A new steroidal 5,7-diene derivative, 3β-hydroxyandrosta-5,7-diene-17β-carboxylic acid, shows potent anti-proliferative activity

Article abstract of DOI:10.1016/j.steroids.2009.12.004

The new steroidal 5,7-diene, 3β-hydroxyandrosta-5,7-diene-17β-carboxylic acid (17-COOH-7DA), was synthesized from 21-acetoxypregnenolone, with the oxidative cleavage of the side chain being dependent on the presence of oxygen. In human epidermal (HaCaT) keratinocytes, 17-COOH-7DA inhibited proliferation in a dose-dependent manner, starting at a dose as low as 10^-11 M. This inhibition was accompanied by decreased expression of epidermal growth factor receptor, bcl2 and cyclin E2 mRNAs and by increased expression of involucrin mRNA. Inhibition of proliferation was associated with slowing of the cell cycle in G1/G0 phases but not with cell death. 17-COOH-7DA was significantly more potent than pregnenolone, 17-COOH-pregnenolone, 17-COOCH₃-7DA and calcitriol. 17-COOH-7DA also inhibited proliferation of normal human epidermal melanocytes and human and hamster melanoma lines, however, with lower potency than for keratinocytes. In normal human dermal fibroblasts 17-COOH-7DA stimulated proliferation in serum-free media but inhibited it in the presence of 5% serum. 17-COOH-7DA inhibited cell colony formation of human and hamster melanoma cells, and induced monocyte-like differentiation of human HL60 leukemia cells. Thus, the new steroidal 5,7-diene, 17-COOH-7DA, can serve as an inhibitor of proliferation of normal keratinocytes and normal and malignant melanocytes, as a condition-dependent regulator of fibroblast proliferation and a stimulator of leukemia cell differentiation.

Full text of DOI:10.1016/j.steroids.2009.12.004

Steroids 75 (2010) 230–239
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
A new steroidal 5,7-diene derivative,
3␤-hydroxyandrosta-5,7-diene-17␤-carboxylic acid, shows potent
anti-proliferative activity
Tae-Kang Kima, Jianjun Chenb, Wei Lib, Jordan Zjawionyc,d, Duane Millerb,
Zorica Janjetovica, Robert C. Tuckeye, Andrzej Slominskia,∗
a Department of Pathology and Laboratory Medicine, Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA
c Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA
d Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA
e School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2009
Received in revised form 7 December 2009
Accepted 8 December 2009
Available online 16 December 2009
The new steroidal 5,7-diene, 3␤-hydroxyandrosta-5,7-diene-17␤-carboxylic acid (17-COOH-7DA), was
synthesized from 21-acetoxypregnenolone, with the oxidative cleavage of the side chain being dependent
on the presence of oxygen. In human epidermal (HaCaT) keratinocytes, 17-COOH-7DA inhibited prolifer-
ation in a dose-dependent manner, starting at a dose as low as 10−11 M. This inhibition was accompanied
by decreased expression of epidermal growth factor receptor, bcl2 and cyclin E2 mRNAs and by increased
expression of involucrin mRNA. Inhibition of proliferation was associated with slowing of the cell cycle in
G1/G0 phases but not with cell death. 17-COOH-7DA was significantly more potent than pregnenolone,
17-COOH-pregnenolone, 17-COOCH3-7DA and calcitriol. 17-COOH-7DA also inhibited proliferation of
normal human epidermal melanocytes and human and hamster melanoma lines, however, with lower
potency than for keratinocytes. In normal human dermal fibroblasts 17-COOH-7DA stimulated prolif-
eration in serum-free media but inhibited it in the presence of 5% serum. 17-COOH-7DA inhibited cell
colony formation of human and hamster melanoma cells, and induced monocyte-like differentiation of
human HL60 leukemia cells. Thus, the new steroidal 5,7-diene, 17-COOH-7DA, can serve as an inhibitor of
proliferation of normal keratinocytes and normal and malignant melanocytes, as a condition-dependent
regulator of fibroblast proliferation and a stimulator of leukemia cell differentiation.
Keywords:
3␤-Hydroxyandrosta-5,7-diene-17␤-
carboxylic
acid
5,7-Dienes
Skin
Keratinocytes
Melanoma
Melanocytes
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
tives from 7DHC has also been described in normal adrenal glands
incubated ex vivo [6].
Pregna-5,7-dienes and their hydroxylated derivatives can be
formed in vivo when there is a deficiency in 7-dehydrocholesterol
(7DHC) ꢀ-reductase, as seen in the Smith–Lemli–Opitz syndrome
(SLOS) [1–4]. 7DHC ꢀ-reductase is the enzyme responsible for the
conversion of 7DHC to cholesterol. The lack of this key enzyme leads
to the accumulation of 5,7-dienes and 5,7,9(11)-trienes which is
believed to result in multiple metabolic defects. In addition, recent
data have shown that steroidal 5,7-dienes are generated through
the P450scc-mediated cleavage of the side chain of 7DHC [4,5].
Production of pregna-5,7-dienes and their hydroxylated deriva-
In the skin 7DHC is converted to vitamin D3 following
the absorption of photons of ultraviolet light B (wavelength
290–320 nm) [7–9]. Vitamin D3 undergoes 25-hydroxylation
in the liver then 1␣-hydroxylation in the kidney producing
1␣,25-dihydroxyvitamin D3 (calcitriol; 1,25(OH)2D3)), the active
regulator of calcium metabolism. 1,25(OH)2D3 can also be pro-
duced locally in the skin [7,8]. 1,25(OH)2D3 and its precursors have
immune and neuroendocrine activities, and tumorostatic and anti-
carcinogenic properties, affecting proliferation, differentiation and
apoptosis in cells of different lineages, and protecting DNA against
oxidative damage [7,10–13].
Benign or malignant hyperproliferative skin disorders such as
psoriasis, solar keratosis, basal and squamous cell carcinoma as well
as melanoma represent a significant clinical problem. Melanoma
especially, has the fastest growing incidence of any cancer in the
United States. While early stage melanoma can be cured by surgery,
advanced melanoma is notoriously resistant to all existing treat-
∗
Corresponding author at: Department of Pathology and Laboratory Medicine,
Center for Cancer Research, University of Tennessee Health Science Center, 930
Madison Avenue, RM525, Memphis, TN 38163, USA. Tel.: +1 901 448 3741;
fax: +1 901 448 6979.
E-mail address: aslominski@uthsc.edu (A. Slominski).
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.12.004

T.-K. Kim et al. / Steroids 75 (2010) 230–239
231
ments. Dacarbazine (DTIC) is the only FDA approved drug to treat
metastatic melanoma in terms of chemotherapy but the complete
remission rate is less than 5% in patients [14,15]. Tremendous
efforts have been made in recent years to search for more effec-
tive agents for the treatment of advanced melanoma [16]. However,
none of them has clearly demonstrated better efficacy than DTIC.
