Concentration of endogenous estrogens and estrogen metabolites in the NCI-60 human tumor cell lines

Abstract

Endogenous estrogens and estrogen metabolites play an important role in the pathogenesis and development of human breast, endometrial, and ovarian cancers. Increasing evidence also supports their involvement in the development of certain lung, colon and prostate cancers.
In this study we systemically surveyed endogenous estrogen and estrogen metabolite levels in each of the NCI-60 human tumor cell lines, which include human breast, central nerve system, colon, ovarian, prostate, kidney and non-small cell lung cancers, as well as melanomas and leukemia. The absolute abundances of these metabolites were measured using a liquid chromatography-tandem mass spectrometry method that has been previously utilized for biological fluids such as serum and urine.
Endogenous estrogens and estrogen metabolites were found in all NCI-60 human tumor cell lines and some were substantially elevated and exceeded the levels found in well known estrogen-dependent and estrogen receptor-positive tumor cells such as MCF-7 and T-47D. While estrogens were expected to be present at high levels in cell lines representing the female reproductive system (that is, breast and ovarian), other cell lines, such as leukemia and colon, also contained very high levels of these steroid hormones. The leukemia cell line RMPI-8226 contained the highest levels of estrone (182.06 pg/106 cells) and 17β-estradiol (753.45 pg/106 cells). In comparison, the ovarian cancer cell line with the highest levels of these estrogens contained only 19.79 and 139.32 pg/106 cells of estrone and 17β-estradiol, respectively. The highest levels of estrone and 17β-estradiol in breast cancer cell lines were only 8.45 and 87.37 pg/106 cells in BT-549 and T-47D cells, respectively.
The data provided evidence for the presence of significant amounts of endogenous estrogens and estrogen metabolites in cell lines not commonly associated with these steroid hormones. This broad discovery of endogenous estrogens and estrogen metabolites in these cell lines suggest that several human tumors may be beneficially treated using endocrine therapy aimed at estrogen biosynthesis and estrogen-related signaling pathways.

Full text

RESEARCH Open Access
Concentration of endogenous estrogens and
estrogen metabolites in the NCI-60 human tumor
cell lines
Xia Xu and Timothy D Veenstra
*
Abstract
Background: Endogenous estrogens and estrogen metabolites play an important role in the pathogenesis and
development of human breast, endometrial, and ovarian cancers. Increasing evidence also supports their
involvement in the development of certain lung, colon and prostate cancers.
Methods: In this study we systemically surveyed endogenous estrogen and estrogen metabolite levels in each of
the NCI-60 human tumo r cell lines, which include human breast, central nerve system, colon, ovarian, prostate,
kidney and non-small cell lung cancers, as well as melanomas and leukemia. The absolute abundances of these
metabolites were measured using a liquid chromatography-tandem mass spectrometry method that has been
previously utilized for biological fluids such as serum and urine.
Results: Endogenous estrogens and estrogen metabolites were found in all NCI-60 human tumor cell lines and
some were substantially elevated and exceeded the levels found in well known estrogen-dependent and estrogen
receptor-positive tumor cells such as MCF-7 and T-47D . While estrogens were expected to be present at high
levels in cell lines representing the female reproductive system (that is, breast and ovarian), other cell lines, such as
leukemia and colon, also contained very high levels of these steroid hormones. The leuke mia cell line RMPI-8226
contained the highest levels of estrone (182.06 pg/10
6
cells) and 17b-estradiol (753.45 pg/10
6
cells). In comparison,
the ovarian cancer cell line with the highest levels of these estrogens contained only 19.79 and 139.32 pg/10
6
cells
of estrone and 17b-estradiol, respectively. The highest levels of estrone and 17b-estradiol in breast cancer cell lines
were only 8.45 and 87.3 7 pg/10
6
cells in BT-549 and T-47D cells, respectively.
Conclusions: The data provided evidence for the pres ence of significant amounts of endogenous estrogens and
estrogen metabolites in cell lines not commonly associated with these steroid hormones. This broad discovery of
endogenous estrogens and estrogen metabolites in these cell lines suggest that several human tumors may be
beneficially treated using endocrine therapy aimed at estrogen biosynthesis and estrogen-related signaling
pathways.
Background
Endogenous estrogens and estrogen metabolites (EMs)
have long been associated with carcinogenesis and devel-
opment of several hormone-dependent human carcino-
mas, such as breast, endometrial, and ovarian cancers
[1,2]. Increasing evidence suggests that these metabolites
may be involved in the pathogenesis and development of
human lung [3,4] and colon [5] cancers as well as prostate
cancer [6]. Historically, the major primary function of 17b
estradiol (E
2
) was the development of female secondary
sexual characteristics and regulation of reproductive func-
tion. Today it is recognized that E
2
exerts some effect on
almost every organ in the body [7]. The effects of E
2
and
other estrogens have expanded to include roles in neurolo-
gical function [8], retinal degenerative disease [9], cardio-
vascular health [10], and even sleep regulation [11].
Given the well documented mitogenic and possible gen-
otoxic nature of endogenous estrogens and EMs [2,12,13],
potential involvement of EMs in the carcinogenesis of
an eve n greater va riety of human tumors is conceivable.
* Correspondence: veenstrat@mail.nih.go v
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc.,
National Cancer Institute at Frederick, Frederick, MD 21702, USA
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reproduction in any medium, provi ded the original work is prope rly cit ed.

For example, some studies have suggested that estrogen
may be involved in the development of skin cancer as skin
keratinocytes possess estrogen receptors (ERs) [14], and
oral contraceptives and hormone therapy decrease acne
[15] and skin aging [16], respectively. Epidemiological stu-
dies examining assoc iations between hormone therapy
and melanoma risk have not been entirely conclusive,
although some studies hav e shown a link between hor-
mone use and increased risk of melanoma [17,18]. Epide-
miologically, estrogens have also been linked to colon
cancer, as men are more likely to develop this disease and
hormon e replacement therapy has been shown to reduce
the risk of this cancer in women [19].
