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the Nutritional Prevention of Cancer Trial. Cancer Epidemiol. Biomarkers Prev., 11, 630–639.[Abstract/Free Full Text]
  7. Rayman,M.P. (2000) The importance of selenium to human health. Lancet, 356, 233–241.[CrossRef][ISI][Medline]
  8. Klein,E.A., Thompson,I.M., Lippman,S.M., Goodman,P.J., Albanes,D., Taylor,P.R. and Coltman,C. (2001) SELECT: the next prostate cancer prevention trial. Selenium and Vitamin E Cancer Prevention Trial. J. Urol., 166, 1311–1315.[ISI][Medline]
  9. Hoque,A., Albanes,D., Lippman,S.M. et al. (2001) Molecular epidemiologic studies within the Selenium and Vitamin E Cancer Prevention Trial (SELECT). Cancer Cause Control, 12, 627–633.[CrossRef][ISI][Medline]
  10. El-Bayoumy,K. (2001) The protective role of selenium on genetic damage and on cancer. Mutat. Res., 475, 123–139.[ISI][Medline]
  11. El-Bayoumy,K. (1991) The role of selenium in cancer prevention. In DeVita,V.T., Hellman,S. and Rosenberg,S.A. (eds), Cancer Prevention. JB Lippincott Co., Philadelphia, PA, pp. 1–15.
  12. El-Bayoumy,K., Rao,C.V. and Reddy,B.S. (2001) Multiorgan sensitivity to anticarcinogenesis by the organoselenium 1,4-phenylenebis(methylene) selenocyanate. Nutr. Cancer, 40, 18–27.[ISI][Medline]
  13. Ganther,H.E. (1999) Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase. Carcinogenesis, 20, 1657–1666.[Abstract/Free Full Text]
  14. El-Bayoumy,K., Upadhyaya,P., Sohn,O.S., Rosa,J.G. and Fiala,E.S. (1998) Synthesis and excretion profile of 1,4-[14C]phenylenebis(methylene) selenocyanate in the rat. Carcinogenesis, 19, 1603–1607.[Abstract]
  15. Rao,C.V., Wang,C.Q., Simi,B., Rodriguez,J., Cooma,I., El-Bayoumy,K. and Reddy,B.S. (2001) Chemoprevention of colon cancer by a glutathione conjugate of 1,4-phenylenebis(methylene)selenocyanate, a novel organoselenium compound with low toxicity. Cancer Res., 61, 3647–3652.[Abstract/Free Full Text]
  16. Medina,D., Thompson,H., Ganther,H. and Ip,C. (2001) Se-methylselenocysteine: a new compound for chemoprevention of breast cancer. Nutr. Cancer, 40, 12–17.[ISI][Medline]
  17. El-Bayoumy,K., Chae,Y.-H., Upadhyaya,P., Meschter,C., Cohen,L.A. and Reddy,B.S. (1992) Inhibition of 7,12-dimethylbenz[a]anthracene-induced tumors and DNA adduct formation in the mammary glands of female Sprague–Dawley rats by the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate. Cancer Res., 52, 2402–2407.[Abstract]
  18. Russo,J., Russo,I.H., Rogers,A.E., van Zwietan,M.J. and Gusterson,B. (1990) Pathology of tumours in laboratory animals. Tumors of the rat. Tumour of the mammary gland. IARC Scientific Publications, IARC, Lyon, 99, 47–78.
  19. Kaplan,E.L. and Meier,P. (1978) Nonparametric estimation from incomplete observation. J. Am. Stat. Assoc., 53, 457–481.