With the fast rising melanoma occurrences in the United States and
the world, searching for more effective agents is becoming a press-
ing task for drug makers. Furthermore, development of new drugs
that could be used in treatment of psoriasis, solar keratosis or squa-
mous or basal cell carcinomas represents a challenge in clinical and
experimental dermatology.
Here we report the structure, synthesis and biological testing
of 3␤-hydroxyandrosta-5,7-diene-17␤-carboxylic acid (17-COOH-
7DA), a new steroidal 5,7-diene resulting from our search for
new therapeutic agents for melanoma and hyperproliferative skin
disorders. This is the first report of the synthesis and biological
evaluation of this compound.
were labeled along with the purity determination (Supplementary
materials).
2.2. NMR analysis
All NMR measurements were performed using an inverse triple-
resonance probe on a Varian Unity Inova 500-MHz spectrometer
(Varian, Inc., Palo Alto, CA, USA). Samples were dissolved in DMSO-
d6. Temperature was regulated at 22 ◦C and was controlled to an
accuracy of 0.1 ◦C. Chemical shifts were referenced to residual
solvent peaks for DMSO-d6 (2.50 ppm for proton and 40.0 ppm for
carbon). Standard 2D NMR experiments (1H–1H TOCSY, 1H–13
C
HSQC, 1H–1H COSY or 1H–13C HMBC, Supplementary materials)
were acquired to fully elucidate the structures of the new com-
pounds. All data were processed using Varian’s VNMR 6.1C
software, with zero-filling in the direct dimension and linear pre-
diction in the indirect dimension. Full structural assignments were
performed described before [4,17,18].
2. Experimental
2.2.1. Pregn-5-en-20-one-3ˇ,21-diyl diacetate
(3ˇ,21-dihydroxypregn-5-en-20-one diacetate) (2)
2.1. Chemical synthesis
The acetylation of compound 1 was carried out following a
known procedure [19]. A mixture of steroid 1 (3.74 g, 10 mmol) in
a small amount of acetic anhydride (20 ml) and catalytic amount of
pTSA·H2O (38 mg, 0.2 mol) was placed in an open glass tube. This
mixture was irradiated in the microwave-assisted synthesizer for
10 min. After completion of the reaction, the mixture was brought
to room temperature, mixed with ice-cold water and stirred until
solid product precipitated out. The solid was collected by filtration
then washed with saturated sodium bicarbonate solution (30 ml)
and water (40 ml). The dried material was used for next step with-
out further purification Yield: 95%. 1H NMR (500 MHz, CDCl3):
␦ 5.38–5.41 (m, 1H), 4.75 (d, J = 18.0 Hz, 1H), 4.60–4.66 (m, 1H),
4.56 (d, J = 18.0 Hz, 1H),2.54 (t, J = 9.8 Hz, 1H), 2.33–2.36 (m, 2H),
2.22–2.26 (m, 1H), 2.18 (s, 3H), 2.06 (s, 3H), 2.01–2.08 (m, 2H),
1.87–1.90 (m, 2H), 1.40–1.76 (m, 10H), 1.28–1.34 (m, 1H), 1.14–1.22
(m, 1H), 1.04 (s, 3H), 0.70 (s, 3H). ESI-MS: calculated for C25H36O5,
416.3, found 439.3 [M+Na]+
The synthesis of compounds 5, 6,
Schemes 1 and 2.
8
are shown in
2.1.1. General methods
All reagents were purchased from Sigma–Aldrich Chemical Co.
(St. Louis, MO), Fisher Scientific (Pittsburgh, PA), Alfa Aesar (Ward
Hill, MA) or Steraloids Inc. (Newport, RI), and were used without
further purification. The solvents for moisture sensitive reactions
were freshly distilled and the reactions were carried out under
an argon atmosphere. Routine thin layer chromatography (TLC)
was performed on aluminum backed Uniplates (Analtech, Newark,
DE). Melting points were measured with Fisher–Johns melting
point apparatus (uncorrected). Mass spectra were recorded using a
Bruker Esquire-LC/MS spectrometer equipped with an electrospray
ionization (ESI) source as described previously [17]. The purity of
the final compounds was determined using a Supelco AscentisTM
5 ␮M RP-Amide column (250 × 4.6 mm) at ambient temperature
on a Waters 2695 HPLC system equipped with a Photodiode
Array Detector. Individual HPLC conditions were optimized and
2.2.2. 3ˇ, 21-Dihydroxypregna-5,7-dien-20-one diacetate (3)
Compound
3 was synthesized according to a literature-
described procedure [20]. To a solution of compound 2 (2.08 g,
Scheme 1. Synthesis of 17-COOH-7DA and its methyl ester. Reagents and conditions: (a) Ac2O, microwave, p-toluenesulfonic acid monohydrate; (b) Dibromantin, 2,2ꢀ-
azobisisobutyronitrile, benzene/hexane (1:1), 100 ◦C, reflux; (c) Bu4NBr, Bu4NF, THF, room temperature; (d) K2CO3, O2, MeOH-THF, room temperature, overnight; (e) DBU,
MeI, THF, room temperature.
Scheme 2. Synthesis of 3␤-hydroxyandrost-5-ene-17␤-carboxylic acid. Reagents and conditions: (d) K2CO3, O2, MeOH-THF, room temperature, overnight.

232
T.-K. Kim et al. / Steroids 75 (2010) 230–239
5.0 mmol) in benzene–hexane (120 ml, 1:1 in volume) was added
dibromantin (0.86 g, 3.0 mmol) and 2,2ꢀ-azobisisobutyronitrile
(33 mg, 0.2 mmol). The mixture was refluxed under argon for
30 min in a preheated oil bath (100 ◦C) and then placed in an ice
bath to cool. The insoluble material was removed by suction filtra-
tion, followed by evaporation of the filtrate to yield a yellow-brown
solid. To a solution of this yellow-brown material in anhydrous
tetrahydrofuran (40 ml) was added tetrabutylammonium bromide
(0.4 g, 1.25 mmol), the resulting solution was stirred for 75 min
under argon at room temperature. To this reaction mixture was
added tetrabutylammonium fluoride (10 ml of 1.0 M solution in
tetrahydrofuran, 10 mmol) and the resulting dark brown solution
was stirred for an additional 50 min to yield a brown solid after
removing solvent. A solution of this solid in ethyl acetate (200 ml)
was washed with water 3 times (3 × 100 ml) and dried over anhy-
drous Na2SO4. Solvent was removed to give crude compound 3. The
crude compound 3 was subjected to flash chromatography (column
eluted with hexane–ethyl acetate 20:1, 10:1, 5:1, 1:1 in order).
