Methylseleninic acid, a potent growth inhibitor of synchronized mouse mammary epithelial tumor cells in vitro

Article abstract of DOI:10.1016/S0006-2952(00)00545-1

Selenium compounds have been shown to be effective chemopreventive agents in several animal models and in cultured cells in vitro. It has been proposed that compounds able to generate monomethyl Se have an increased potential to inhibit cell growth. To test this hypothesis, methylseleninic acid (MSeA) and other compounds that could generate methylselenol rapidly were compared with Se compounds that do not generate monomethyl Se, using a well-characterized synchronized TM6 mouse mammary epithelial tumor model in vitro. MSeA at a low micromolar concentration inhibited TM6 growth after 10- to 15-min treatment times. Cells resumed growth after 24 hr but remained sensitive to the fresh addition of monomethyl Se-generators. Dimethyl selenide (DMSe), a putative metabolite of methylselenol, was inactive. Cells treated with 5 μM MSeA were arrested in G₁. The effects of 5 μM MSeA on gene expression were evaluated using the Atlas mouse cDNA expression array. A 10-min exposure with MSeA caused a 2- to 3-fold change in the expression of three genes: laminin receptor 1 (decreased), integrin beta (decreased), and Egr-1 (increased). The results provide experimental support for the hypothesis that monomethylated forms of Se are the critical effector molecules in Se-mediated growth inhibition in vitro.

Full text of DOI:10.1016/S0006-2952(00)00545-1

Biochemical Pharmacology 61 (2001) 311–317
Methylseleninic acid, a potent growth inhibitor of synchronized mouse
mammary epithelial tumor cells in vitro
Raghu Sinhaa,*, Emmanual Unnia, Howard E. Gantherb, Daniel Medinaa
aDepartment of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
bDepartment of Nutritional Sciences, University of Wisconsin, Madison, WI 53706, USA
Received 5 April 2000; accepted 13 July 2000
Abstract
Selenium compounds have been shown to be effective chemopreventive agents in several animal models and in cultured cells in vitro.
It has been proposed that compounds able to generate monomethyl Se have an increased potential to inhibit cell growth. To test this
hypothesis, methylseleninic acid (MSeA) and other compounds that could generate methylselenol rapidly were compared with Se
compounds that do not generate monomethyl Se, using a well-characterized synchronized TM6 mouse mammary epithelial tumor model in
vitro. MSeA at a low micromolar concentration inhibited TM6 growth after 10- to 15-min treatment times. Cells resumed growth after 24
hr but remained sensitive to the fresh addition of monomethyl Se-generators. Dimethyl selenide (DMSe), a putative metabolite of
methylselenol, was inactive. Cells treated with 5 ␮M MSeA were arrested in G1. The effects of 5 ␮M MSeA on gene expression were
evaluated using the Atlas mouse cDNA expression array. A 10-min exposure with MSeA caused a 2- to 3-fold change in the expression
of three genes: laminin receptor 1 (decreased), integrin beta (decreased), and Egr-1 (increased). The results provide experimental support
for the hypothesis that monomethylated forms of Se are the critical effector molecules in Se-mediated growth inhibition in vitro. © 2001
Elsevier Science Inc. All rights reserved.
Keywords: Methylseleninic acid; Mouse mammary tumor; Growth inhibition
1. Introduction
generating monomethylated Se as it can be converted di-
rectly to methylselenol [CH3SeH] via a cysteine conjugate
␤ -lyase reaction [16]. MSC may also be oxidized to MSC
selenoxide, which in the presence of cysteine conjugate ␤
-lyase can give rise to methylselenenic acid (unstable) lead-
ing to methylselenenylsulfide and finally to methylselenol
(Fig. 1). MSC is reported to be a better chemopreventive
agent than either selenomethionine or selenite at 1–3 ppm
[14,15]. In contrast, DMSeO undergoes rapid reduction to
DMSe, and it has a low chemopreventive activity [17] since
DMSe is rapidly expired in breath [18].