  20. Peto,R. and Peto,J. (1972) Asymptomatically efficient rank invariant procedure. J. R. Stat. Soc., 135A, 185–207.[ISI]
  21. Miller,R.G. (1981) Simultaneous Statistical Inference. Springer-Verlag, New York, p. 37.
  22. Hochberg,Y. and Tamhane,A.C. (1987) Multiple Comparisons Procedures. John Wiley & Sons, New York, pp. 3–5.
  23. Narayanan,B.A., Narayanan,N.K., Simi,B. and Reddy,B.S. (2003) Modulation of inducible nitric oxide synthase and related proinflammatory genes by the omega-3 fatty acid docosahexaenoic acid in human colon cancer cells. Cancer Res., 63, 972–979.[Abstract/Free Full Text]
  24. Narayanan,B.A., Narayanan,N.K., Stoner,G.D. and Bullock,B.P. (2002) Interactive gene expression pattern in prostate cancer cells exposed to phenolic antioxidants. Life Sci., 70, 1821–1839.[CrossRef][ISI][Medline]
  25. Kaminski,N., Allard,J.D., Pittet,J.F., Zuo,F., Griffiths,M.J., Morris,D., Huang,X., Sheppard,D. and Heller,R.A. (2000) Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc. Natl Acad. Sci. USA, 97, 1778–1783.[Abstract/Free Full Text]
  26. Eisen,M.B., Spellman,P.T., Brown,P.O. and Botstein,D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA, 96, 14863–14868.[CrossRef]
  27. Guengerich,F.P. (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem. Res. Toxicol., 14, 611–650.[ISI][Medline]
  28. Morrison,V.M., Burnett,A.K. and Craft,J.A. (1991) Metabolism of 7,12-dimethylbenz[a]anthracene in hepatic microsomal membranes from rats treated with isoenzyme-selective inducers of cytochromes P450. Biochem. Pharmacol., 41, 1505–1512.[CrossRef][ISI][Medline]
  29. Lambard,S.E., Burnett,A.K., Wolf,C.R. and Craft,J.A. (1991) The role of specific cytochromes P450 in the formation of 7,12-dimethylbenz(a) anthracene-protein adducts in rat liver microsomes in vitro. Biochem. Pharmacol., 42, 1529–1535.[CrossRef][ISI][Medline]
  30. McCord,A., Burnett,A.K., Wolf,C.R., Morrison,V. and Craft,J.A. (1988) Role of specific cytochrome P-450 isoenzymes in the regio-selective metabolism of 7,12-dimethylbenz[a]anthracene in microsomes from rats treated with phenobarbital or Sudan III. Carcinogenesis, 9, 1485–1491.[Abstract]
  31. Liu,J.Z., Zhang,B.Z. and Milner,J.A. (1994) Dietary selenite modifies glutathione metabolism and 7,12-dimethylbenz(a)anthracene conjugation in rats. J. Nutr., 124, 172–180.[ISI][Medline]
  32. Day,B.W., Sahali,Y., Hutchins,D.A., Wildschutte,M., Pastorelli,R., Nguyen,T.T., Naylor,S., Skipper,P.L., Wishnok,J.S. and Tannenbaum,S.R. (1992) Fluoranthene metabolism: human and rat liver microsomes display different stereoselective formation of the trans-2,3-dihydrodiol. Chem. Res. Toxicol., 5, 779–786.[ISI][Medline]
  33. Stowers,S.J. and Anderson,M.W. (1984) Ubiquitous binding of benzo[a]pyrene metabolites to DNA and protein in tissues of the mouse and rabbit. Chem.-Biol. Interact., 51, 151–166.[CrossRef][ISI][Medline]
  34. Ginsberg,G.L. and Atherholt,T.B. (1990) DNA adduct formation in mouse tissues in relation to serum levels of benzo(a)pyrene-diol-epoxide after injection of benzo(a)pyrene or the diol-epoxide. Cancer Res., 50, 1189–1194.[Abstract]
