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Current Medicinal Chemistry, 2015, 22, ????-????

Dendrogenin A: A Mammalian Metabolite of Cholesterol with Tumor

Suppressor and Neurostimulating Properties
Florence Dalenc1,2, Marc Poirot1,2,* and Sandrine Silvente-Poirot1,2,*
1

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corresponding author(s)
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Sterol Metabolism and Therapeutic Innovations, INSERM UMR 1037,

University of Toulouse, Cancer Research Center of Toulouse, Toulouse, France; 2Institut Claudius Regaud, Institut Universitaire du
Cancer, Toulouse, France

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Abstract: Cholesterol-5,6-epoxide hydrolase (ChEH) in mammals is a heterooligomeric complex of two cholesterogenic enzymes that control mammalian
developmental programs. Following the identification of this complex, it was
Marc Poirot
S. Silvente-Poirot
hypothesized that a new metabolic pathway existed that centered on 5,6-epoxy
cholesterols (5,6-EC). Conjugation products of 5,6-EC with biogenic amines known to interact with ChEH
subunits were synthesized. According to their structures, these steroidal alkaloids showed the specific potency
to induce cell differentiation at low doses, suggesting their possible existence as metabolites. One of these
compounds, named dendrogenin A (DDA), was recently discovered in mammalian tissues. It was shown that
DDA arises from the stereoselective enzymatic conjugation of 5,6-epoxy-cholesterol with histamine by an
as-yet-unidentified enzyme. DDA was detected in normal tissues from several organs but not in cancer cells
and its level was decreased in breast tumors from patients, evidencing a deregulation of DDA metabolism
during carcinogenesis. DDA was also able to control the growth of tumor cells implanted in mice and improve animal survival. In addition, DDA efficiently restored hearing in a preclinical model of deafness. These
biological properties of DDA, as well as its decreased levels in tumors, suggest a physiological function in
maintaining cell integrity and differentiation. DDA is the first steroidal alkaloid found to date in mammals. Its
discovery reveals the existence of a new metabolic pathway in mammals at the crossroads of cholesterol and
histamine metabolism that leads to the production of a metabolic tumor suppressor and neuroprotective agent.

Keywords: AEBS, cancer, cholesterol, cholesterol-5,6-epoxide hydrolase, dendrogenin, steroidal alkaloid,

tamoxifen.
1. INTRODUCTION
Cancer is one of the major causes of death in the
world and many aspects of its diagnosis and treatment
remain a global unmet medical need. Great progress
has been made in our understanding of cancers, which
has led to significant progress in cancer management
through the development of targeted therapies and the
emergence of personalized medicine. Tamoxifen (Tam)
was one of the first targeted therapies that was developed in the seventies. Professor V. Craig Jordan first
demonstrated that Tam blocked the mitogenic effects
of 17 estradiol (E2) and protected rodents against the
*Address correspondence to these authors at the Department of
Sterol Metabolism, Cancer Research Center of Toulouse, INSERM
UMR 1037-University of Toulouse, P.O. Box: 31017, Toulouse,
France; Tel/Fax: +33-582741626; E-mails: marc.poirot@inserm.fr
and sandrine.poirot@inserm.fr.
0929-8673/15 $58.00+.00

development of estrogen-positive breast cancers (ER(+)

BC) [1-4]. This led to the clinical development of Tam
for the treatment and chemo-prevention of ER(+) BC.
Interestingly, an enormous effort to develop Tam analogues based on the rationale that modulating ER activity could improve the efficacy of hormonotherapy has
been underway since the eighties and several novel
antiestrogenic compounds have been produced, such as
afimoxifene, lasofoxifene and bazedoxifene (see Fig. 1)
[5].
However, despite good efficacies none of them have
been able to supplant Tam in the clinic [5]. Tam displays a complex pharmacology and several additional
off-targets have been identified [6-14]. Along with
other selective estrogen receptor modulators (SERM),
it was shown to interact with high affinity to a membranous saturable and intracellular binding site named
2015 Bentham Science Publishers

2 Current Medicinal Chemistry, 2015, Vol. 22, No. 1

Dalenc et al.

