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dihydroxycholesterol [37]. In our hands, treatment of cholesterol
with SeO2 (1.5 equiv) in CHCl3 produced 4 -hydroxycholesterol 1
and the 7 -hydroxycholesterol [38] 4 in 62% and 17%, respectively.
On transformed and tumoral cells (158N cells, C6 cells, SK-N-
BE cells), under treatment with 7 -OHC and 7KC, as previously
observed on different cells of the vascular wall [13], the most
potent effects, both on cell viability and cell growth, were
b
b
a
It is important to mention that the formation of compound 4 from
cholesterol by SeO2 has not been reported previously. In contrast,
observed with 7KC and 7
b
-OHC, and at a lower extend with 7
a-
treatment of cholesteryl acetate 5 with the SeO2 furnished 4
b-hy-
OHC (Figs. 2e4AeC; Figs. 2e4FeH). Interestingly, 7a
-OHC which
droxylated 6 and 6
[39e41].
a
-hydroxylated 7 in 50% and 25% respectively
is not toxic on human monocytic leukemia cells (U937) [47] was
cytotoxic on 158N and C6 cells but not on SK-N-BE cells. Previ-
Surprisingly, treatment of cholesteryl acetate 5 with SeO2 in the
presence of N-methylmorpholine (pKb ¼ 7.38) at 70 ꢁC decreases
the formation of compound 7 to 5% and increases the amount of
compound 6 to 60% yield, whereas no reaction occurred in the
presence of triethylamine (pKb ¼ 11). These results show that the
formation of 7 is dependent on the reaction medium (SeO2,
ously, 7
human neuroblastoma bone marrow derived cell line (SH-SY5Y)
[25]. Noteworthy, under treatment with 4 -OHC and 4 -OHC, no
effect on cell viability, and more or less pronounced effects on
cell growth were found on 158N and C6 cells in a range of con-
b-OHC was also reported to induce cell death on the
a
b
centrations from 5 to 160
mM (Figs. 2e4 DE; Figs. 2e4 IJ), sup-
pKa ¼ 2.62), and it seems that the formation of 6
was produced from 4 -hydroxylated 6 rather than by oxidation of
cholesteryl acetate 5 with SeO2 (the mechanism of the formation of
-hydroxylated 7 from cholesteryl acetate 5 is currently under
study in our laboratory).
Finally, the 4 -hydroxycholesterol 2 was obtained as followed:
catalytic oxidation of compound 6 with tetrapropylammonium
perruthenate (TPAP) [42] in the presence of 4-methylmorpholine
N-oxide (NMO) afforded the expected ketone 8 in very good
yields, which was reduced selectively by NaBH4 and in situ hy-
a
-hydroxylated 7
porting potential cytostatic activity of these molecules
a
b
(Table 1). These later data are in agreement with those obtained
on HT-29 human colon carcinoma cells and ARPE-19 human
retinal pigmentary epithelial cells [24,25]. On mixed primary
culture of murine glial cells (astrocytes and oligodendrocytes), no
6
a
a
effects of 7
range of the LD50 and IC50 of 7
the cell type considered, whereas pronounced cytotoxic effects
were detected with 7 -OHC (marked decrease of cell viability
a
-OHC, 4
a
-OHC and 4
b-OHC (used at 50 mM; in the
b
-OHC) were revealed whatever
b
and inhibition of cell growth), and at a lower extend with 7KC
(Fig. 5A and B).
drolysis of the remaining acetate gave the 4a-hydroxyl cholesterol
2 [43] (Fig. 1).