Compound 3 is a white solid. Yield: 40–50%. 1H NMR (500 MHz,
CDCl3): ␦ 5.60 (dd, J = 9.6 Hz, 2.8 Hz, 1H), 5.44–5.47 (m, 1H), 4.78 (d,
J = 16.0 Hz, 1H), 4.70–4.76 (m, 1H), 4.58 (d, J = 16.0 Hz, 1H), 2.64 (t,
J = 9.6 Hz, 1H), 2.52–2.56 (m, 1H), 2.39 (t, J = 14.8 Hz, 1H), 2.25–2.32
(m, 1H), 2.20 (s, 3H), 2.12–2.15 (m, 1H), 2.08 (s, 3H), 2.04–2.10
(m, 2H), 1.50–1.96 (m, 8H), 1.50 (dt, J = 14.8 Hz, 8.0 Hz, 1H),1.40
(dt, J = 14.0 Hz, 5.0 Hz, 1H), 0.96 (s, 3H), 0.65 (s, 3H). ESI-MS: calcu-
lated for C25H34O5, 414.2, found 437.3 [M+Na]+, m.p. 139–141 ◦C,
consistent with the value (141–142) reported in the literature [21].
␦ 11.90 (s, 1H), 5.24 (d, J = 7.0 Hz, 1H), 4.60 (s, 1H), 3.21–3.30
(m, 1H), 2.24 (t, J = 15.0 Hz, 1H), 2.05–2.11 (m, 2H), 1.89–1.95 (m,
3H), 1.03–1.76 (m, 14H), 0.90 (s, 3H), 0.59 (s, 3H). Table 2 sum-
marized detailed chemical shifts for all atoms of this compound.
ESI-MS: calculated for C20H30O3, 318.2, found 317.0 [M−H]−.
HPLC: isocratic, water:methanol = 30:70, flow rate 0.6 mL/min,
tR = 17.03 min, purity 98.7%, m.p. 271–273 ◦C.
2.3. Cell culture
Immortalized human epidermal keratinocytes (HaCaT) were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supple-
mented with 5% charcoal-treated FBS (Hyclone, Logan, UT) and 1%
antibiotics (penicillin/streptomycin/amphotericin, Sigma–Aldrich,
St. Louis, MO). Human SKMEL-188 and hamster AbC1-melanoma
cells were grown in Ham’s F10 supplemented with 5% charcoal-
treated fetal bovine serum (ctFBS) and 1% antibiotics. Human
WM35- and WM1341-melanoma cells were grown in DMEM sup-
plemented with 5% ctFBS, 1% antibiotics, and 5 ␮g/ml insulin.
Normal melanocytes were cultured in MBM-4 medium (Lonza,
Walkersville, MD) containing MGM-4 (Lonza, Walkersville, MD).
Fibroblast cells were cultured in DMEM containing 5 ␮g/ml insulin,
1% antibiotics, and with or without ctFBS as indicated in the figure
legend. All cultures were performed at 37 ◦C in 5% CO2.
2.4. DNA synthesis
Incorporation of [3H]-thymidine into DNA was used as a mea-
sure of cell proliferation [23]. Cells were inoculated into 24-well
plates at 5000–25,000 cells/well, depending on cell type. After
overnight incubation at 37 ◦C, the cultures were placed in serum-
free media to synchronize cells at G0/G1 phase of the cell cycle
[23,24]. After 24 h 17-COOH-7DA (dissolved in DMSO and diluted
in culture medium) was added with fresh media containing growth
supplements (as indicated in t figures legends) and incubated for
an additional 24–72 h. After a defined period of time (see above),
[3H]-thymidine (specific activity 88.0 Ci/mmol; GE Healthcare, Pis-
cataway, NJ, USA) was added to a final concentration of 0.5 ␮Ci/ml
in medium. After 4 h of incubation at 37 ◦C, media were discarded,
cells precipitated with 10% TCA in PBS (phosphate-buffered saline)
for 30 min, washed twice with 1 ml PBS and then incubated with 1 N
NaOH/1% SDS (250 ␮l/well) for 30 min at 37 ◦C. The extracts were
collected in scintillation vials and 5 ml of scintillation cocktail were
added. 3H-radioactivity incorporated into DNA was measured with
a beta-counter (Direct Beta-Counter Matrix 9600; Packard, USA).
2.2.3. 3ˇ-Hydroxyandrosta-5,7-diene-17ˇ-carboxylic acid (5)
Compound 5 was synthesized as shown in Scheme 1. To a solu-
tion of compound 3 (414 mg, 1 mmol) in THF:MeOH (60 ml, 1:2
in volume) was added potassium carbonate (414 mg, 3 mmol) and
the solution stirred for 2 days in the presence of air. HCl (1% aque-
ous solution) was added to the reaction mixture to make it slightly
acidic. The precipitated solid was filtered and washed by methanol
to give a white solid. Yield: 49%. 1H NMR (500 MHz, DMSO-d6): ␦
12.00 (s, 1H), 5.49 (dd, J = 3.5 Hz, 1.6 Hz, 1H), 5.35–5.37 (m, 1H), 4.66
(s, 1H), 3.32–3.42 (m, 1H), 2.38 (t, J = 9.0 Hz, 1H), 2.09–2.15 (m, 1H),
1.92–2.06 (m, 4H), 1.72–1.81 (m, 5H), 1.59–1.61 (m, 2H), 1.43–1.47
(m, 1H), 1.30–1.35 (m, 2H), 1.21–1.25 (m, 1H), 0.85 (s, 3H), 0.56
(s, 3H). Table 2 summarized detailed chemical shifts for all atoms
of this compound. ESI-MS: calculated for C20H28O3, 316.2, found
315.0 [M−H]−; HPLC: isocratic, water:methanol = 20:80, flow rate
1.0 mL/min, tR = 8.58 min, purity 96.8%, m.p. 237–239 ◦C.
2.2.4. Methyl 3ˇ-hydroxyandrosta-5, 7-diene-17ˇ-carboxylate
(6)
2.5. Colony forming assay
Compound 6 was synthesized following a known procedure
[22]. To a solution of compound 5 (63.2 mg, 0.2 mmol) in THF (5 ml)
was added DBU (46 mg, 0.3 mmol) and methyl iodide (156 mg,
0.22 mmol). The mixture was stirred at room temperature for 3 h.
Water was added to the mixture and the precipitate collected by
filtration and washed with ethyl acetate to provide a white solid.
Yield: 95%. 1H NMR (500 MHz, DMSO-d6): ␦ 5.49 (dd, J = 4.0 Hz,
2.0 Hz, 1H), 5.36–5.37 (m, 1H), 4.66 (s, 1H), 3.36–3.40 (m, 1H), 3.60
(s, 3H), 2.30–2.33 (m, 1H), 1.91–2.18 (m, 5H), 1.71–1.85 (m, 4H),
1.59–1.62 (m, 2H), 1.45–1.49 (m, 1H), 1.19–1.38 (m, 4H), 0.85 (s,
3H), 0.52 (s, 3H). Table 2 summarized detailed chemical shifts for
all atoms of this compound. ESI-MS: calculated for C21H30O3, 330.2,
found 353.3 [M+Na]+. HPLC: isocratic, water:methanol = 10:90,
flow rate 1.0 mL/min, tR = 6.11 min, purity 98.8%, m.p. 210–212 ◦C.