Cl
HO
4-Hydroxytamoxifen
4OHTam, afimoxifene

Tamoxifen
ICI 46,474, Nolvadex

Clomiphene
clomid
N

N
O

OH

S
Cl
HO
Raloxifene
keoxifen

Toremifene
Fareston, Acapodene

HO
Lazofoxifene

OH

N
O
N

HO

HO
Bazedoxifene

HO

HN

Dendrogenin A
DDA

N
N
H

Fig. (1). Chemical structure of some selective estrogen receptor modulators (SERM) and dendrogenin A.

the antiestrogen binding site (AEBS). The AEBS was

first described by Robert Sutherland in the eighties [15]
and identification of its molecular structure was
achieved 25 years later [12]. Characterization of the
AEBS established the importance of cholesterol (cholest-5-en-3-ol) metabolism in the pharmacology of
Tam [16] and permitted the identification of a new
metabolic pathway leading to the production of dendrogenin A (DDA) (see Fig. 1), the first steroidal alkaloid identified in mammals [17]. Importantly, DDA is a
cholesterol metabolite with tumor suppressing properties whose production is impaired during oncogenesis
[18]. This has opened up new promising opportunities
for cancer treatments and new routes to understand the
etiology of cancers. This review describes the path of
investigation over the last 20 years that has led from

studies on the effects of Tam to the identification of

ChEH and, more recently, to the exciting discovery of
DDA. Along the way the biochemical and molecular
biology background behind the different compounds
involved are discussed and should explain why the discovery of DDA provides much hope for future therapeutics.
2. MOLECULAR CHARACTERIZATION OF AN
OFF-TARGET OF TAM
2.1. The Microsomal Antiestrogen Binding Site
(AEBS)
In order to study the AEBS, diphenyl methane
(DPM) compounds were developed. These are selective AEBS ligands with no affinity for the ER [19-21].

Dendrogenin A is a Tumor Suppressor

Current Medicinal Chemistry, 2015, Vol. 22, No. 1

Tesmilifene and PBPE are prototypical DPM with high

affinities for the AEBS (see Fig. 2) [19-21]. The use of
these tools helped to determine that the AEBS was involved in the cytotoxicity of its cognate ligands in cancer cells [20, 22-25] and led to the development of
tesmilifene for anticancer applications in the clinic
even before a clear understanding of its mechanism of
action was established [26-30]. Two synthesized
ligands specifically targeting the AEBS [19, 31, 32]
made evaluation of the molecular composition of the
AEBS possible using photo-affinity labeling [31-35].
These were named azido-MBPE and MBOPE (see Fig.
2). Tam and PBPE present structural similitudes to triparanol and boxidine, respectively (see Fig. 2). These
two drugs display antiproliferative and anticancer
properties [36-40] and are inhibitors of cholesterol biosynthesis [41]. Triparanol is a potent inhibitor of 3hydroxysteroid-24-reductase (DHCR24), inducing the
accumulation of desmosterol [42-44] and boxidine is
an inhibitor of 3-hydroxysteroid-7-reductase
(DHCR7) (DHCR7) [38, 39], inducing the accumulation of 7-dehydrocholesterol (see Fig. 3).

N
O

In addition to their estrogen receptor modulating activity, both Tam and Toremifene (see Fig. 1) are reported to induce zymostenol accumulation, suggesting
that Tam inhibits cholesterol biosynthesis in patients at
the 3-hydroxysteroid-8,7-isomerase (D8D7I/EBP)
step [45, 46].
The impact of AEBS ligands on cholesterol biosynthesis was studied in breast cancer (BC) cell lines
where it was found that both Tam and PBPE induced a
significant accumulation of zymostenol in BC cells
[14]. These data suggested that Tam and PBPE inhibited D8D7I in BC cells, which was in accordance with
data published by other groups studying yeast and other
cell lines [47-49]. In addition, it was found that PBPE
inhibited 3-hydroxysteroid-7-reductase (DHCR7),
thereby
inducing
the
accumulation
of
7dehydrocholesterol as expected based on its structural
similarity to boxidine [12]. D8D7I and DHCR7 are
involved in the late stages of cholesterol biosynthesis
Fig. 3). Depending on the chemical structure of the
AEBS ligand and the different cell lines used [8, 11,
12, 48, 50-52], additional cholesterol precursors