Owing to th e pervasive nature of the functions of EMs,
their effect on cancers may be more pronounced than pre-
viously thought. Determining EM roles in various cancers
requires a lot of information, including tumor receptor
status, aromatase activities, and the levels of these com-
pounds within cells. Towards this aim, we systemically
surveyed human tumor cell EM levels by using NCI-60
human tumor cell lines, including human b reast, central
nervous system (CNS), colon, ovarian, prostate, kidney,
melanoma, leukemia, and non-small cell lung cancers.
Detailed EM profiles in NCI-60 human tumor cell lines
are summarized and reported in this manuscript.
Materials and methods
Reagents and materials
Cell pellets from NCI-60 human tumor cell lines were
obtained from Developmental Therapeutics Program,
NCI/NIH. Fifteen estrogens and EMs, including estro ne
(E
1
), estradiol (E
2
), estriol (E
3
), 16-epiestriol (16-epiE
3
),
17-epiestriol (17-epiE
3
), 16-ketoestradiol (16-ketoE
2
),
16a-hydroxyestrone (16a-OHE
1
), 2-methoxyestrone
(2-MeOE
1
), 4-methoxyestrone (4-MeOE
1
), 2-hydroxyes-
trone-3-methyl ether (3-MeOE
1
), 2-methoxyestradiol
(2-MeOE
2
), 4-methoxyestradiol (4-MeOE
2
), 2-hydro-
xyestrone (2-OHE
1
), 4-hydr oxyestrone (4-OHE
1
), and
2-hydroxyestradiol (2-OHE
2
)wereobtainedfromStera-
loids, Inc. (Newport, RI, USA). Stable isotope-labeled
estrogens (SI-EM), including estradiol-13,14,15,16,17,18-
13
C
6
(
13
C
6
-E
2
) and estrone-13,14,15,16,17,18-
13
C
6
(
13
C
6
-E
1
) were purchased from Cambridge Isotope
Laboratories, Inc. (Andover, MA, USA); estriol-2,4,17-d
3
(d
3
-E
3
), 2-hydroxyestradiol-1,4,16,16,17 -d
5
(d
5
-2-OHE
2
),
and 2-methoxyestradiol-1,4,16,16,17-d
5
(d
5
-2-MeOE
2
),
were obtained from C/D/N Isotopes, Inc. (Pointe-Claire,
Quebec, Canada). 16-Epiestriol-2,4,16-d
3
(d
3
-16-epiE
3
)
was purchased from Medical Isotopes, Inc. (Pelham,
NH, USA). All EM a nd SI-EM analytical standards have
reported chemical and isotopic purity ≥98%, and were
used without further purification. Dichloromethane,
methanol and formic acid were obtained from EM
Science (Gibbstown, NJ, USA). Glacial acetic acid,
sodium bicarbonate, and L-ascorbic acid were purchased
from JT Baker (Phillipsburg, NJ, USA) and sodium
hydroxide and sodium acetate were purchased from
Fisher Scientific (Fair Lawn, NJ, USA). Dansyl chloride
and acetone were purchased from Aldrich Chemical Co.
(Milwaukee, WI, USA). All chemicals and solvents used
in this study were HPLC or reagent grade unless other-
wise noted.
Preparation of stock and working standard solutions and
calibration standards
Stock solutions o f EMs and SI-EMs were each pr epared
at 80 μg/ml by dissolving 2 mg of the estrogen powders
in met hanol with 0.1% L-ascorbic acid to a final volume
of 25 ml in a volumetric flask. The stock solutions are
stable for at least two months while stored at - 20°C.
Stock solutions were analyzed at the beginning of each
analysis to verify no time-dependent degradation of the
EM and SI-EM standards had occurred. Working stan-
dards of EMs and SI-EMs at 8 ng/ml were prepared b y
dilutions of the stock solutions using methanol wit h
0.1% L-ascorbic acid.
MCF-10A cell lysate with no detectable levels of EMs
was employed for preparation of calibration standards
and quality control samples. Each calibration standard
contained lysate from approximately 50,000 MCF-10A
cellsandwaspreparedbyadding2μloftheSI-EM
working internal standard solution (16 pg of each SI-
EM) to various volumes of the EM working standard
solution. These calibration standards typically contain
0.2 to 200 pg of each EM in 0.5 ml of MCF-10A cell
lysate and were assayed in duplicate. The calibration
standards cover three orders of magnitude.
Sample preparation procedure
Samples were prepared and analyzed following a pre-
viously published method [20,21]. Briefly, each tumor
cell pellet contained approximately 1 million cells. They
were first suspended in 2 ml ice cold 12.5 mM
NH
4
HCO
3
solution. Cell lyses were prepared by tip
sonication on ice in five cycles of 10-second pulses and
10-second breaks followed by 30-minute water bath
sonication. To 0.5 ml of each cell lysate, 0.5 ml o f
freshly prepared 0.15 M sodium acetate buffer (pH 4.6)
containing 16 pg of each SI-EM and 2 mg of L-ascorbic
acid was added. Sa mples then underwent slow inverse
extraction at 8 rpm (RKVSD™ ,ATR,Inc.,Laurel,MD,
USA) with 5 ml dichloromethane for 30 minutes. After
extraction, the organic solvent p ortion was transferred
into a clean glass tube and evaporated to dryne ss at 60°
C under nitrogen gas (Reacti-Vap III™, Pierce, Rock-
ford, IL, USA).
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To each dried sample, 32 μl of 0.1 M sodium acetate
buffer (pH at 9.0) and 32 μl of dansyl c hloride s olution
(1 mg/ml in acetone) were added. After vortexing, the
samplewasheatedat60°C(Reacti-ThermIII™ Heating
Module, Pierce, Rockford, IL, USA) for 10 minutes to
form the EM and SI-EM dansyl derivatives (EM-Dansyl
and SI-EM-Dansyl, respectively). Calibration standards
and quality control samples were hydrolyzed, extracted,
and derivatized following the same procedure used for
unknown cell samples. After derivatization, all samples
were analyzed by capillary liquid chromatography (LC)-
tandem mass spectrometry (MS
2
).
