MSA induces striking dose- and time-dependent changes in gene expression in LNCaP cells, suggesting that selenium acts by diverse mechanisms as a putative prostate cancer preventive agent. MSA decreases proliferation of LNCaP cells, possibly by causing cells to exit the cell cycle, alters the expression of many genes in the androgen axis, including AR and many androgen-responsive genes, and induces expression of phase 2 detoxification enzymes, an effect that could be particularly relevant to human prostate cancer chemoprevention. Our findings support the hypothesis that monomethylated selenium may be responsible, at least in part, for the potential anticancer activity of selenium supplements.
Several reports using a variety of model systems have shown that selenium inhibits cell proliferation, and this inhibition is thought to underlie selenium chemoprevention (Ip et al., 2000a
; Combs, 2001
; Ganther, 2001
; Lu, 2001
). Decreased proliferation has been attributed to cell cycle arrest, although in prostate cancer cell lines no consistent pattern of arrest has been observed. After treatment with sodium selenite or selenomethionine, growth arrest has been reported in the G1 and G2/M phases of the cell cycle, depending on the prostate cancer cell line in which these compounds were tested (Redman et al., 1998
; Menter et al., 2000
; Venkateswaran et al., 2002
; Bhamre et al., 2003
). This lack of consistency may be due to innate differences between the cell lines or to differences in metabolism of the forms of selenium used in these studies. Based on compelling evidence that methylselenol is largely responsible for the chemopreventive activities of selenium compounds, we used MSA in our studies because it can be converted directly into methylselenol in vitro (Ip et al., 2000b
). MSA produced a dose-dependent inhibition of cell growth of LNCaP with an accumulation of cells in G0/G1 phase. Similar inhibition of proliferation and accumulation of cells in G0/G1 has been observed in breast cancer and endothelial cells treated with MSA (Sinha et al., 2001
; Wang et al., 2001
; Dong et al., 2002
We noted that a striking decrease in expression of many cell cycle-regulated genes from all phases of the cell cycle accompanied growth inhibition in LNCaP cells. Microarray analysis has been used in mammary cancer cells and PC-3 prostate cancer cells, and down-regulation of cell cycle-regulated genes has been observed along with increased expression of CDK inhibitors (Dong et al
). In these reports, decreased proliferation had been attributed to cell cycle arrest due to modulation of key regulators of the cell cycle, many of which are seen in our data set. Comparison of our data set to genes whose expression varies periodically as HeLa cells pass through the cell cycle provides a broader view of the effects of MSA on the cell cycle. The coordinate, decreased expression of genes involved in all phases of the cell cycle coupled with the increased expression of CDK-inhibitors (CDKN1A, CDKN2D, and CDKN1C) suggest MSA causes LNCaP cells to exit the cell cycle, rather than inducing an arrest at a specific phase in the cell cycle. Whether this is the primary mechanism by which selenium compounds inhibit cell growth awaits further study. Certainly, assessment of the effects of other forms of selenium on the expression of cell cycle genes in prostate cells could provide additional information on the means by which selenium compounds inhibit prostate cancer growth. Ultimately, it will be necessary to evaluate the effects of selenium on prostate cancer growth in vivo, and the cell cycle-regulated genes identified in this and other studies could serve as biomarkers of response.
Perhaps the most striking observation from our microarray experiments is that MSA produced changes in transcript levels of AR and AR-regulated genes. Androgens are critical to prostate carcinogenesis, and androgen deprivation therapy is a mainstay of prostate cancer treatment. MSA suppresses the expression of AR at both mRNA and protein levels, decreases transcript levels of PSA, and decreases PSA protein excretion into the media. A small set of well-characterized androgen-regulated genes, including those with androgen response regulatory elements, show expression changes that are reciprocal to those induced by androgen. Comparison of the MSA data set with a large data set of genes modulated in response to androgens shows that many, but not all, androgen-regulated genes show expression changes opposite to what is seen after treatment with androgens. Some genes were regulated similarly in the two data sets, suggesting that MSA has mixed effects on the transcription of AR-regulated genes. It is possible that genes that are regulated similarly by MSA and androgens are not direct targets of androgen signaling pathways. For instance, androgen treatment of LNCaP cells is known to produce cellular stress by inducing an oxidative burst, and induction of stress response genes has been observed with expression profiling after androgen treatment (Xu et al., 2001
; DePrimo et al., 2002
). Therefore, the transcripts regulated similarly by androgens and MSA (DNAJB9, ATF3, and VEGF) might reflect cellular stress or other pathways that have been activated secondarily.
Effects of selenium on AR and AR-regulated genes in prostate cancer cell lines have not been observed with other selenium compounds; in fact, two reports have shown that selenomethionine does not have an effect on AR function or PSA secretion in LNCaP cells (Zhang et al., 2002
; Bhamre et al., 2003
). One possible explanation for the lack of effect of selenomethionine on androgen-regulated genes is its poor conversion to methylselenol in vitro. Intriguingly, men supplemented with selenized yeast do show small but significant decreases in their serum PSA levels compared with control subjects, suggesting the possibility that selenium compounds can affect AR-regulated genes in vivo where they can be metabolized to methylselenol (El-Bayoumy et al., 2002
). In addition, effects of MSA on AR-regulated genes in PC-3 cells were not observed by Dong et al
), suggesting that MSA may affect transcription of AR-regulated genes through AR.
It is tempting to speculate that MSA blocks proliferation in prostate cells through its effects on AR and AR-regulated genes. Consistent with our findings, Venkateswaran et al.
(200) observed that selenomethionine did not affect the growth of wild-type (AR-null) PC-3 prostate cancer cell lines, but did inhibit growth of PC-3 cells stably expressing AR. However, three other groups have observed growth inhibition by selenium compounds in prostate cancer cell lines that do not express AR (Redman et al., 1998
; Menter et al., 2000
; Dong et al., 2003
). Additional work will be necessary to understand the role of MSA on androgen signaling pathways and cell growth.
Our studies suggest that enhancement of detoxification is another mechanism that underlies the chemopreventive effects of MSA. MSA up-regulates mRNA levels of several phase 2 enzymes, including EPHX1, NQO1, NAT2, and members of the UGTB family, as well as the enzymatic activity of NQO1. We have observed similar induction of NQO1 enzymatic activity in LNCaP cells treated with sodium selenite and selenium dioxide (Brooks et al., 2002
), demonstrating that several forms of selenium are capable of inducing phase 2 enzymatic activity in prostate cells. Induction of phase 2 enzymatic activity has been proposed as a promising avenue of prostate cancer prevention after the discovery that virtually all human prostate cancers and precursor lesions (PIN) lose expression of the phase 2 enzyme glutathione S-transferase π (GSTP1) (DePrimo et al., 2001
; Nelson et al., 2001
). Global induction of phase 2 enzymes by selenium compounds might compensate for the loss of GSTP1 expression that occurs early in prostate carcinogenesis thereby and protect vulnerable prostatic epithelial cells against genome damage.
In summary, we have characterized the global transcriptional response program of LNCaP to MSA. The expression changes we observed imply that MSA exerts its anticancer activity through diverse mechanisms, including inhibition of cell proliferation, modulation of the expression of AR and its regulated genes, and induction of enzymes involved in carcinogen detoxification. Therefore, this data set provides a potential resource for understanding the modes of action of MSA and serves as a source for candidate biomarkers of selenium's effects that could be measured in vivo. Discovery of such markers could help in the design and interpretation of selenium intervention trials currently in progress.