O
O

N3

N
O

PBPE

DPPE, tesmilifene

Azido-MBPE

MBOPE

N
Cl

OH
C F
F F
Triparanol, Mer 29

Boxidine

Fig. (2). Chemical structure of selective AEBS ligands and cholesterol biosynthesis inhibitors. DPPE and PBPE are prototypical selective AEBS ligands in the DPM series. Azido-MBPE and MBOPE were synthesized to allow photoaffinity labeling
of the AEBS. Triparanol is an analogue of the triphenylethylenic antiestrogens depicted in Fig. (1). Boxidine is a diphenyl analog of PBPE.

4 Current Medicinal Chemistry, 2015, Vol. 22, No. 1

Dalenc et al.

HO

lanosterol

DHCR24
HO

Tam
PBPE
tesmilifene

HO

zymostenol
D8D7I/EBP

lathosterol

raloxifene

D8D7I/EBP

boxidine
PBPE
tesmilifene

7-dehydrocholesterol

raloxifene

DHCR24
HO
SC5D

SC5D

HO

zymosterol

HO

DHCR24

HO

7-dehydrodesmosterol
DHCR7

DHCR7

DHCR24
HO

cholesterol

HO

desmosterol

triparanol
4OHTam
Fig. (3). Scheme describing some of the enzymatic steps in the post-lanosterol cholesterol biosynthesis pathway. Steps
inhibited by the AEBS ligands, triparanol and boxidine, are specified.

such as desmosterol or zymosterol were also found to

accumulate in cells. For example, 4OHTam was
shown to exclusively induce desmosterol accumulation, while raloxifene induced zymosterol accumulation in BC cells [11, 12, 51]. Following these reports,
it was found that inhibition of the AEBS subunits was
non-competitive and did not correlate with the affinity
of drugs for the AEBS. A shift of two Logarithms was
found between their affinity and their inhibition of
cholesterol biosynthesis due to an undirect effect.
However, high-affinity ligands inhibited post-

lanosterol cholesterol biosynthesis enzymes, leading

to the accumulation of free sterols in cancer cells. For
all AEBS ligands, this accumulation of free sterols
was linked to the induction of a protective autophagy
in MCF-7 cells regardless of the nature of the free
sterol that had accumulated. These data showed that
this AEBS-dependent effect impaired the cytotoxicity
induced by AEBS ligands including DPM and selective ER modulators (SERMs) such as Tam, 4OHTam,
raloxifene or bazedoxifene [11, 12, 50, 51].

Dendrogenin A is a Tumor Suppressor

Current Medicinal Chemistry, 2015, Vol. 22, No. 1

2.2. The Discovery that AEBS Carried Out Cholesterol-5,6-Epoxide Hydrolase Activity

5,6-EC (the substrates of ChEH). These results paralleled the typical behavior of substrates and product
for their enzyme. To determine the level of similarity
between AEBS and ChEH, ChEH inhibition was
measured for a number AEBS ligands with affinities
ranging from nM to M. A series of steroids was also
tested. A positive correlation between AEBS affinity
and inhibition of ChEH activity was established,
which showed the pharmacological identity between
ChEH and AEBS [9]. In addition, knock down of
each AEBS sub-unit in the breast cancer cell line
MCF-7 led to a concomitant loss of AEBS binding
capacity for Tam and a loss in ChEH activity. Conversely, the over expression of each subunit in COS
cells led to the reconstitution of ChEH activity. Altogether, these results established that ChEH was
molecularly identical to the AEBS (see Fig. 5) [7].