The assay followed standard methodology used in our labora-
tory as described previously [24,25]. Briefly, cells were plated in
six-well plates at a density of 20 cells/9.6 cm2 in medium containing
5% ctFBS, 1% antibiotic solution, and 17-COOH-7DA at graded con-
centrations or DMSO (vehicle control). Cells were cultured at 37 ◦C
for 7–17 days with media being changed every 3 days. Colonies
were fixed with 4% paraformaldehyde in PBS overnight at 4 ◦C,
washed, stained with 2% crystal violet in PBS for 15 min, rinsed,
and air-dried. The number and size of the colonies were measured
using an ARTEK counter 880 (Dynex Technologies Inc., Chantilly,
VA). Colony forming units were calculated by dividing the number
of colonies by the number of cells plated and then multiplying by
100.
2.2.5. 3ˇ-Hydroxyandrost-5-ene-17ˇ-carboxylic acid (8)
2.6. Reverse transcription polymerase chain reaction (RT-PCR)
Compound 8 was synthesized following the procedure shown
in Scheme 2 by using the same method as described for the syn-
thesis of compound 5. Yield: 55%. 1H NMR (500 MHz, DMSO-d6):
RNA from cells was extracted using the Absolutely RNA RT–PCR
Miniprep kit (Stratagene, La Jolla, CA). Reverse transcription was

T.-K. Kim et al. / Steroids 75 (2010) 230–239
233
Table 1
Table 2
Primer sequences for real-time PCR.
NMR chemical shift assignments for 17-COOH-DA, 17-COOCH3-7DA and 17-COOH-
A by analysis of their 2D NMR spectra (solvent: DMSO-d6).
Gene
Primer sequences
Atom
17-COOH-7DA(5)
17-COOCH3-7DA(6)
17-COOH-A(8)
Cyclophilin B
5ꢀ-TGTGGTGTTTGGCAAAGTTC-3ꢀ
5ꢀ-GTTTATCCCGGCTGTCTGTC-3ꢀ
5ꢀ-TGCCTCAGCCTTACTGTGAGT-3ꢀ
5ꢀ-TCATTTGCTCCTGATGGGTA-3ꢀ
5ꢀ-GGCCAAAATCGACAGGAC-3ꢀ
5ꢀ-GGGTCTGCACAGACTGCAT-3ꢀ
5ꢀ-AGTACCTGAACCGGCACCT-3ꢀ
5ꢀ-GGCCGTACAGTTCCACAAA-3ꢀ
5ꢀ-ACACAGAATCTATACCCACCAGAGT-3ꢀ
5ꢀ-ATCAACTCCCAAACGGTCAC-3ꢀ
1H
13C
1H
13C
1H
13C
Involucrin
Cyclin E1
Bcl-2
1␣
1␤
2␣
2␤
1.22
1.80
1.74
1.33
3.37
4.66
2.32
2.12
NA
38.35
38.35
32.27
32.27
68.97
NA
41.18
41.18
141.41
119.05
117.13
NA
1.24
1.81
1.73
1.34
3.38
4.66
2.32
2.14
NA
38.47
38.47
32.27
32.27
69.17
NA
41.27
41.27
141.77
119.03
117.26
NA
0.98
1.77
1.68
1.34
3.26
4.60
2.14
2.09
NA
5.27
1.53
1.93
1.41
0.91
NA
1.37
1.54
1.23
1.98
NA
1.09
1.62
1.20
1.69
1.95
2.26
0.64
0.95
11.90
NA
37.47
37.47
31.96
31.96
70.60
NA
3
3-OH
4␣
4␤
5
6
7␣
7␤
8
9
EGFR
42.67
42.67
142.18
120.73
31.87
31.87
32.12
50.24
37.64
20.97
20.97
38.16
38.16
43.60
56.08
24.63
24.63
23.72
23.72
55.11
13.54
19.67
175.18
NA
5.49
5.36
NA
5.51
5.39
NA
performed using Transcriptor First Strand cDNA Synthesis Kit
(Roche Applied Science, Indianapolis, IN). The reaction was per-
formed with LightCycler 480 Probes Master (Roche Applied Science,
Indianapolis, IN). The primers and probes were designed with the
Universal Probe Library (Roche Applied Science, Indianapolis, IN).
The primer sequences are shown in Table 1. Real-time PCR was
performed using TaqMan PCR Master Mix at 50 ◦C for 2 min, 95 ◦C
for 10 min, and then performing 45 cycles (95 ◦C for 15 s, 60 ◦C for
1 min). The data were collected with a Roche Light Cycler 480. The
amounts of mRNA were normalized by comparative Ct method,
using cyclophilin B as a housekeeping gene.
NA
1.93
NA
139.96
46.12
37.16
20.90
20.90
37.74
37.74
44.18
53.55
23.39
23.39
23.60
23.60
54.54
13.44
16.53
175.15
NA
NA
1.94
NA
1.62
1.62
1.36
2.01
NA
139.72
46.14
37.10
20.97
20.97
37.39
37.39
44.50
53.37
23.34
23.34
23.63
23.63
54.35
13.54
16.51
174.06
51.87a
10
11␣
11␤
12␣
12␤
13
1.61
1.61
1.33
2.05
NA
1.97
1.74
1.45
1.78
2.00
2.39
0.56
0.86
11.96
NA
14
2.00
1.77
1.48
1.84
2.08
2.49
0.53
0.86
NA
15␣
15␤
16␣
16␤
17
18
19
20-COOH
21-CH3
2.7. Cell cycle analysis
HaCaT keratinocytes were cultured in DMEM media contain-
ing 5% ctFBS to 40% confluence followed by synchronizing for 24 h
in serum-free media. Then, cells were growing in the media with
10−7 M 17-COOH-7DA for 72 h. The cells were washed with PBS
and fixed in 70% ethanol at −20 ◦C for 3 days. The fixed cells were
washed with PBS to remove ethanol and stained in PBS containing
0.05 mg/ml propidium iodide (Sigma–Aldrich, St. Louis, MO) and
0.1 mg/ml RNase for 1.5 h at 37 ◦C. After incubation, cell cycle was
determined using flow cytometry (UTHSC Flow Cytometry and Cell
Sorting Laboratory) with 10,000 cells scored.
3.61a
NA: not applicable (ternary carbons); 7␣ and 7␤ is only applicable for 17-COOH-A.
a
Chemical shifts are for the methyl group in 17-COOCH3-7DA.