Liquid chromatography-tandem mass spectrometry
LC-MS
2
analysis was performed using an Agilent 1200
series nanoflow LC system (Agilent Technologies, Palo
Alto, CA, USA) coupled to a TSQ™ Quantum Ultra tri-
ple quadrupole m ass spect rometer ( Thermo Electron,
San Jose, CA, USA). The LC separation was carried out
on a 150 mm long × 300 μm internal diameter column
packed with 4 μm Synergi Hydro-RP particles (Phenom-
enex, Torrance, CA, USA) and maintained at 40°C. A total
of 8 μl of each sample was injected onto the column. The
mobile phase, operating at a flow rate of 4 μl/minute, con-
sists of methanol as solvent A and 0.1% (v/v) formic ac id
in water a s solvent B. A lin ear gradient from 72 to 85%
solvent B in 75 minutes was employed for separation of
EMs and SI-EMs. The mass spectrometry conditions were:
source, ESI; ion polarity,positive; spray voltage, 3200 V;
sheath and auxiliary gas, nitrogen; sheath gas pressure, 10
arbitrary units; ion transfer capillary temperature, 270 °C;
scan type, selected reaction monitoring; collision gas,
argon; collision gas pressure, 1.5 mTorr; scan width, 0.7 u;
scan time, 0.30 s; Q1 peak width, 0.70 u full-width half-
maximum (FWHM ); Q3 peak width, 0.70 u FWHM. The
optimized selected reaction monitoring conditions for the
protonated molecules [MH]
+
of EM-Dansyl and SI-EM-
Dansyl were similar to those previously described [9,10].
Quantification of estrogen metabolites
Quantification of EMs was carried out using Xcalibur ™
Quan Browser (Thermo Electron) as previously described
[20,21]. Brie fly, calibration curves for the each EM w ere
constructed by plotting EM-Dansyl/SI-EM-Dansyl peak
area ratios obtained from calibration standards versus
amounts of the EM injected on the column and fitting
these data using linear regression with 1/X weighting. The
amounts of EMs in cells were then interpolated using this
linear function. Based on their similarity of structures and
retention times,
13
C
6
-E
2
was used as the internal standard
for E
2
;
13
C
6
-E
1
for E
1
;d
3
-E
3
for E
3
, 16-ketoE
2
,and16a-
OHE
1
;d
3
-16-epiE
3
for 16-epiE
3
and 17-epiE
3
;d
5
-2-
MeOE
2
for 2-MeOE
2
, 4-MeOE
2
, 2-MeOE
1
, 4-MeOE
1
,and
3-MeOE
1
;d
5
-2-OHE
2
for 2-OHE
2
, 2-OHE
1
, and 4-OHE
1
.
Results and discussion
The levels of endogenous estrogens and EMs were mea-
sured in the NCI-60 cell lines, which comprise breast (n =
5), CNS (n = 6), colon (n = 7), leukemia (n = 6), melanoma
(n = 9), non-small cell lung (n = 9), ovarian (n = 9), pros-
tate (n = 2), and renal (n = 8) cancers. This study focused
on measuring only the unconjugated, active forms o f the
EMs. Glucoronidated and sulfated forms of the EMs were
not included in the analysis. All NCI-60 human tumor cell
lines showed significant levels of E
1
,E
2
, 16-ketoE
2
,16a-
OHE
1
,E
3
,2-MeOHE
1
,2-MeOHE
2
,and2-OHE
1
.The
chromatograms showing the eight quantified endogenous
EMs for an ovarian (SK-OV-3) and colon cance r cell line
(HCC-2998) are shown in Figure 1. The peaks were gener-
ally well resolved and had good signal-to-noise ratios for
all cell lines analyzed. While undetectable in all the others,
2-OHE
1
was found i n the non-small cell lung cancer cell
line NCI-H460.
Within the same type of cancer, different tumor cell
lines had substantially different levels o f EMs (Table 1).
For example, SF-539 and SNB-75 cells produced greater
amounts of estrogens than the other CNS lines tested.
HCC-2998 colon ca ncer cells, RMPI-8226 leukemia
cells, SK-MEL-28, UACC-257, UACC-62, MALME-3M
melanoma cells, EKVX, NCI-H23, NCI-H226 non-small
cell lung (NSCL) cancer cells, OVCAR-4, OVCAR-5,
SK-OV-3 ovarian cancer cells, and CAKI-1 renal ca ncer
cells all produced greater amou nts of estrogens than the
other cell line s within thei r category. Furthermore,
estrogen levels in these tumor cell lines were substan-
tially elevated and even exceeded the levels typically
found in well characterized estrogen-dependent and ER-
positive tumor cells such as MCF-7 and T-47D.
Within each tumor cell line, E
2
was by far the most
abundant unconjugated estrogen followed by E
1
and 2-
OHE
2
(Table 1). For the five b reast cancer cell lines, E
2
represented 75 to 85% of the tot al amount of unconju-
gated EMs measured. For six of the ovarian cancer cell
lines, E
2
represented 77 to 87% of the total estrogen con-
tent, while this percentage was only 62% for OVCAR-3
cells. T-47D and MCF-7 cells are both estrogen-dependent
and ER-positive human breast cancer cells and have E
2
levels at 87 and 81 pg/10
6
cells, which accounted for 85%
and 82% of their total unconjugated estrogens, respe c-
tively. MDA-MB-231 is an estroge n-independent, ER-
negative, HER2-positive human breast cancer cell line and
still has E
2
levels of about 37 pg/10
6
cells, which accounts
for about 75% of its total unconjugated estrogen levels.
The cell lines with the highest E
2
levels are shown in
Figure 2a. Although estrogens are commonly associated
with breast cancer, none of these cel l lines were among
those that contained the highes t levels of E
2
. Co nsistent
with evidence linking estrogen level s with cancers of the
reproductive system in general, three ovarian canc er cell
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lines (OVCAR- 4, OVCAR-5, and SK-OV-3) were
amongst thos e with the highest E
2
levels. OVCAR-4 and
-5 are both ERa-negative, ERb-positive ovarian cell lines
who’sgrowthisinsensitivetoE
2
treatment [22]. While
SK-OV-3 cells do express ERa, their growth is also
insensitive to treatment with E
2
[22]. The leukemia cell
line RMPI-8226 possessed the highest levels of E
2
(753
pg/10
6
cells). In fact, its E
2
levels were more than 3.5-
fold higher than the colon cell line HCC-2998, which
contained the next highest level of E2 (209 pg/10
6
cells).