A monomethylated form of Se that is not too volatile is
needed for in vitro studies that examine mechanisms of Se
anti-cancer action. Therefore, MSeA [CH3SeO2H], an oxi-
dized monomethylated form of Se, may be suitable for use
directly or as a precursor of methylselenol (Fig. 1). The free
acid ionizes above pH 5 to the anion, methylseleninate
[CH3SeO2Ϫ], which is practically odorless. A stock solution
of methylseleninate at pH 7, therefore, is convenient for use
and is quite stable if protected from microbiological action
or reducing agents. It is a fairly strong oxidizing agent, like
sodium selenite, and will readily undergo reduction, giving
Selenium compounds have been shown to inhibit tumor-
igenesis in experimental animals [1–5], and recent studies
indicate that supplemental Se reduces cancer risk in humans
[6]. Several organic Se compounds have been recognized as
effective chemopreventive agents against the development
of tumors in the mammary gland, lung, colon, and prostate
[2,7–13]. The chemopreventive ability of Se as an anti-
tumor agent depends on its chemical form [14], and metab-
olism of the parent compound is critical to provide the
reactive form. Monomethylated forms of Se have significant
effects on carcinogenesis [15]. MSC is a good precursor for
* Corresponding author. Tel.: ϩ1-713-798-8561; fax: ϩ1-713-790-
0545.
E-mail address: rsinha@bcm.tmc.edu (R. Sinha).
Abbreviations: DMEM:F12, Dulbecco’s Modified Eagle’s Medium:
Nutrient mixture F12; DMSe, dimethyl selenide; DMSeO, dimethyl sel-
enoxide; DMSeS, dimethyl selenenylsulfide; DMSeSe, dimethyl dis-
elenide; DMSS, dimethyl disulfide; MSC, Se-methylselenocysteine;
MSeA, methylseleninic acid; and MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5,diphenyltetrazolium bromide.
0006-2952/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0006-2952(00)00545-1

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R. Sinha et al. / Biochemical Pharmacology 61 (2001) 311–317
0.42. Reduction with excess borohydride gave a UV peak
(252 nm, millimolar extinction coefficient 6.35) correspond-
ing in wavelength and intensity to that of aliphatic seleno-
lates. DMSeS was synthesized by reacting methylselenenyl
bromide (prepared by reacting DMSeSe with bromine) with
methane thiol [20]. Analysis of the reaction mixture by C18
reversed phase HPLC (75% methanol) using a diode array
detector showed successive peaks having UV spectra cor-
responding to S-S, Se-S, and Se-Se derivatives [20].
DMSeS was obtained in pure form by separating it from the
symmetrical products using preparative HPLC in 75%
methanol. The isolated product was stable when stored at
Ϫ20°.
2.2. Cell culture and synchronization
The TM6 tumor cell line was derived from the COM-
MA-D mouse mammary epithelial cell line [21]. TM6 cells
were maintained in DMEM:F12 (1:1) medium supple-
mented with 5 ␮g/mL of insulin, 5 ng/mL of epidermal
growth factor, 2% adult bovine serum, and 5 ␮g/mL of
gentamicin at 37° in a humidified atmosphere in the pres-
ence of 5% CO2. For experiments, cells were seeded at
6.6 ϫ 103/cm2 and were synchronized as described earlier
[9]. Cells were released from arrest by feeding them com-
plete medium (containing growth factors and serum) for 6
hr at which time these cells were treated separately with
each of the five selenium compounds: MSeA, DMSeSe,
DMSeS, DMSe, and DMSeO (all at a 5 ␮M concentration
for [Se] for all the experiments). Untreated cells were taken
as control cells and were analyzed in the following assays at
various times after Se exposure.
Fig. 1. Schematic presentation for the formation of various methylse-
lenides. MSeA forms methylselenol through a series of reactions with
methylselenenylsulfide as an intermediate compound. MSC, on the other
hand, can be converted to methylselenol by the cysteine conjugate ␤-lyase.