3. Results
3.1. Synthesis of 3ˇ-hydroxyandrosta-5,7-diene-17ˇ-carboxylic
acid (17-COOH-7DA) and related compounds
2.8. Cell differentiation of leukemia cell line
The new steroidal 5,7-diene, 17-COOH-7DA, was synthesized
following Scheme 1 (details of the synthesis are in Section 2;
HL-60 human promyelocytic cells were cultured in RPMI
1640 medium containing 10% fetal bovine serum (FBS) (Atlanta
Biologics) and 1% penicillin/streptomycin/amphotericin antibiotic
solution (Sigma) at 37 ◦C. The compounds to be tested were dis-
solved in DMSO and added to the culture to a final concentrations
of 10−7 M. Differentiation of HL-60 cells toward monocytes-like
morphology and NBT-reduction was assessed after 5 days. Cells
(2 × 106) were washed with PBS four times and resuspended in
200 ␮l of NBT solution (4 mg/ml) in water. After the addition of
200 ␮l of TPA solution (2 ␮g/ml) in PBS cells were incubated at 37 ◦C
for 60 min in 24-well plates. The cell differentiation was assessed
by intracellular blue formazan deposits. The NBT-positive and -
negative cells were scored under light microscopy examination
(20×) with a minimum of 200 cells scored [26].
2.9. Statistical analysis
Data are presented as means S.E.M. and have been analyzed
with Student’s t-test (for two groups) or one-way analysis of vari-
ance, and appropriate post hoc test (for more than two groups),
using Microsoft Excel and Prism 4.00 (GraphPad Software, San
Diego, CA). Statistically significant differences are denoted by
roman numerals as follows: I, P < 0.05; II, P < 0.01; III, P < 0.001; IV,
P < 0.0001 in student’s t-test. IA, P < 0.05; IIA, P < 0.01 in one-way
ANOVA analysis.
Fig. 1. 17-COOH-7DA inhibits DNA synthesis in HaCaT keratinocytes and has greater
effect than pregnenolone. HaCaT cells were incubated with drugs for 72 h in DMEM
containing 5% charcoal-treated FBS. [3H]-thymidine was added for last 4 h of incuba-
tion. DNA synthesis was measured by counting the radioactivity incorporated into
TCA precipitable material. Data are presented as means S.E.M. (n = 4). The dose-
dependent inhibition was analyzed by one-way ANOVA with IA, P < 0.05 and IIA,
P < 0.01. The differences between 17-COOH-7DA and pregnenolone was analyzed
with student’s t-test where II, P < 0.01 and III, P < 0.001.

234
T.-K. Kim et al. / Steroids 75 (2010) 230–239
Fig. 2. 17-COOH-7DA stimulates involucrin and represses cyclin E1, bcl-2 and EGFR mRNA expression in HaCaT keratinocytes. HaCaT keratinocytes were treated with 17-
COOH-7DA for 6 (involucrin) or 24 h (cyclin E1, bcl-2 and EGFR), total RNA was extracted and subjected to real-time RT-PCR analysis with cyclophilin B used as a housekeeping
gene. Data are presented as means S.E.M. (n = 3). III, P < 0.001; IV, P < 0.0001 in student’s t-test.
NMR and UV spectra as well as proof of purity by HPLC are in
Supplementary material). The NMR spectra of 17-COOH-7DA and
its methyl ester are shown in Supplementary material, while chem-
ical shift assignments in Table 2.
The synthesis of 3 was performed similarly, as described pre-
viously for pregna-5,7-dienes [17] (Scheme 1 and Section 2 for
details). The 17-COOH-7DA (5) was obtained from 3 by hydrolysis
of the 21-acetoxy group accompanied by base-catalyzed oxida-
tive cleavage of the side chain by molecular oxygen present in the
reaction mixture [27–29]. The exclusion of oxygen from the reac-
tion mixture (reaction under argon) prevented the formation of 5
(not shown). The 17-COOH-7DA (5) was further converted to 3␤-
hydroxyandrosta-5,7-diene-17␤-carboxylic acid methyl ester (6;
17-COOCH3-7DA) by treating it with methyl iodide and DBU.
Fig. 4. Comparison of the effects of 17-COOH-7DA, 17-COOH-pregnenolone (17-
COOH-A) and 17-COOCH3-7DA on DNA synthesis in HaCaT keratinocytes. Data are
presented as means S.E.M. (n = 4).The dose-dependent inhibition was analyzed
by one-way ANOVA with IA, P < 0.05 and IIA, P < 0.01. The differences between 17-
COOH-7DA and 17-COOH-A or 17-COOCH3-7DA were analyzed with student’s t-test
where II, P < 0.01 and III, P < 0.001.
Fig. 3. Cell cycle analysis with flow cytometry. HaCaT keratinocytes were cultured
in DMEM plus 5% ctFBS and 10−7 M 17-COOH-7DA for 72 h. The cells were fixed with
70% ethanol and stained with propidium iodide as described in Section 2. Cell cycle
was determined using flow cytometry with 10,000 cells scored. Veh., DMSO control;
17-D, 17-COOH-7DA. I, P < 0.05 in student t-test.

T.-K. Kim et al. / Steroids 75 (2010) 230–239
235
Fig. 5. 17-COOH-7DA inhibits DNA synthesis in human keratinocytes (HaCaT), normal melanocytes, melanoma cells (SKMEL-188, WM35, WM1341) and hamster AbC1-
melanoma cells. Data are presented as means S.E.M. (n = 4). I, P < 0.05; II, P < 0.01; III, P < 0.001 in student’s t-test. IA, P < 0.05; IIA, P < 0.01 in one-way ANOVA analysis.
The above reaction (d) is analogous to the conversion of
21-acetoxypregnenolone (1) to 3␤-hydroxyandrost-5-ene-17␤-
carboxylic acid (8) (Scheme 2). Its NMR spectra, UV and purity
assessment by HPLC are shown in Supplementary material, while
full chemical shift assignments in Table 2.
associated with an increase of involucrin mRNA and inhibition of
cyclin E1, bcl-2 and EGFR mRNA expression (Fig. 2). Morphological
evaluation of the cells and measurement of the LDH activity (an
assay for cytotoxicity) showed a lack of any cytotoxic effect (not
shown). Flow cytometry studies have confirmed the above find-
ings and showed that that 17-COOH-7DA slows cycling of HaCaT
cells through an increase of cells at G1/G0 and a decrease in G2/M,
but with no effect on subG1 (Fig. 3).
3.2. 17-COOH-7DA inhibits proliferation of HaCaT epidermal
keratinocytes
To further define the specificity of 17-COOH-7DA, 17-
COOH-pregnenolone (17-COOH-A) and 17-COOCH3-7DA were
synthesized according to Scheme 2 and their effect on DNA synthe-
sis in HaCaT keratinocytes were compared to that of 17-COOH-7DA.
Fig. 4 shows that 17-COOH-7DA is significantly more potent than
17-COOH-A and 17-COOCH3-7DA, with the latter having no effect
on proliferation (Fig. 4). Thus there is a strong specificity for 17-
COOH-7DA in the inhibition of cell proliferation.