The AEBS was first reported as an oxysterol binding protein [53-55]. Oxysterols are oxygenation
products of cholesterol and some are known to be
produced by Reactive Oxygen Species (ROS) [53].
Among the oxysterol family of compounds, 6- and 7oxocholestanol (see Fig. 4a) were reported to bind
competitively to Tam with a high affinity for the
AEBS [54, 55]. Interestingly, these oxysterols were
also known to be competitive inhibitors of the microsomal cholesterol-5,6-epoxide hydrolase (ChEH)
[56], an unidentified enzyme at that time. ChEH catalyzed the hydration of 5,6-epoxy-cholesterols (5,6EC) into cholestane-3,5,6-triol (CT) (see Fig. 4b).
These oxysterols were tested in AEBS binding assays
and it was found that 5,6-EC, the substrates of ChEH,
were potent and competitive inhibitors of Tam [9]. At
the same time, CT (the product of ChEH activity),
was found to have a weaker affinity for AEBS than

Moreover it was shown that the D8D7I was the

catalytic subunit of ChEH and DHCR7 was its regulatory subunit.

(a)

HO
O
6-ketocholestanol

HO

O
7-ketocholestanol

(b)

HO

O
5,6!-epoxycholesterol
5,6!-EC

ChEH

HO

HO

O
5,6"-epoxycholesterol
5,6"-EC

HO

OH

cholestane-3",5!,6"-triol
CT

Fig. (4). Some AEBS ligands from the oxysterol series and inhibitors of ChEH activity. a) Chemical structure of 6- and 7ketocholestanol. b) Cholesterol-5,6-epoxide hydrolase catalyzes the hydration of 5,6-EC and 5,6-EC.

6 Current Medicinal Chemistry, 2015, Vol. 22, No. 1

Dalenc et al.

mary amines and thiols. It was found that 5,6-EC and

5,6-EC were both totally unreactive, as opposed to
styrene oxide that reacted spontaneously in the presence of ethanolamine or mercaptoethanol to give conjugation products [63].

Fig. (5). AEBS and ChEH activities are carried out by

EBP/D8D7I and DHCR7. AEBS ligands are inhibitors of
ChEH.

Since cytotoxicity of AEBS ligands was shown to

be associated with ROS stimulation and was inhibited
by the antioxidant vitamin E [11], it was suspected that
AEBS ligands stimulated cholesterol epoxidation. Indeed, vitamin E is known to inhibit cholesterol-5,6epoxidation [57]. Analyses of the oxysterol profile of
BC cells treated by Tam or PBPE showed, as expected,
that they induced 5,6-EC accumulation in cells and that
this stimulation was inhibited by vitamin E. 5,6-EC
was not metabolized into CT because of the inhibition
of ChEH by the drugs. Finally, 5,6-EC was shown to
mediate BC cell differentiation, and both 5,6-EC and
5,6-EC were found to be required for cell death induction in BC cells treated with AEBS ligands. These data
established the importance of 5,6-EC in the pharmacology of Tam and related drugs [6].
3. CHEMICAL TRANSFORMATION OF 5,6EPOXYCHOLESTEROL
3.1. Reactivity of 5,6-Epoxycholesterol and 5,6Epoxycholesterol
The accumulation of 5,6-EC was at first intriguing
because epoxide-bearing substances are well known to
chemists as being extremely reactive towards nucleophiles (see Fig. 6a) and thus might have been alkylating substances such as styrene oxide [58].
This property made them potential carcinogenic
substances [59]. However, although 5,6-EC were first
reported to display mutagenic properties [60], this was
always a matter of debate [61] until in vivo tests failed
to demonstrate any carcinogenicity in rodents [62].
This led to investigations into the reactivity of 5,6-EC
and 5,6-EC with nucleophiles such as hydroxyls, pri-