  35. Boyiri,T., Leszczynska,J., Desai,D., Amin,S., Nixon,D.W. and El-Bayoumy,K. (2002) Metabolism and DNA binding of the environmental pollutant 6-nitrochrysene in primary culture of human breast cells and in cultured MCF-10A, MCF-7 and MDA-MB-435 s cell lines. Int. J. Cancer, 100, 395–400.[CrossRef][ISI][Medline]
  36. Sohn,O.S., Fiala,E.S., Upadhyaya,P., Chae,Y.-H. and El-Bayoumy,K. (1999) Comparative effects of phenylenebis(methylene)selenocyanate isomers on xenobiotic metabolizing enzymes in organs of female CD rats. Carcinogenesis, 20, 615–621.[Abstract/Free Full Text]
  37. Perou,C.M., Jeffrey,S.S., van de Rijn,M. et al. (1999) Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc. Natl Acad. Sci. USA, 96, 9212–9217.[Abstract/Free Full Text]
  38. Perou,C.M., Sorlie,T., Eisen,M.B. et al. (2000) Molecular portraits of human breast tumours. Nature, 406, 747–752.[CrossRef][ISI][Medline]
  39. Wang,Y., Hu,L., Yao,R., Wang,M., Crist,K.A., Grubbs,C.J., Johanning,G.L., Lubet,R.A. and You,M. (2001) Altered gene expression profile in chemically induced rat mammary adenocarcinomas and its modulation by an aromatase inhibitor. Oncogene, 20, 7710–7721.[CrossRef][ISI][Medline]
  40. Kuramoto,T., Morimura,K., Yamashita,S., Okochi,E., Watanabe,N., Ohta,T., Ohki,M., Fukushima,S., Sugimura,T. and Ushijima,T. (2002) Etiology-specific gene expression profiles in rat mammary carcinomas. Cancer Res., 62, 3592–3597.[Abstract/Free Full Text]
  41. Shan,L., He,M., Yu,M., Qiu,C., Lee,N.H., Liu,E.T. and Snyderwine,E.G. (2002) cDNA microarray profiling of rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and 7,12-dimethylbenz[a]anthracene. Carcinogenesis, 21, 1561–1568.[CrossRef]
  42. Janocko,L.E., Lucke,J.F., Groft,D.W., Brown,K.A., Smith,C.A., Pollice,A.A., Singh,S.G., Yakulis,R., Hartsock,R.J. and Shackney,S.E. (1995) Assessing sequential oncogene amplification in human breast cancer. Cytometry, 21, 18–22.[ISI][Medline]
  43. Escot,C., Theillet,C., Lidereau,R., Spyratos,F., Champeme,M.H., Gest,J. and Callahan,R. (1986) Genetic alteration of the c-myc protooncogene (MYC) in human primary breast carcinomas. Proc. Natl Acad. Sci. USA, 83, 4834–4838.[Abstract]
  44. Watson,P.H., Safneck,J.R., Le,K., Dubik,D. and Shiu,R.P. (1993) Relationship of c-myc amplification to progression of breast cancer from in situ to invasive tumor and lymph node metastasis. J. Natl Cancer Inst., 85, 902–907.[Abstract]
  45. Li,J., Papa,D., Davis,M.F., Weroha,S.J., Aldaz,C.M., El-Bayoumy,K., Ballenger,J., Tawfik,O. and Li,S.A. (2002) Ploidy differences between hormone- and chemical carcinogen-induced rat mammary neoplasms: comparison to invasive human ductal breast cancer. Mol. Carcinogen., 33, 56–65.[CrossRef][ISI][Medline]
  46. Felsher,D.W. and Bishop,J.M. (1999) Transient excess of MYC activity can elicit genomic instability and tumorigenesis. Proc. Natl Acad. Sci. USA, 96, 3940–3944.[Abstract/Free Full Text]
  47. Matsushime,H., Roussel,M.F., Ashmun,R.A. and Sherr,C.J. (1991) Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell, 65, 701–713.[ISI][Medline]
  48. Oyama,T., Kashiwabara,K., Yoshimoto,K., Arnold,A. and Koerner,F. (1998) Frequent overexpression of the cyclin D1 oncogene in invasive lobular carcinoma of the breast. Cancer Res., 58, 2876–2880.[Abstract]