This result correlates with a prev ious study showi ng
that RMPI-8226 cells possess the highest ER levels com-
pared to other leukemia and myeloid cell lines tested
[23]. Previous studies have shown that the HL60 leuke-
mia cell line possesses ERs and its proliferation is sensi-
tive to E
2
treatment. When the cells are maintained in
a medium containing physiological concentrations
(10
-9
M, 10
-8
M, 10
-7
M) of E
2
, cell growth is stimulated;
however, pharmacological concentrations (10
-6
M) of E
2
inhibit their growth [24]. Adding tamoxifen inhibited
the stimu lating effect of the est rogens by binding to and
blocking the ER. The effect of estrogen was therefore
associated with the presence of ERs in the human leuke-
mic cell line HL60 and may be important in the prolif-
eration of other leukemic cell lines.
Cell lines with the lowest E
2
levels are shown in
Figure 2b. Four of these were colon cell lines (HCT-116,
HCT-15, KM12, and SW-620). Their E
2
levels ranged
from 1.31 to 12.25 p g/10
6
cells. To put into perspective
the range of E
2
values found in all the cell lines, SW-
620 colon cells contained almost 575- fold less E
2
than
RMPI-8226 cells. The finding that colon cell lines gener-
ally contain low levels of E
2
is consistent with a previous
study that found ERs are present in colorectal tumors
and human colonic cancer cell lines at very low levels
[25].
Figure 1 Chromatograms showing the eight quantified endogenous estrogen metabolites for (a) the ovarian cancer cell line SK-OV-3
and (b) the colon cancer cell line HCC-2998. 16-ketoE
2
, 16-ketoestradiol; 16a-OHE
1
,16a-hydroxyestrone; 2-MeOE
1
, 2-methoxyestrone; 2-
MeOE
2
, 2-methoxyestradiol; 2-OHE
2
, 2-hydroxyestradiol; E
1
, estrone; E
2
, estradiol; E
3
, estriol.
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Table 1 Levels of unconjugated endogenous estrogens (picograms) found in NCI-60 cell lines
Cancer Cell Line E
3
16aOHE
1
16-epiE
3
2MeOE
1
2MeOE
2
E
1
E
2
2OHE
1
2OHE
2
Total
Breast T-47D 0.80 0.03 0.33 2.70 0.54 3.65 87.37 NF 7.30 102.72
(0.07) (0.00) (0.04) (0.34) (0.10) (0.06) (2.69) NF (1.22) (3.18)
Breast Hs-578T 0.43 0.03 0.22 0.62 0.94 8.13 74.11 NF 5.86 90.35
(0.06) (0.00) (0.05) (0.07) (0.17) (0.98) (9.94) NF (0.83) (11.95)
Breast BT-549 0.58 0.02 0.06 0.65 0.45 8.45 58.97 NF 6.03 75.22
(0.10) (0.00) (0.03) (0.10) (0.02) (1.20) (11.51) NF (1.26) (14.15)
Breast MDA-MB-231 0.90 0.02 0.57 1.78 0.35 6.30 36.73 NF 2.55 49.21
(0.07) (0.00) (0.05) (0.24) (0.05) (0.37) (2.41) NF (0.33) (2.71)
Breast MCF-7 0.40 0.04 0.28 2.12 5.38 7.70 80.75 NF 1.76 98.42
(0.03) (0.00) (0.06) (0.23) (0.47) (0.71) (14.44) NF (0.06) (13.04)
CNS SF-295 0.24 0.04 0.12 0.93 0.81 6.79 41.51 NF 7.01 57.44
(0.03) (0.00) (0.01) (0.07) (0.12) (0.93) (2.57) NF (1.03) (3.25)
CNS SF-539 0.82 0.11 NF 1.73 1.22 26.24 175.50 NF 27.11 232.78
(0.10) (0.02) NF (0.13) (0.08) (2.01) (13.14) NF (2.78) (14.43)
CNS SF-268 0.35 0.04 0.31 0.43 0.26 2.69 24.68 NF 7.67 36.42
(0.01) (0.00) (0.03) (0.05) (0.01) (0.10) (1.33) NF (0.96) (2.21)
CNS U251 0.35 0.29 0.21 1.19 0.31 4.54 41.06 NF 12.61 60.55
(0.04) (0.03) (0.02) (0.11) (0.04) (0.21) (4.27) NF (1.22) (5.01)
CNS SNB-19 0.31 0.02 0.08 1.27 0.55 5.09 29.34 NF 3.63 40.29
(0.05) (0.00) (0.01) (0.14) (0.09) (0.64) (2.31) NF (0.25) (3.45)
CNS SNB-75 0.30 0.04 0.10 1.46 0.56 13.15 94.94 NF 2.23 112.79
(0.04) (0.00) (0.01) (0.23) (0.03) (0.57) (1.97) NF (0.24) (1.80)
Colon SW-620 0.75 0.08 0.28 1.70 1.39 0.44 1.31 NF 2.68 8.63
(0.11) (0.01) (0.05) (0.14) (0.23) (0.05) (0.09) NF (0.31) (0.61)
Colon HCT-116 0.12 0.02 0.27 1.40 0.80 0.45 2.17 NF 0.33 5.58
(0.02) (0.00) (0.04) (0.06) (0.08) (0.04) (0.13) NF (0.03) (0.11)
Colon COLO-205 0.33 0.07 0.19 0.57 0.76 1.12 50.44 NF 15.20 68.69
(0.02) (0.01) (0.02) (0.07) (0.07) (0.08) (4.57) NF (0.77) (5.22)
Colon KM12 0.55 0.04 0.31 1.09 0.44 0.14 1.77 NF 0.73 5.06
(0.06) (0.00) (0.04) (0.10) (0.09) (0.01) (0.12) NF (0.13) (0.49)
Colon HT29 0.51 0.03 0.51 1.12 0.28 1.90 30.71 NF 0.51 35.56
(0.01) (0.00) (0.06) (0.12) (0.03) (0.10) (1.12) NF (0.05) (0.90)
Colon HCT-15 28.10 0.40 1.08 0.87 0.29 0.88 12.25 NF 3.75 47.62
(1.54) (0.06) (0.04) (0.09) (0.03) (0.10) (1.22) NF (0.36) (3.05)