DMSe can be reduced to methylselenol, while DMSeO does not form
methylselenol.
rise to methylselenol. Methylselenol then undergoes oxida-
tion or methylation to give DMSeSe and DMSe, respec-
tively. According to the hypothesis of Ip and Ganther [14,
15,19], simple monomethylated forms of Se should have
strong growth inhibitory activity at low concentrations and
exert effects rapidly. We sought to test this hypothesis by
examining the effects of MSeA in a well-defined in vitro
system, using TM6 mammary epithelial cells.
2.3. Cell cycle analysis
Synchronized TM6 cells were treated with MSeA (5
␮M) for various time intervals followed by enzymatic dis-
sociation and fixation in 95% alcohol overnight. The un-
treated control and MSeA-treated cells were stained with
propidium iodide (50 ␮g/mL), incubated with RNase (100
␮g/mL) at 37° for 30 min, and examined by flow cytometry.
2. Materials and methods
2.1. Synthesis of Se compounds
2.4. [3H]Thymidine assay
DMSeO was synthesized as described earlier [17].
MSeA was prepared by combining DMSeSe (Aldrich
Chemical Co.) in methanol with hydrogen peroxide (3%) at
65° until the yellow color of the diselenide had disappeared.
The solution was adjusted to pH 7 with KOH, and then
applied to a column of Dowex 1 (chloride). After washing
with water until a negative starch/iodide test was obtained,
MSeA was eluted with 0.01 N HCl. The main starch/iodide-
positive fractions were pooled, adjusted to pH 7 with KOH,
and analyzed. TLC on cellulose in butanol:acetic acid:water
(5:2:3) showed a single starch/iodide positive spot of Rf ϭ
[3H]Thymidine incorporation into DNA was used to
measure cell proliferation activity. Synchronized TM6 cells
were treated with MSeA, DMSeSe, DMSeS, DMSe, and
DMSeO (all at 5 ␮M), rinsed with DMEM:F12 medium,
and pulsed with [methyl-3H]thymidine (1 ␮Ci/well) for 1 hr
following varied time intervals for treatment, as described in
Results. Untreated TM6 cells were taken as controls. Ra-
dioactivity incorporated into acid-precipitable material was
counted as described previously [22]. The data are depicted
as means Ϯ SEM of three observations.

R. Sinha et al. / Biochemical Pharmacology 61 (2001) 311–317
313
2.5. MTT assay
TM6 cells were plated in 12-well plates. Following syn-
chronization, the cells were treated with MSeA and
DMSeSe (both at 5 ␮M) for 1, 2, and 3 hr. The cells were
rinsed once with DMEM:F12 medium (without phenol red,
Sigma) and incubated in DMEM:F12 medium (without phe-
nol red) containing MTT at 1 mg/mL for 4 hr at 37° in the
dark. The MTT solution was aspirated from the wells,
DMSO (3 mL/well) was added, and the optical density was
read at 570 nm taking DMSO as the blank [23]. Data are
depicted as means Ϯ SEM of three observations.
2.6. cDNA array analysis
Treating synchronized TM6 cells with MSeA for 5–15
min showed a significant inhibition in [3H]thymidine incor-
poration when measured 3 hr later; therefore, we wanted to
investigate if an early treatment with MSeA resulted in
alteration of gene expression in these cells. For this exper-
iment, synchronized TM6 cells were treated with MSeA (5
␮M) for 10 min, and RNA was isolated from them using
TRIzol (Gibco BRL). Untreated cells were included as con-
trols. After treatment with DNase, poly(Aϩ) RNA was pre-
pared using the Oligotex mRNA kit (Qiagen). Labeled first
strand cDNA probes were prepared from both the MSeA
and control poly(Aϩ) RNA, and each was hybridized with
individual membranes spotted with 588 genes (Clontech,
mouse cDNA array). Following washings according to the
manufacturer, the membranes were exposed to X-ray film as
well as to a PhosphoImager for quantitation. The signals
were normalized against ␤-actin (housekeeping gene), and
the data were represented as fold increase or decrease in
expression compared with the untreated control cells.