  49. Gillett,C., Fantl,V., Smith,R., Fisher,C., Bartek,J., Dickson,C., Barnes,D. and Peters,G. (1994) Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res., 54, 1812–1817.[Abstract]
  50. Zhu,Z., Jiang,W. and Thompson,H.J. (1999) Effect of energy restriction on the expression of cyclin D1 and p27 during premalignant and malignant stages of chemically induced mammary carcinogenesis. Mol. Carcinogen., 24, 241–245.[CrossRef][ISI][Medline]
  51. Wang,T.C., Cardiff,R.D., Zukerberg,L., Lees,E., Arnold,A. and Schmidt,E.V. (1994) Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature, 369, 669–671.[CrossRef][ISI][Medline]
  52. Zhu,Z., Jiang,W., Ganther,H.E. and Thompson,H.J. (2002) Mechanisms of cell cycle arrest by methylseleninic acid. Cancer Res., 62, 156–164.[Abstract/Free Full Text]
  53. Hall,M., Bates,S. and Peters,G. (1995) Evidence for different modes of action of cyclin-dependent kinase inhibitors: p15 and p16 bind to kinases, p21 and p27 bind to cyclins. Oncogene, 11, 1581–1588.[ISI][Medline]
  54. Fredersdorf,S., Burns,J., Milne,A.M. et al. (1997) High level expression of p27(kip1) and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27(kip1) and degree of malignancy in human breast and colorectal cancers. Proc. Natl Acad. Sci. USA, 94, 6380–6385.[Abstract/Free Full Text]
  55. Baretton,G.B., Klenk,U., Diebold,J., Schmeller,N. and Lohrs,U. (1999) Proliferation- and apoptosis-associated factors in advanced prostatic carcinomas before and after androgen deprivation therapy: prognostic significance of p21/WAF1/CIP1 expression. Br. J. Cancer, 80, 546–555.[CrossRef][ISI][Medline]
  56. Reed,J.C. (1994) Bcl-2 and the regulation of programmed cell death. J. Cell. Biol., 124, 1–6.[ISI][Medline]
  57. Reed,J.C. (2000) Mechanisms of apoptosis. Am. J. Pathol., 157, 1415–1430.[Abstract/Free Full Text]
  58. Korsmeyer,S.J. (1999) BCL-2 gene family and the regulation of programmed cell death. Cancer Res., 59 (suppl. 7), 1693s–1700s.[ISI][Medline]
  59. Hori,M., Nogami,T., Itabashi,M., Yoshimi,F., Ono,H. and Koizumi,S. (1997) Expression of Bcl-2 in human breast cancer: correlation between hormone receptor status, p53 protein accumulation and DNA strand breaks associated with apoptosis. Pathol. Int., 47, 757–762.[ISI][Medline]
  60. Silvestrini,R., Veneroni,S., Daidone,M.G., Benini,E., Boracchi,P., Mezzetti,M., Di Fronzo,G., Rilke,F. and Veronesi,U. (1994) The Bcl-2 protein: a prognostic indicator strongly related to p53 protein in lymph node-negative breast cancer patients. J. Natl Cancer Inst., 86, 499–504.[Abstract]
  61. Joensuu,H., Pylkkanen,L. and Toikkanen,S. (1994) Bcl-2 protein expression and long-term survival in breast cancer. Am. J. Pathol., 145, 1191–1198.[Abstract]
  62. Dramsi,S., Scheid,M.P., Maiti,A., Hojabrpour,P., Chen,X., Schubert,K., Goodlett,D.R., Aebersold,R. and Duronio,V. (2002) Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity. J. Biol. Chem., 277, 6399–6405.[Abstract/Free Full Text]
  63. Thompson,H.J., Wilson,A., Lu,J., Singh,M., Jiang,C., Upadhyaya,P., El-Bayoumy,K. and Ip,C. (1994) Comparison of the effects of an organic and an inorganic form of selenium on a mammary carcinoma cell line. Carcinogenesis, 15, 183–186.[Abstract]
Received March 31, 2003; revised June 2, 2003; accepted June 14, 2003.