Colon HCC-2998 0.88 0.10 0.45 3.10 0.92 17.10 209.34 NF 4.29 236.18
(0.05) (0.01) (0.07) (0.05) (0.17) (0.08) (13.95) NF (0.14) (14.05)
Leukemia HL-60 3.89 0.03 0.29 1.58 3.15 18.78 36.61 NF 16.50 80.83
(0.02) (0.00) (0.01) (0.11) (0.10) (2.39) (2.70) NF (2.81) (2.39)
Leukemia CCRF-CEM 0.41 0.04 0.06 0.66 1.33 1.18 3.48 NF 1.16 8.31
(0.07) (0.00) (0.02) (0.04) (0.10) (0.12) (0.50) NF (0.10) (0.91)
Leukemia K562 0.42 0.02 0.21 0.57 2.23 0.56 13.88 NF 1.16 33.14
(0.07) (0.00) (0.04) (0.06) (0.07) (0.06) (1.75) NF (0.11) (1.24)
Leukemia MOLT-4 0.30 0.08 0.13 0.55 0.77 2.04 7.77 NF 0.75 12.37
(0.04) (0.01) (0.01) (0.04) (0.07) (0.37) (1.14) NF (0.09) (1.53)
Leukemia RMPI-8226 0.93 0.11 0.59 2.04 3.60 182.06 753.45 NF 2.93 945.71
(0.07) (0.02) (0.11) (0.30) (0.16) (5.19) (48.20) NF (0.22) (52.18)
Leukemia SR 4.67 0.06 0.80 3.24 1.45 6.67 26.39 NF 1.22 44.51
(0.53) (0.01) (0.15) (0.51) (0.22) (0.47) (1.38) NF (0.19) (2.00)
Melanoma MDA-MB-435 0.37 0.22 0.09 0.61 3.65 1.77 14.24 NF 2.82 23.77
(0.03) (0.04) (0.02) (0.11) (0.76) (0.18) (0.91) NF (0.32) (1.05)
Melanoma SK-MEL-28 0.82 0.05 0.27 3.83 5.41 21.50 146.59 NF 27.77 206.24
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Table 1 Levels of unconjugated endogenous estrogens (picograms) found in NCI-60 cell lines (Continued)
(0.03) (0.01) (0.02) (0.33) (0.99) (2.12) (15.34) NF (2.17) (16.78)
Melanoma UACC-257 1.20 0.36 0.37 0.74 7.00 36.36 130.70 NF 2.50 179.23
(0.23) (0.07) (0.05) (0.10) (0.78) (2.41) (4.37) NF (0.22) (6.37)
Melanoma LOX IMVI 0.93 0.07 0.44 1.33 1.01 0.35 12.10 NF 8.26 24.49
(0.11) (0.01) (0.07) (0.06) (0.07) (0.02) (0.55) NF (0.64) (0.50)
Melanoma UACC-62 0.20 0.30 0.06 1.41 1.47 17.49 98.71 NF 1.62 121.27
(0.03) (0.04) (0.01) (0.06) (0.25) (0.21) (4.61) NF (0.30) (4.82)
Melanoma SK-MEL-2 1.56 1.60 0.38 2.21 0.62 6.66 50.93 NF 2.90 66.86
(0.15) (0.08) (0.07) (0.05) (0.08) (0.40) (5.84) NF (0.33) (6.40)
Melanoma SK-MEL-5 0.38 0.38 0.13 14.84 0.52 11.64 72.02 NF 3.83 103.75
(0.03) (0.05) (0.02) (0.47) (0.08) (0.31) (4.06) NF (0.50) (5.25)
Melanoma MALME-3M 0.36 0.06 0.32 3.88 0.45 1.26 94.27 NF 1.30 101.90
(0.03) (0.01) (0.05) (0.26) (0.06) (0.05) (0.88) NF (0.13) (1.13)
Melanoma M14 1.33 0.05 0.18 1.24 0.69 1.21 13.95 NF 1.90 20.55
(0.06) (0.00) (0.02) (0.11) (0.11) (0.09) (0.50) NF (0.28) (0.91)
NSCL A549 0.56 0.04 0.54 1.04 1.18 2.95 17.01 NF 2.19 25.51
(0.06) (0.00) (0.05) (0.05) (0.07) (0.27) (0.86) NF (0.24) (1.18)
NSCL EKVX 1.70 0.31 0.92 1.10 7.16 15.16 125.95 NF 4.53 156.83
(0.06) (0.05) (0.01) (0.12) (0.72) (1.13) (13.92) NF (0.47) (12.21)
NSCL HOP-62 1.48 0.08 0.78 2.37 4.70 6.19 25.45 NF 13.29 54.33
(0.24) (0.01) (0.14) (0.40) (0.44) (1.18) (1.85) NF (1.56) (5.04)
NSCL NCI-H23 1.08 0.29 0.39 6.10 0.92 8.03 90.47 NF 15.79 123.07
(0.05) (0.04) (0.08) (0.43) (0.05) (0.64) (2.81) NF (2.87) (3.46)
NSCL NCI-H460 0.18 0.05 0.15 1.36 1.15 4.03 59.76 27.89 2.21 96.78
(0.01) (0.01) (0.00) (0.23) (0.04) (0.06) (3.33) (0.32) (0.12) (3.69)
NSCL NCI-H226 0.29 0.07 0.13 0.50 0.61 25.40 181.13 NF 21.63 229.75
(0.02) (0.01) (0.01) (0.02) (0.07) (1.05) (7.48) NF (0.69) (8.87)
NSCL HOP-92 0.33 0.03 0.43 0.55 0.24 4.87 34.58 NF 2.20 43.24
(0.01) (0.00) (0.08) (0.03) (0.04) (0.28) (1.51) NF (0.24) (1.43)
NSCL NCI-H522 0.18 0.03 0.06 1.38 0.31 4.25 32.36 NF 1.55 40.12
(0.02) (0.00) (0.01) (0.08) (0.04) (0.36) (2.39) NF (0.31) (2.58)
NSCL NCI-H322M 0.19 0.04 0.13 0.80 0.45 0.72 7.01 NF 1.29 10.62
(0.02) (0.00) (0.01) (0.06) (0.04) (0.08) (0.27) NF (0.12) (0.13)
Ovarian OVCAR-3 0.50 0.07 0.34 0.97 3.70 5.37 26.60 NF 5.47 43.02
(0.05) (0.01) (0.04) (0.09) (0.54) (0.73) (1.63) NF (0.81) (3.21)
Ovarian OVCAR-5 0.16 0.03 0.13 1.26 0.33 12.85 107.47 NF 1.57 123.79
(0.00) (0.00) (0.02) (0.17) (0.05) (0.33) (5.55) NF (0.14) (5.89)
Ovarian IGR-OV1 0.15 0.04 0.11 0.73 2.54 4.00 29.72 NF 0.66 37.94
(0.01) (0.00) (0.00) (0.01) (0.36) (0.15) (0.70) NF (0.06) (1.10)
Ovarian NCI/ADR-RES 1.33 0.15 0.44 2.54 2.92 10.46 75.10 NF 4.61 97.56
(0.08) (0.02) (0.04) (0.28) (0.14) (0.31) (0.47) NF (0.28) (0.06)
Ovarian SK-OV-3 1.78 0.13 0.39 0.73 0.55 12.26 132.33 NF 4.18 152.36
(0.23) (0.02) (0.03) (0.09) (0.10) (0.25) (9.43) NF (0.63) (9.94)
Ovarian OVCAR-8 0.44 0.13 0.15 2.10 0.35 5.12 38.83 NF 3.61 50.73