Fig. 2. Effects of MSeA and DMSeSe (5 ␮M Se concentration) on the
growth of synchronized TM6 cells. (A) MTT assay measuring growth and
viability of TM6 cells. (B) [3H]Thymidine incorporation for the DNA
synthesis of TM6 cells. Values are means Ϯ SEM; N ϭ 3 at each time
point for both assays. Key: (*) indicates significance at P Ͻ 0.05 when
compared with the untreated control.
for 5, 10, 15, 30, 60, 120, and 180 min with either MSeA or
DMSeSe (5 ␮M), rinsed with incomplete medium, and
allowed to incubate with complete medium without Se com-
pounds. A [3H]thymidine incorporation assay was per-
formed for all the treatments and control cells at 3 hr. Both
MSeA and DMSeSe inhibited [3H]thymidine incorporation
significantly in TM6 cells following 15 min of exposure
(Fig. 3).
The synchronized TM6 cells incorporated high levels of
[3H]thymidine at the 16-hr time point (Fig. 4A). This cor-
responds to the maximum number of cells in S phase, as
reported earlier [9]. When these cells were allowed to cycle
in the presence of MSeA, DMSeS, or DMSeSe, the data
showed that these compounds could inhibit [3H]thymidine
incorporation (91–97%) up to the 24-hr time point, which is
the end of a complete cell cycle (Fig. 4A). Thereafter, it
seemed that the compounds had been metabolized com-
pletely, as the treated cells resumed proliferation at the
34-hr time point. In fact, these cells were able to achieve the
same extent of [3H]thymidine incorporation as untreated
3. Results
Preliminary concentration–response experiments with
MSeA demonstrated that 0.001, 0.5, 1, and 5 ␮M induced
13, 27, 50, and 81% inhibition in [3H]thymidine incorpora-
tion performed after 10 hr of treatment, respectively (data
not shown); thus, the experiments described herein were
performed using a 5 ␮M concentration for all the com-
pounds.
Synchronized TM6 cells treated with either MSeA or
DMSeSe showed a decreased optical density in the MTT
assay within 2–3 hr of treatment (Fig. 2A) as compared with
untreated control cells (P Ͻ 0.05). [3H]Thymidine incorpo-
ration was inhibited significantly (P Ͻ 0.05) in the cells
treated with either 5 ␮M MSeA (1, 2, and 3 hr) or DMSeSe
(2 and 3 hr) when compared with untreated control cells
(Fig. 2B).
The cells were subjected to treatments with MSeA and
DMSeSe over a range of time intervals. Cells were treated

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R. Sinha et al. / Biochemical Pharmacology 61 (2001) 311–317
Fig. 3. [3H]Thymidine incorporation in synchronized TM6 cells. Cells
were treated with MSeA and DMSeSe (5 ␮M Se concentration) for the
indicated time duration, and [3H]thymidine incorporation was performed
after 3 hr. Untreated TM6 cells served as the control. Values are means Ϯ
SEM; N ϭ 3 for each treatment. Key: (**) P Ͻ 0.05, and (*) P Ͻ 0.01,
compared with the control.
cells. In a separate experiment, DMSS, which is the sulfur
analogue of DMSeSe, induced only 9.8% inhibition of
growth compared with 80% inhibition induced by DMSeSe
(data not shown).
TM6 cells previously treated with MSeA and DMSeSe
were exposed to the respective compounds again at the
24-hr time point to investigate if reversibility of the effect
was due to loss of chemical over time in culture. The cells
exposed to these Se compounds again were inhibited at the
34-hr time point, and started to recover by the 48-hr time
point (Fig. 4A). These results confirm that the cells remain
sensitive to methylselenol generators.