We tested the effect of 17-COOH-7DA on proliferation of HaCaT
keratinocytes by measuring [3H]-thymidine incorporation into
DNA (Fig. 1). 17-COOH-7DA inhibited DNA synthesis in a dose-
dependent manner with significant inhibitory effect seen at a
concentration as low as 10−11 M. 17-COOH-7DA was more potent
than pregnenolone (Fig. 1), indicating a degree of specificity for
17-COOH-7DA. The anti-proliferative effect of 17-COOH-7DA was

236
T.-K. Kim et al. / Steroids 75 (2010) 230–239
3.3. The effect of 17-COOH-7DA on DNA synthesis in normal and
malignant skin cells
We tested the responses of normal epidermal melanocytes
and melanoma cells to 17-COOH-7DA at concentrations rang-
ing from 10−11 to 10−7 M and compared them to those of
HaCaT keratinocytes (Fig. 5). Dose-dependent inhibition curves
for DNA replication were constructed and the EC50 values were
4.34 × 10−12, 4.60 × 10−11, 6.36 × 10−10, 9.13 × 10−10, 6.60 × 10−12
,
and 4.82 × 10−12 M for HaCaT keratinocytes, neonatal melanocytes
and SKMEL-188-, AbC1-, WM35- and WM1341-melanoma cells,
respectively (Fig. 5). Thus potency was dependent on cell type
with HaCaT keratinocytes and human melanoma cells being more
sensitive to 17-COOH-7DA than normal melanocytes or hamster
(AbC1) melanoma cells. The latter were the least sensitive to the
growth inhibition. The magnitude of the inhibition was also cell-
type dependent with WM1341-melanoma cells showing the largest
effect and AbC1 cells the smallest.
For normal human dermal fibroblasts, the effect of 17-COOH-
7DA on DNA synthesis was dependent on the presence or absence
of serum (Fig. 6). When fibroblasts were cultured in serum-free
media or in the presence of 5% or 1% of charcoal-treated FBS, 17-
COOH-7DA increased, inhibited or had no effect on DNA synthesis,
respectively (Fig. 6).
3.4. 17-COOH-7DA effects on melanoma and leukemia cells
To further define the effect of 17-COOH-7DA on cell colony
formation, SKMEL-188-, WM35- and AbC1-melanoma cells were
plated at a density of 20 cells/9.6 cm2 in medium containing 5%
charcoal-treated FBS and incubated at 37 ◦C for 7 (SKMEL-188 and
AbC1) or 17 days (WM35), in the presence of graded concentra-
tions of the drug (Fig. 7). 17-COOH-7DA inhibited colony formation
with an effect that was dependent on the cell line. The strongest
inhibitory effect on SKMEL-188 and AbC1 cells was seen for the
largest colonies (>1 mm), while significant inhibition of colony for-
mation by 17-COOH-7DA (10−11 and 10−9 M) was seen for small
colonies (>0.2 mm) of WM35 melanoma cells. The parent steroid
for the chemical synthesis of the 17-COOH-7DA, pregnenolone
was used as a control and again showed no significant effect
(Fig. 7).
Next we investigated the effect of 17-COOH-7DA on the human
HL-60 cell line. Differentiation toward monocytes-like morphology
and NBT-reduction was assessed after 5 days of treatment. Fig. 8
shows that 17-COOH-7DA stimulated monocytes-like differentia-
tion of HL-60 cells. In contrast, 17-COOCH3-7DA and 17-COOH-A
slightly but significantly decreased cell differentiation induced by
the DMSO vehicle.
Fig. 6. The effect of 17-COOH-7DA on DNA synthesis in normal human dermal
fibroblasts is dependent on the presence or absence of serum. Normal human dermal
fibroblasts were cultured in DMEM containing 5 ␮g/ml insulin and 17-COOH-7DA
in the absence (serum-free media) or presence of 5% or 1% of charcoal-treated
FBS. After 68 h, [3H]-thymidine was added and after 4 h the incorporation of [3H]-
thymidine into DNA was measured using a beta-counter. A, 5% FBS; B, 1% FBS; C,
serum-free media plus 0.1% BSA. Data are presented as means S.E.M. (n = 4). II,
P < 0.01; III, P < 0.001 in student’s t-test. IA, P < 0.05; IIA, P < 0.01 in one-way ANOVA
analysis.
3.5. 17-COOH-7DA is more potent inhibitor of cell proliferation
than 1,25(OH)2D3, the hormonally active form of vitamin D3
Since active form of vitamin D3 (1,25(OH)2D3) has well doc-
umented anti-proliferative activity against normal and malignant
skin cells [30–32] and is used topically in the therapy of psoriasis
[33], we compared the anti-proliferative effects of 17-COOH-
7DA and 1,25(OH)2D3. 17-COOH-7DA caused significantly more
inhibition of proliferation of HaCaT epidermal keratinocytes, and
SKMEL-188 and AbC1-melanoma cells, than 1,25(OH)2D3 (Fig. 9).
mixture. We have found that 17-COOH-7DA is biologically active
in normal (keratinocytes, melanocytes, and fibroblasts) and malig-
nant (melanoma and leukemia) cells, is non-toxic, and has higher
potency than its methyl derivative (17-COOCH3-7DA), its 5-diene
steroidal counterpart (17-COOH-preg), as well as pregnenolone and
1,25(OH)2D3.
In vivo steroidal 5,7-dienes are detected in patients with
Smith–Lemli–Opitz syndrome, a pathological condition charac-
terized by deficiency of the 7-ꢀ-reductase [1–4], or in horses
under physiological conditions during synthesis of equilin (delta-
7-estrone) [34]. Furthermore, recent findings documenting that
P450scc can transform 7DHC to 7DHP with a possible process-
ing to other steroidal 5,7-dienes, depending on the activity and
availability of steroigogenic enzymes [4,6], raises an intriguing
4. Discussion
We report for the first time on the steroidal 5,7-diene, 3␤-
hydroxyandrosta-5,7-diene-17␤-carboxylic acid (17-COOH-7DA).
We have devised an efficient and reproducible method for its syn-
thesis that is dependent on the presence of oxygen in the reaction

T.-K. Kim et al. / Steroids 75 (2010) 230–239
237
Fig. 7. 17-COOH-7DA inhibits colony formation of SKMEL-188 (A), WM35 (B) and AbC1 (C) melanoma cells. The plates were incubated with drugs for 7 (SKMEL-188 and
AbC1) or 17 days (WM35) and colonies were stained with crystal violet. The number of formed colony units (CFU) was counted for all colonies (>0.2 mm), medium size
colonies (>0.5 mm) or large colonies (>1 mm). Data are presented as means S.E.M. for colony forming assay (n = 4). I, P < 0.05; II, P < 0.01; III, P < 0.001 in student’s t-test.