(0.02) (0.03) (0.03) (0.17) (0.06) (0.22) (1.01) NF (0.58) (1.56)
Ovarian OVCAR-4 0.43 0.06 0.31 3.90 0.43 19.79 139.32 NF 4.69 168.93
(0.02) (0.01) (0.02) (0.06) (0.07) (1.06) (2.84) NF (0.46) (2.82)
Prostate PC-3 0.15 0.03 0.20 1.05 2.33 7.63 41.94 NF 0.63 53.96
(0.02) (0.00) (0.03) (0.05) (0.14) (0.56) (2.30) NF (0.07) (2.90)
Prostate DU145 0.55 0.04 0.42 2.22 0.71 4.94 27.78 NF 2.55 39.22
(0.05) (0.00) (0.05) (0.16) (0.10) (0.29) (1.02) NF (0.23) (1.06)
Renal UO-31 0.30 0.13 0.33 1.36 1.66 5.05 22.94 NF 2.45 34.22
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As with E
2
, the leukemic cell line RPMI-8226 con-
tained the highest levels of E
1
of the NCI-60 cell lines
(Figure 3a). The amount measured in this cell line was
more than five-fold higher than that f ound in the cell
line (melanoma UACC-257) cont aining the ne xt highest
levels of E
1
. Again, none of the five brea st ca ncer cell
lines tested was amongst the top ten E
1
-containing cells.
The ovarian cancer cell line OVCAR-4 (19.79 pg/10
6
cells) was sixth on the list of cell lines containing the
most E
1
. Two non-sma ll cell lung carci noma cell l ines
(EKVX and NCI-H226) were amongst the top ten in
both E
1
and E
2
levels. This result is interesting consider-
ing that females that never smoked are far more likely
to develop lung carcinoma than never-smoked males,
suggesting a gende r differenc e exists in the clinical and
pathophysiology of lung cancer [21]. Recent studies
showing aromatase-dependent synthesis of estrogens in
situ in male and female lun g cancers sugges t that estro-
gens may contribute to the manifestation and progres-
sion of lung carcinoma [26-28].
Cell lines with the lowest E
1
levels are shown in
Figure 3b. COLO-205 was included along with the four
colon cancer cell lines that were amongst the ten con-
taining the lowest E
1
levels (HCT-116, HCT-15, KM12,
and SW-620). Their E
1
levels ranged from 0.14 to 1.12
pg/10
6
cells. The levels of E
1
found in KM12 colo n can-
cer cells was approximately 1,300-fold less than that
found in RPMI-8226 leukemia cells. T wo leukemia cell
lines, CC RF-CE M and K562, whic h were amongst those
possessing the lowest E
2
levels, also contained low E
1
levels.
To identify general trends within the various cell lines
tested, the means and standard deviations (SDs) of the
total EM levels found in the cell types analyzed. As
shown in Table 2, the leukemia cel l lines had the hi ghest
overall mean total EM values ( 187.5 pg/10
6
cells). This
value was almost twice as h igh as the cell types with the
next highest total EM levels. Ovarian (96.33 pg/10
6
cells)
andbreast(83.18pg/10
6
cells) cancer cells, which are
commonly associated with estrogens, c ontained the sec-
ond and sixth highest levels of total EMs. In fact, five of
the cancer cell lines (ovarian, melanoma, CNS, NSCL,
and breast) had t otal EM levels between 80 and 100 pg/
10
6
cells. A noticeable feature of Table 2 are the very
highSDs,butinparticularthatfortheleukemiacell
lines. T o further explore what contributed to this high
SD, the highest and lowest total EM levels measure d for
the individual cell lines in the different cell types w ere
eliminated and the mean and SD values were recalcu-
lated. The mea n E M levels for the leukemia cell lines
after eliminating the cell lines with the highest and lowest
concentrations was 42.71 pg/10
6
cells with a SD of 28.68;
dropping their overall rank from first to sixth. The cell
types having the three highest EM levels after eliminating
the cell lines with the highest and lowest concentrations
were (in order) ovarian (93.29 pg/10
6
cells, SD = 46.84),
melanoma (88.75 pg/10
6
cells, SD = 55.53), and breast
(88.00 pg/10
6
cells, SD = 11.78).
The finding that the melanoma cell lines contained
relatively high levels of endogenous estro gen and EMs is
interesting. Two melanoma cell lines in particular, SK-
MEL-28 and UACC-257, were among those containing
the highest levels of E
1
(21.50 a nd 36.36 pg/10
6
cells,
respectively) and E
2
(146.59 and 130.70 pg/10
6
cells,
respectively). Only four other cell lines, SF-539 (CNS),
NCI-H226 (NSCL), RMPI-8226 (leukemia) and HCC-
2998 (colon), contained h igher levels of total estrogens.