Fig. 4. [3H]Thymidine incorporation in synchronized TM6 cells treated
with various Se compounds at the 6-hr time point. (A) Treatments with
methylselenol-generating Se compounds (all at a 5 ␮M concentration);
dashed lines indicate repeat treatment at the 24-hr time point. (B) Treat-
ment with non-methylselenol-generating Se compounds. Methanol was the
carrier solvent. Values are means Ϯ SEM, N ϭ 3 for both experiments at
each time point. An asterisk (*) indicates P Ͻ 0.05, compared with
untreated TM6 cells that served as controls.
In another experiment, DMSe and DMSeO, theoretically
two inactive metabolites of methylselenol, were compared
(Fig. 4B). The synchronized TM6 cells were treated with
DMSe and DMSeO at a 6-hr time point, and [3H]thymidine
incorporation was measured at 12-, 24-, and 48-hr time
points. These compounds, including the solvent methanol,
did not inhibit [3H]thymidine incorporation at the 12-hr
time point. [3H]Thymidine incorporation was high in the
DMSe- and solvent-treated cells at the 24-hr time point but
was not statistically significant from the untreated controls.
At the 48-hr time point, DMSeO-treated cells showed a
slight reduction of 8% (P Ͻ 0.05) of growth, but this
decrease was also observed in the solvent control and was
not significant.
control cells by acridine orange and ethidium bromide stain-
ing.1
To determine any initial changes occurring at the gene
level in the MSeA-treated cells, cDNA array analysis was
1
Henry Thompson, personal communication.
The majority of TM6 cells synchronized and treated with
5 ␮M MSeA were arrested in the G1 phase of the cell cycle
(Fig. 5) with no appreciable apoptosis observed by FACS
analysis, using an adherent cell population of MSeA-treated
cells. A 10–15% apoptosis, however, was observed in the
floating cell population of MSeA (5 ␮M)-treated TM6 cells
at the 24-hr time point as compared with 5% apoptosis in
Fig. 5. FACS analysis of MSeA-treated cells. TM6 cells were mainly
confined to G1 after treatment with 5 ␮M MSeA, with no appreciable
apoptosis.

R. Sinha et al. / Biochemical Pharmacology 61 (2001) 311–317
315
Fig. 6. cDNA array analysis of a 10-min treatment of TM6 cells with 5 ␮M MSeA. The three genes marked with an asterisk indicate greater than 2-fold change
in expression. ␤-Actin (housekeeping gene) was used to normalize the change in fold expression when control (C) and MSeA-treated cells were compared.
The numbers in the brackets depict the GenBank Accession Number.
performed using the Atlas mouse cDNA expression array
(Clontech). Several genes were differentially expressed in
the cDNA array when cells treated for 10 min with MSeA
(5 ␮M) were compared with control cells. The majority of
the cells were in G1 within that given hour (data not shown)
and beyond the 9-hr time point (Fig. 5). The differential
expression of only three genes was notable. Egr-1, a Zn-
finger regulatory protein, was almost 2-fold higher in cells
treated with MSeA (Fig. 6), whereas integrin beta and
laminin receptor 1 were decreased several fold compared
with control cells (Fig. 6). The expression of other genes
showed less than 1.3X difference when control and MSeA-
treated samples were compared, and these differences were
not significant. The expression of several genes including
protein tyrosine phosphatase (Accession No. D83966), in-
sulin-like growth factor binding protein 2 (Accession No.
X81580), and vascular endothelial growth factor (Accession
No. M95200) were not different when control and MSeA
treatments were compared (Fig. 6). Of interest was the
observation that the screen identified no cell cycle genes,
growth factors, or kinases.
to give rise to methylselenol (MSeA and DMSeSe) were
demonstrated to be effective inhibitors of cell growth in
synchronized TM6 cells. In contrast, compounds that were
predicted to not yield methylselenol (DMSS, DMSeO) were
ineffective inhibitors of cell growth. MSC has been reported
to be more efficacious than either selenite or selenomethi-
onine in cancer chemoprevention in the range of 1–3 ppm
Se [14,15]. Our results show that precursor Se compounds
that are able to produce a steady stream of monomethylated
metabolite are likely to have good chemopreventive activity.