Overall, our data underline that the hydroxyl substrate in C7, on
ring B of the sterol nucleus, provides the highest cytotoxicity, even
on immortalized and normal glial cells calling thus into question its
selective cytotoxicity [25,48] (Table 1). They also show that hy-
droxyl position at C4, on ring A of the sterol nucleus, has no effect
on cell viability, and either more or less pronounced effects on cell
growth of immortalized or tumoral cells (158N and C6 cells,
respectively), or no effects on cell proliferation of normal glial cells
2.2. Structure activity relationship on cell viability and cell growth
On the basis of the oxidation state at C4 and C7 on ring A and
B, respectively, of the sterol nucleus and on the oxygenated
groups known to be essential for the side effects of different
oxysterols, various oxysterols were evaluated for their cytotox-
icity (effects on cell viability and cell growth). The purpose of this
study was to evaluate the effects of 4
are not well known, comparatively to oxysterols with well
described biological activities (7KC, 7
cells of the CNS.
Therefore, to evaluate the impact of the hydroxyl at C4, and the
incidence of its position ( or ), a panel of immortalized, tumoral
and normal cells of the CNS was used: murine oligodendrocytes
158N immortalized with the SV-40 large T-antigen [44]; C6 rat
glioma cells; SK-N-BE human neuroblastoma cells; mixed murine
primary culture of astrocytes and oligodendrocytes. The neuro-
blastoma derived cell line (SK-N-BE) was chosen because of the role
of cholesterol and of a particular oxysterol, 24S-hydroxycholesterol,
in the normal brain cellular functions and also because of the in-
fluence of oxysterol imbalance in neurodegenerative processes
[45]. Moreover, neuroblastoma as well as glioma (taken in
consideration in the present study with the use of C6 rat glioma
cells) are common malignancies in childhood [46] with generally
low cure rates due to inefficient therapies as a result of the
impermeable specific characteristics of the blood brain barrier.
Oxysterols, with amphiphilic properties and rapid exchange rates
between membranes are expected to cross easily the blood brain
and the brain hematotumoral barriers being potentially useful as a
chemotherapeutic alternative for neuroblastoma and glioma
treatment. Murine oligodendrocytes (158N) cells with some char-
acteristics of well differentiated oligodendrocytes [44], and mixed
primary culture of astrocytes and oligodendrocytes were used as
model of noncancerous cells to take in consideration the
potential side effects on myelin synthesis (oligodendrocytes are
myelin synthetizing cells) and on cholesterol homeostasis, which is
tightly regulated in the neurons via tight connections with the
astrocytes [45].
(astrocytes, oligodendrocytes) (Table 1). Noteworthy, to date, 4
OHC and 4 -OHC are the only oxysterols identified with cytostatic
properties suggesting that these molecules, whereas not cytotoxic,
may have some interests to counteract cell proliferation. As 4 -OHC
b-
a
-OHC and 4
b
-OHC, which
a
b
-OHC, and 7
a
-OHC) [13] on
b
is a quantitatively dominating oxysterol in human circulation
[10,11], it is suggested that it could be involved in the control of cell
a
b
proliferation. In addition, as 4
whatever the cell type considered, whereas it is a potent LXR
LXR agonist [29,30], this oxysterol or some of its derivatives may
have some pharmacological interests to trigger LXR and/or LXR
b
-OHC has no cytotoxic effects,
a
and
b
a
b
associated metabolic pathways without interference on cell
viability.
3. Conclusions
We described and validated a new methodology for 4
a-OHC and
4b-OHC synthesis, and we bring new information establishing the
absence of effects of these oxysterols on cell viability as well as
more or less pronounced effects on cell growth (cytostasis) of
immortalized and tumoral cells, and no effect on normal cells of the
CNS. The cytostatic properties of 4
some pharmacological interests.
a-OHC and 4b-OHC may have
4. Experimental methods
4.1. Materials and general methods
7-Ketocholesterol (7KC) was from SigmaeAldrich (St. Louis, MO,
U.S.A.). The following oxysterols, 7 -hydroxycholesterol (7 -OHC)
and 7 -hydroxycholesterol (7 -OHC) were either from Sigmae
b
b
a
a
Aldrich or a generous gift from Prof. M. Samadi (Département de
Chimie, LCP e A2MC, Université de Lorraine, Metz, France). The