High-affinity E
2
receptors have been reported for primary
Table 1 Levels of unconjugated endogenous estrogens (picograms) found in NCI-60 cell lines (Continued)
(0.03 (0.02 (0.02 (0.26 (0.26 (0.60 (1.18 NF (0.15 (0.92
Renal 786-0 0.15 0.02 0.11 0.58 0.16 2.11 23.40 NF 1.54 28.07
(0.02) (0.00) (0.01) (0.05) (0.02) (0.16) (0.14) NF (0.19) (0.17)
Renal SN12C 0.14 0.05 0.27 6.47 1.92 4.02 25.50 NF 0.89 39.25
(0.01) (0.01) (0.02) (0.29) (0.17) (0.07) (0.97) NF (0.05) (0.96)
Renal CAKI-1 0.54 0.16 0.13 4.76 0.49 7.57 81.60 NF 6.52 101.77
(0.05) (0.03) (0.03) (0.42) (0.07) (0.61) (1.42) NF (0.79) (2.29)
Renal RXF-393 0.27 0.03 0.09 0.47 0.44 0.34 14.48 NF 1.41 17.54
(0.04) (0.00) (0.02) (0.09) (0.04) (0.04) (0.39) NF (0.13) (0.38)
Renal TK-10 0.65 0.25 0.42 2.54 0.58 7.87 37.29 NF 6.25 55.86
(0.06) (0.04) (0.02) (0.24) (0.06) (0.22) (3.96) NF (0.60) (4.27)
Renal A498 0.53 0.02 0.16 0.29 0.50 3.05 37.53 NF 2.58 44.65
(0.04) (0.00) (0.00) (0.03) (0.07) (0.29) (1.09) NF (0.37) (1.45)
Renal ACHN 0.43 0.08 0.19 1.25 0.39 6.17 39.27 NF 3.40 51.17
(0.06) (0.01) (0.02) (0.16) (0.06) (0.74) (1.96) NF (0.28) (2.51)
Data are expressed as mean (standard deviation) of three replicated analyses of 10
6
cells. E
3
, estriol; 16aOHE
1
,16a-hydroxyestrone; 16-epiE
3
, 16-epiestriol;
2MeOE
1
, 2-methoxyestrone; 2MeOE
2
, 2-methoxyestradiol; E
1
, estrone; E
2
,17b-estradiol; 2OHE
1
, 2-hydroxyestrone; 2OHE
2
, 2-hydroxyestradiol; CNS, central nervous
system; NF, not found; NSCLC, non-small cell lung carcinoma.
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human melanomas [29] and patients expressing these
receptors seem to have a better prognosis, suggesting E
2
may inhibit the growth of t hese m elanoma t umors [30].
While previous studies have identified the cl assical ER in
only a small percentage of human melanomas via immu-
nohistochemistry [30], the low affinity type II ER has
been characterized in a variety of human melanomas
[31]. This receptor has the same affinity as the classical
receptor and also binds tamoxifen. Treating SK-Mel 23
melanoma cells with E
2
has been shown to inhibit their
growth, while pre-treating the cells with tamoxifen (an
anti-estrogen) blocks the effects of E
2
[32]. The preva-
lence of E
2
within the melanoma cell lines may prevent
uncontrolled cell proliferation by acting back upon the
cells and binding to the type II ER.
In general, unconjugated E
3
,16aOHE
1
, and 16-epiE
3
were less abundant except for in HCT-15 colon tumor
cells, which had a greater amount of E
3
than E
2
.The
catechol estrogen 2-OHE
2
was the only catechol estro-
gen detected in the tumor cell lines, except for the
NSCL cancer cell line NCI-H460, which also contains a
relatively high level of 2-OHE
1
(Table 1). N o unconju-
gated 4-hydroxy catechol estrogens were detected in any
of the NCI-60 tumor cells. This result is likely due to
the fa ct that 4-hydroxy catechol estrogens are quickly
transformed into other reactive species such as their
quinones and semi-qui nones, which could damage DNA
and l ead to tumor initiation [2,33,34]. In contrast, 2-
hydroxy-catechol estrogens largelyformstableconju-
gates such as 2-MeOHE
1
and 2 -MeOHE
2
. S ignificant
Figure 2 Cell lines containing the highest and lowest 17b-estradiol (E
2
) levels within the NCI-60 panel. (a) Cell lines with the highest E
2
levels; (b) cell lines with the lowest E
2
levels.
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Figure 3 Cell lines containing the highest and lowest estrone (E
1
) levels within the NCI-60 panel. (a) Cell lines with the highest E
1
levels;
(b) cell lines with the lowest E
1
levels.
Table 2 Means and standard deviations of total estrogen levels measured in cell types within the NCI-60 cell line
panel
Mean SD Mean (-high/low)* SD (-high/low)*
Breast 83.18 21.70 88.00 11.78
CNS 90.05 75.07 67.77 31.31
Colon 58.19 82.12 33.21 26.64
Leukemia 187.48 372.4 42.71 28.67
Melanoma 94.23 67.69 88.75 55.49
NSCL 86.69 71.98 77.13 49.25
Ovarian 96.33 54.00 93.49 46.84
Prostate 46.59 10.42 NA NA
Renal 46.57 25.49 42.20 10.44
Mean and standard deviation (SD) are presented for all values within specific cell types and for values after eliminating cell lines with the highest and lowest
total estrogen levels. CNS, central nervous system; NSCLC, non-small cell lung carcinoma. *Mean and standard deviation calculated after removing cell lines
containing highest and lowest total measured estrogen levels. NA, only two prostate cell lines were included in NCI-60 cell lines analyzed.
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levels of b oth of these EMs were found in all NCI-60
cell lines tested in this study.