It is intriguing that a 15-min treatment with MSeA and
DMSeSe can significantly inhibit [3H]thymidine incorpora-
tion measured 3 hr later. Earlier reports have shown that Se
compounds can decrease thymidine kinase activity [24,25].
The [3H]thymidine incorporation in MSeA- and DMSeSe-
treated cells remained inhibited until the 24-hr time point,
which coincides with the end of a complete TM6 cell cycle,
as depicted by the incorporation in control cells. The recov-
ery of cells from the effects of MSeA, DMSeSe, and
DMSeS after 34 hr shows that the compounds are no longer
interacting with the cellular components of the cell.
After recovery, exposure of the previously treated cells
to a fresh concentration of MSeA and DMSeSe again in-
hibited [3H]thymidine incorporation. Thus, cells that have
recovered remain sensitive to methylselenol generators. Al-
though MSC also reduces growth of TM6 cells, inhibition
occurs much later at the 48-hr time point as described earlier
[9]. Possibly the metabolism of MSC to methylselenol is
much slower; therefore, a longer time period is required to
4. Discussion
The results provide experimental support for the hypoth-
esis of Ip and Ganther [14,15,19] that monomethylated
forms of Se are critical effector molecules in Se-mediated
growth inhibition in vitro. Compounds that were predicted

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attain a critical level of the methylselenol form. The mech-
anism of action of MSeA and MSC could be different at the
molecular level, and further investigation is needed.
Treatment with a relatively low concentration (5 ␮M) of
MSeA arrested the TM6 cells, primarily in the G1 phase of
the cell cycle. The cells also underwent apoptosis when
treated with 50 ␮M MSC as reported earlier [9] but only at
48 hr, whereas 50 ␮M MSeA could induce apoptosis as
early as the 16-hr time point (data not shown). If a high level
of methylselenol induces apoptosis, the time difference may
be due to slow conversion of the MSC to methylselenol.
Since a rather brief (10–15 min) treatment with MSeA
was able to inhibit [3H]thymidine incorporation, we decided
to perform cDNA array analysis to determine early changes
due to MSeA at the gene level, using the poly(Aϩ) prepared
from control and MSeA-treated cells. No cell cycle genes,
growth factors, or kinases were affected by the treatment.
Expressions of two genes (integrin beta and laminin recep-
tor 1) were reduced by a 10-min MSeA treatment at a 5 ␮M
concentration. These genes are mainly involved in cell at-
tachment. One gene (Egr-1) was up-regulated by MSeA
treatment. The Egr-1 gene product is a transcription factor
with roles in differentiation and growth [26,27]. Egr-1 has
been implicated in the induced expression of platelet-de-
rived growth factor (PDGF)-A chain [28], PDGF-B chain
[29], TGF␤ [30], and fibronectin [31]. A 45-min MSeA
treatment at a 5 ␮M concentration in the TM6 cells resulted
in a 10-fold induction in Egr-1 expression (cDNA array data
not shown) as compared with untreated controls. There were
no changes in the PDGF-A and TGF␤ expression at either
10- or 45-min MseA treatments. Further evaluation is re-
quired to see if cellular effects of MSeA are associated with
any of these genes, especially in a time–course study. The
preliminary cDNA array analysis suggests that the effect of
MSeA may be on specific protein activity and not ultimately
the amount of protein that may be modified. This is sup-
ported by the reversibility of the inhibitory effect of MSeA.
MSeA can react directly with sulfhydryl groups of proteins
or undergo metabolism to other monomethylated forms of
Se (as shown in Fig. 1) that can react with sulfhydryl groups
or disulfide bonds in target molecules. Possible chemical
reactions relevant to the regulation of cellular metabolism
by monomethylated Se derivatives are described elsewhere
[32]. In summary, MSeA is useful for the direct provision in
vitro of highly reactive monomethylated Se metabolites that
are implicated in Se inhibition of tumor cell growth and
other chemopreventive mechanisms.
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