Lower right corner shows representative plates of SKMEL-188 and AbC1 treated with vehicle control (−) or 10−9 M 17-COOH-7DA (+).
question whether 17-COOH-7DA can be produced in vivo. Interest-
ingly, hydrolysis of the 21-acetoxy group of the 21-acetoxy-7DHP
is accompanied by a spontaneous oxidative cleavage of the side
chain when molecular oxygen is present leading to generation of
17-COOH-7DA. In contrast, 21(OH)7DHP is produced when oxy-
gen is excluded (see above), and this steroid has indeed been
detected in clinical samples from patients with SLOS [2]. Although
we are currently unable to answer the question on in vivo produc-
tion of 17-COOH-7DA, the information above encourages future
experimental and clinical studies aimed at the detection of 17-
COOH-7DA in biological fluids in SLOS patients or fetal horse
tissues.
found that it inhibits proliferation of normal (keratinocytes and
melanocytes) and malignant (melanoma) cells with an effect that is
seen at a concentration as low as 10−11 M for HaCaT keratinocytes,
Inhibition of proliferation was found to be associated with an
increased fraction of cells arrested at G1/G0 phase of cell cycle but
with no induction of apoptosis or necrosis. Studies on melanoma
cells also showed that 17-COOH-7DA inhibits colony formation,
indicating an anti-tumor potential. Importantly, 17-COOH-7DA
was more potent than pregnenolone, 17-COOH-A or 17-COOCH3-
7DA indicating not only structural selectivity for a free carboxyl
group attached to C17 but also the presence of the double bond
at the 7-position of the 17-COOH-7DA for full biological activity.
This specificity was also seen in leukemia cells where 17-COOH-
7DA stimulated monocyte-like differentiation, while 17-COOH-A
Of particular interest and potential clinical importance is
the biological activity of 17-COOH-7DA. Specifically, we have

238
T.-K. Kim et al. / Steroids 75 (2010) 230–239
Fig. 8. Effects of 17-COOH-7DA, 17-COOH-pregnenolone (17-COOH-A) and 17-
COOCH3-7DA on monocyte-like differentiation of HL-60 leukemia cells. After 5 days
of treatment, the cells were resuspended in NBT/TPA solution and incubated at
37 ◦C for 60 min. The differentiated cells were identified by their intracellular blue
formazan deposits. The NBT-positive and -negative cells were scored under light
contrast microscopy (20×) with a minimum of 200 cells scored. N, untreated con-
trol; D, DMSO treated control; 17-D, 17-COOH-7DA; 17-Ac., 17-COOCH3-7DA; 17-A,
17-COOH-A. Data are presented as means S.E.M. (n = 3). I, P < 0.05; III, P < 0.001 in
student’s t-test.
or 17-COOCH3-7DA inhibited it. Also, the cell type and culture con-
ditions were found to affect the activity of 17-COOH-7DA on dermal
fibroblasts. When cultured in serum-free medium, 17-COOH-7DA
stimulated their proliferation whereas with 1% serum there was no
effect and with 5% inhibition occurred. This shows that the bioac-
tivity of this novel compound can be context dependent. These
findings are consistent with recently reported anti-proliferative
activities of selected ꢀ7 unsaturated steroidal compounds in
selected skin cell lines [6,18]. Also, profound behavioral abnor-
malities of SLOS are thought to result from the production of ꢀ7
unsaturated neurosteroid-like compounds [35]. Therefore, care-
ful testing of the biological activity of steroidal 5,7-dienes may
provide clinically and perhaps therapeutically important informa-
tion.
The active form of vitamin D3 (1,25(OH)2D3) has well docu-
mented anti-proliferative activity against normal and malignant
skin cells [30–32]. It is also used topically in therapy of a
benign hyperproliferative skin disorder, psoriasis [33]. Therefore,
our finding that 17-COOH-7DA is significantly more potent than
1,25(OH)2D3 in inhibiting proliferation of human epidermal ker-
atinocytes and human and hamster melanoma cell lines could
have potential therapeutic implications. For example, it could be
a good candidate for a topically applied agent for treatment of
psoriasis or other hyperproliferative skin disorders. Furthermore,
metastatic melanoma is a deadly disease for which there is no
effective therapy. Therefore, any relatively non-toxic compound
showing anti-melanoma activity warrants future animal testing,
and we believe that 17-COOH-7DA is an example of such a com-
pound. Eventually, it could serve as a candidate for a topically
applied adjuvant drug for lentigo maligna or mucosal melanoma
that, because of size or location, cannot be entirely resected.
In summary, we report for first time the structure and synthe-
sis of the new steroidal 5,7-diene, 17-COOH-7DA. We show that it
has specific and potent anti-proliferative or anti-tumor activities
against human keratinocytes, human melanocytes and malignant
melanomas (human and hamster), leukemia cells, and acts as a
condition-dependent regulator (stimulator or inhibitor) of fibrob-
last proliferation.
Fig. 9. 17-COOH-7DA inhibits DNA synthesis in HaCaT keratinocytes, SKMEL-188
and AbC1 and has a larger effect than 1,25(OH)2D3. The cells were incubated with
drugs for 72 h in DMEM (HaCaT) and Ham’s F-10 (SKMEL-188 and AbC1) contain-
ing 5% charcoal-treated FBS. [3H]-thymidine was added for last 4 h of incubation.
DNA synthesis was measured by counting the radioactivity incorporated into TCA
precipitable material. A, HaCaT; B, SKMEL-188; C, AbC1. Data are presented as
means S.E.M. (n = 4). The dose-dependent inhibition was analyzed by one-way
ANOVA with IA, P < 0.05 and IIA, P < 0.01. The differences between 17-COOH-7DA and
1,25(OH)2D3 was analyzed with student’s t-test where I, P < 0.05 and III, P < 0.001.

T.-K. Kim et al. / Steroids 75 (2010) 230–239
239
Acknowledgements
[14] Anderson CM, Buzaid AC, Legha SS. Systemic treatments for advanced cuta-
neous melanoma. Oncology (Williston Park) 1995;9:1149–58.
[15] Serrone L, Zeuli M, Sega FM, Cognetti F. Dacarbazine-based chemotherapy for
metastatic melanoma: thirty-year experience overview. J Exp Clin Cancer Res
2000;19:21–34.
[16] Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted
therapy. Nature 2007;445:851–7.
[17] Zmijewski MA, Li W, Zjawiony JK, Sweatman TW, Chen J, Miller DD, et al. Syn-
thesis and photo-conversion of androsta- and pregna-5,7-dienes to vitamin
D3-like derivatives. Photochem Photobiol Sci 2008;7:1570–6.
[18] Zmijewski MA, Li W, Zjawiony JK, Sweatman TW, Chen J, Miller DD, et al. Photo-
conversion of two epimers (20R and 20S) of pregna-5,7-diene-3beta, 17alpha,
20-triol and their bioactivity in melanoma cells. Steroids 2009;74:218–28.