This study measured the unconjugated levels o f endo-
genous estrogens and EMs in the NCI-60 cell panel. From
our previous experience, if we had measured the conju-
gated levels by adding a sulfatase/glucoronidase enzyme to
deconjugat e sulfat ed and glucoronida ted molecules prior
to LC-MS
2
analysis, we would expect to see a large
increase in the levels of every metabolite that was routinely
detected. We would also expect that 16-epiE
3
,17-epiE
3
,4-
MeOE
1
, 3-MeOE
1
, 4-MeOE
2
,2-OHE
1
, and 4-OHE
1
would
also be detectable. Our studies analyzing serum have
shown that endogenous estrogens and EMs exist primarily
(that is, 90%) in the conjugated forms in the circulation
[20]. While this disparity between conjugated and uncon-
jugated forms of these steroid hormones may not be as
large i n cells, we predict that a large amou nt of endogen-
ous estrogens and EMs exist within cells in their conju-
gated forms. It is interesting to note that when we analyze
serum, only E
1
,E
2
,E
3
,2-MeOHE
1
and 2-MeOHE
2
are
detected in their uncon jugated forms [20]. In th e NCI-60
cell lines we were able to also routinely detect 16-aOHE
1
,
16-epiE
3
,and2-OHE
2
. Unfortunately, it is difficult to
directly compare the estrog en and EM levels as the cell
line concentrations are recorded in pg/10
6
cells, while
those in serum are measured as pg/ml. The fact that more
compounds are detected in their unconjugated forms in
the cell lines, however, sugge sts that, in general, the con-
centrations of estrogens and EMs are higher in cells than
in the circulation.
To determine if ER status correlates with the levels of
estrogensandEMsidentifiedinthevariouscelllines,
we compare our data to that published by Holbeck et
al. [35], who measured the mRNA levels of 48 nuclear
receptors in 51 of the NCI-60 cell lines. The mRNA
levels of ERa for nine of the cell lines that were found
to contain the highest E
2
levels were measured in this
study. Of these, detecta ble ER a levels were found f or
SKOV-3, OVCAR-4, UACC-257, SK-MEL-28, and SF-
539 cell lines. No ERa mRNA was detected f or H CC-
2998, NCI-H226, EKVX, and OVCAR-5 cell lines. The
cell lines with the highest levels of ERa mRNA were
SK-OV-3, and two breast cancer cell lines, MCF-7 and
T-47D.Ofthese,onlySK-OV-3wasamongthecell
lines containing the highest amounts of E
2
.Wealso
compared the ERa and E
2
levels wit hin the nine mela-
noma cell lines analyzed in both studies. In this case,
the six melanoma cell lines with detectable levels of
ERa mRNA (SK-MEL-28, UACC-257, UACC-62, SK-
MEL-2, SK-MEL-5, and MALME-3M) contained the
highest amounts of E
2
within that group. The melanoma
cell lines containing the lowest E
2
concentrations (M14,
LOX IMVI, and MDA-MB-435) did not show detectable
levels of ERa. Overall, there is no obvious correlation
between ERa and E
2
levels; however, only about 25% of
the cell lines had detectable levels of ERa whereas E
2
could be measured in every one.
Conclusions
This study utilized an LC-MS
2
approach with the cap-
ability of measuring up t o 15 different EMs to measure
the l evels o f e ndogenous e strogens within the NCI-60
cell lines. Eight of the measured endogenous estrogens
were consistently observed in all of the NCI-60 cell
lines, providing an unprecedented view of these metabo-
lites within these cancer cell lines. What is particularly
striking is that the levels of EMs in well-known estro-
gen-dependent cancers such as o var ian and breast were
not substantially grea ter than those found in other types
of cancer cell lines. In fact, none of the breast cancer
cell lines were amongst the top ten that contained the
highest levels of E
1
or E
2
. Cell lines not generally asso-
ciated with estrogens, such as leukemia, colon, CNS,
and NSCL, were found to h ave a ppreciable levels of
these m etabolites. The broad presence of EMs within
the NCI-60 cell lines suggests that many cancers outside
of the reproductive system may respond to treatments
with anti-estrogens such as tamoxifen, toremifene, and
fulvestrant. Considering technologies for measuring
estrogen levels in biologicalsamplesisconsiderably
improved, it is now worth the effort to test various
tumors for the levels of these metabolites.
Abbreviations
16-epiE
3
: 16-epiestriol; 16-ketoE
2
: 16-ketoestradiol; 16α-OHE
1
:16α-
hydroxyestrone; 17-epiE
3
: 17-epiestriol; 2-MeOE
1
: 2-methoxyestrone; 2-MeOE
2
:
2-methoxyestradiol; 2-OHE
1
: 2-hydroxyestrone; 2-OHE
2
: 2-hydroxyestradiol; 3-
MeOE
1
: 2-hydroxyestrone-3-methyl ether; 4-MeOE
1
: 4-methoxyestrone; 4-
MeOE
2
: 4-methoxyestradiol; 4-OHE
1
: 4-hydroxyestrone; CNS: central nervous
system; E
1
: estrone; E
2
: estradiol; E
3
: estriol; EM: estrogen metabolite; ER:
estrogen receptor; LC: liquid chromatography; MS
2
: tandem mass
spectrometry; NSCL: non-small cell lung; SD: standard deviation; SI-EM: stable
isotope-labeled estrogen. Bre: breast; Col: colon; Leu: leukemia; Mel:
melanoma; Ovc: ovarian; Pros: prostate; Ren: renal.
Acknowledgements
This project has been funded in whole or in part with federal funds from
the National Cancer Institute, National Institutes of Health, under contract
N01-CO-12400. The content of this publication does not nece ssarily reflect
the views or policies of the Department of Health and Human Services, nor
does mention of trade names, commercial products, or organizations imply
endorsement by the United States Government.
Authors’ contributions
TV participated in the conception, design, and implementation of the
experiments, the analysis and interpretation of the data and drafting of the
manuscript. XX participated in the conception, design, and implementation
of the experiments, conducted the sample preparation and data acquisition
and participated in the analysis and interpretation of the data and drafting
of the manuscript. All authors have read and approved the final version of
the manuscript for publication.
Competing interests
The authors declare that they have no competing interest s.
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Received: 11 January 2012 Revised: 4 April 2012
Accepted: 30 April 2012 Published: 30 April 2012
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doi: 10.1186/gm330
Cite this article as: Xu and Veenstra: Concentration of endogenous
estrogens and estrogen metabolites in the NCI-60 human tumor cell
lines. Genome Medicine 2012 4:31.
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