[19] Marwah P, Marwah A, Lardy HA. Microwave induced selective enolization of
steroidal ketones efficient acetylation of sterols in semisolid state. Tetrahedron
2003;59:2273–87.
[20] Guo LW, Wilson WK, Pang J, Shackleton CH. Chemical synthesis of 7-
and 8-dehydro derivatives of pregnane-3,17alpha,20-triols, potential steroid
metabolites in Smith–Lemli–Opitz syndrome. Steroids 2003;68:31–42.
[21] Antonucci R, Bernstein S, Giancola D, Sax KJ. ꢀ5,7-steroids. VI. The preparation
of ꢀ5,7-steroidal hormones. J Org Chem 1951;16:1126–33.
[22] Dey S, Mal D. Total synthesis of BE-23254, a chlorinated angucycline antibiotic.
Tetrahedron Lett 2005;46:5483–6.
[23] Slominski A, Zbytek B, Pisarchik A, Slominski RM, Zmijewski MA, Worts-
man J. CRH functions as a growth factor/cytokine in the skin. J Cell Physiol
2006;206:780–91.
The project described was supported by Grant Number
AR052190 from NIAMS to AS and 1R15CA125623 from NIH/NCI to
WL. Its contents are solely the responsibility of the authors and do
not necessarily represent the official views of the NIH.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2009.12.004.
References
[1] Tint GS, Irons M, Elias ER, Batta AK, Frieden R, Chen TS, et al. Defective choles-
terol biosynthesis associated with the Smith-Lemli-Opitz syndrome. N Engl J
Med 1994;330:107–13.
[2] Shackleton CH, Roitman E, Kelley R. Neonatal urinary steroids in
Smith–Lemli–Opitz syndrome associated with 7-dehydrocholesterol reductase
deficiency. Steroids 1999;64:481–90.
[3] Shackleton C, Roitman E, Guo LW, Wilson WK, Porter FD. Identification of 7(8)
and 8(9) unsaturated adrenal steroid metabolites produced by patients with
7-dehydrosterol-delta7-reductase deficiency (Smith–Lemli–Opitz syndrome).
J Steroid Biochem Mol Biol 2002;82:225–32.
[4] Slominski A, Zjawiony J, Wortsman J, Semak I, Stewart J, Pisarchik A, et
al. A novel pathway for sequential transformation of 7-dehydrocholesterol
and expression of the P450scc system in mammalian skin. Eur J Biochem
2004;271:4178–88.
[5] Guryev O, Carvalho RA, Usanov S, Gilep A, Estabrook RW. A pathway for the
metabolism of vitamin D3: unique hydroxylated metabolites formed dur-
ing catalysis with cytochrome P450scc (CYP11A1). Proc Natl Acad Sci U S A
2003;100:14754–9.
[6] Slominski AT, Zmijewski MA, Semak I, Sweatman T, Janjetovic Z, Li W, et
al. Sequential metabolism of 7-dehydrocholesterol to steroidal 5,7-dienes
in adrenal glands and its biological implication in the skin. PLoS One
2009;4:e4309.
[7] Holick MF. Vitamin D:
2003;88:296–307.
[8] Holick MF. McCollum award lecture, Vitamin D-new horizons for the 21st cen-
tury. Am J Clin Nutr 1994;60:619–30.
[24] Zbytek B, Janjetovic Z, Tuckey RC, Zmijewski MA, Sweatman TW, Jones E,
et al. 20-Hydroxyvitamin D3,
a product of vitamin D3 hydroxylation by
cytochrome P450scc, stimulates keratinocyte differentiation. J Invest Dermatol
2008;128:2271–80.
[25] Fischer TW, Zmijewski MA, Zbytek B, Sweatman TW, Slominski RM, Wortsman
J, et al. Oncostatic effects of the indole melatonin and expression of its cytoso-
lic and nuclear receptors in cultured human melanoma cell lines. Int J Oncol
2006;29:665–72.
[26] Mahdavi M, Yazdanparast R. Gnidilatimonoein from Daphne mucronata
induces differentiation and apoptosis in leukemia cell lines. Arch Pharm Res
2007;30:177–81.
[27] Li M, Chen B, Monteiro S, Rustum AM. Mechanism of base-catalyzed autooxida-
tion of corticosteroids containing 20-keto-21-hydroxyl side chain. Tetrahedron
Lett 2009;50:4575–81.
[28] Velluz L, Petit A, Pesez M, Berret R. Saponification of the acetate of desoxycor-
ticosterone. Bull Soc Chim France 1947:123–5.
[29] Herzig PT, Ehrenstein M. Investigations on steroids. XIV. Studies in the series
of the adrenal cortical hormones: dehydration of 6,21-diacetoxyallopregnane-
5-ol-3,20-dione. J Org Chem 1951;16:1050–63.
[30] Spina CS, Tangpricha V, Uskokovic M, Adorinic L, Maehr H, Holick MF. Vitamin
D and cancer. Anticancer Res 2006;26:2515–24.
[31] Holick MF. Vitamin D deficiency. N Engl J Med 2007;357:266–81.
[32] Bikle DD, Oda Y, Xie Z. Vitamin D and skin cancer: a problem in gene regulation.
J Steroid Biochem Mol Biol 2005;97:83–91.
[33] Bikle DD. What is new in vitamin D: 2006–2007. Curr Opin Rheumatol
2007;19:383–8.
a
millennium perspective.
J
Cell Biochem
[9] Tian XQ, Holick MF. A liposomal model that mimics the cutaneous production
of vitamin D3. Studies of the mechanism of the membrane-enhanced thermal
isomerization of previtamin D3 to vitamin D3. J Biol Chem 1999;274:4174–9.
[10] Kamradt J, Rafi L, Mitschele T, Meineke V, Gartner BC, Wolfgang T, et al. Analysis
of the vitamin D system in cutaneous malignancies. Recent Results Cancer Res
2003;164:259–69.
[11] Bikle DD. Vitamin D regulated keratinocyte differentiation. J Cell Biochem
2004;92:436–44.
[34] Tait AD, Hodge LC, Allen WR. The biosynthesis of
3 beta-hydroxy-5,7-
[12] Wiseman H. Vitamin D is a membrane antioxidant. Ability to inhibit iron-
dependent lipid peroxidation in liposomes compared to cholesterol, ergosterol
and tamoxifen and relevance to anticancer action. FEBS Lett 1993;326:285–8.
[13] Chen TC, Holick MF, Lokeshwar BL, Burnstein KL, Schwartz GG. Evaluation of
vitamin D analogs as therapeutic agents for prostate cancer. Recent Results
Cancer Res 2003;164:273–88.
androstadien-17-one by the horse fetal gonad. FEBS Lett 1985;182:107–10.
[35] Marcos J, Guo LW, Wilson WK, Porter FD, Shackleton C. The implications
of 7-dehydrosterol-7-reductase deficiency (Smith–Lemli–Opitz syndrome) to
neurosteroid production. Steroids 2004;69:51–60.