This established that 5,6-ECs are epoxides of exceptional stability under non-acidic conditions, ruling out
any direct carcinogenic activity. The same experiments
performed in the presence of a catalyst (Lewis acid or
heat) showed that a reaction was possible but only with
5,6-EC and not with 5,6-EC (Fig. 7) [63]. Importantly, only a single product was obtained from the four
possible (see Fig. 6b), showing a strict stereoselectivity
in the reaction with 5,6-EC (Fig. 7a). The weak reactivity of the epoxides was explained by the location of
the epoxide on the steroid backbone. This stereoselective transformation opened up a possible metabolic
pathway centered on 5,6-EC transformation [17, 18,
57].
3.2. Synthesis of Steroidal Alkaloids from 5,6Epoxycholesterol and Biogenic Amines: The Genesis of Dendrogenins
The AEBS was first described as being tightly associated with a histamine (HA) binding site (HIc) by
Brandes' group in the eighties [64, 65]. After establishing the identity between AEBS and ChEH [7], it
was hypothesized that 5,6-EC could concentrate with
HA and favor their conjugation at the 5,6-epoxyde
group. This hypothesis was chemically evaluated and
the reactivity of 5,6-EC and 5,6-EC with HA in the
presence of a catalyst such as a Lewis acid was tested.
As expected, 5,6-EC but not 5,6-EC reacted with
HA [66].
Interestingly, while three different products of substitution at the C6 were possible (see Fig. 8a), only one
was obtained, with a 6 substitute product resulting
from the trans diaxial ring opening of the epoxide by
the primary () amine of HA (see Fig. 8b). Other 5,6epoxysterols such as 5,6-epoxycholest-7ene-3-ol or
5,6-epoxystigmastan-3-ol were next tested and one
single 6 product of conjugation was obtained in each
case (C15 and C16 respectively, see Fig. 9). Since
other biogenic amines were reported to interact with
this HIc [67], different biogenic amines such as spermine, spermidine and putrescine were used and 6
products of conjugation with the 5,6-ECs were obtained (compounds C21, C23, DDB, C28, C29, see Fig.
9). All conjugation products of 5,6-EC and biogenic
amines are steroids with one or more protonable basic
nitrogen atoms, which belong to the steroidal alkaloid
(SA) class [66].

Dendrogenin A is a Tumor Suppressor

Current Medicinal Chemistry, 2015, Vol. 22, No. 1

#$
O

(a)
#+

#$

#+

Nu

R'

(b)

!
HO

HO

"

"

HO

Nu

Nu

!
OH

:Nu
5
HO

6
?

"
HO

5,6-EC

HO

HO

Nu

Nu

"
OH

Fig. (6). Possible products of the addition of an epoxide to a nucleophile through an SN2 mechanism. a) The two carbons
of the epoxide ring can be attacked by a nucleophile. b) 5,6-EC can give 4 different products by addition via a trans diaxial ring
opening.

(a)

Ethanolamine
HO

Catalyst
5,6!-EC

HO

Ethanolamine
O

HN

OH
5!-Hydroxy-6"-[2-hydroxyethylamino]-cholestan-3"-ol
(HAC)

(b)

HO

HO

"

Catalyst

5,6"-EC

No reaction

Fig. (7). Reactivity of 5,6-EC with ethanolamine. a) 5,6-EC gives a single product when added to ethanolamine in the
presence of a catalyst. b) No reaction occurred with 5,6-EC even under the same conditions.

4. PHARMACOLOGICAL
DENDROGENINS

PROPERTIES

OF

4.1. In vitro Activity of Dendrogenins

These new SA were conceived based on their putative involvement in the control of cell differentiation at
low doses. To study this, a simple screen of tumor cell
differentiation that was mainly based on the morphological modifications of cells was set up. Compounds
were tested on two cell line models: 1) the human

monocytic-like cell line U937 that can first undergo

differentiation into macrophages upon treatment with a
phorbol ester and then into dendritic cells upon one
weak treatment with IL4 and GM-CSF (see Fig. 10a)
[68, 69]; 2) the murine carcinoembryonic cell line P19
that can differentiate into neurons following 6 days of
treatment with 1 M all trans-retinoic acid (ATRA)
(see Fig. 10b). P19 cells are pluripotent cells that can
also differentiate into muscular cells and astrocytes
(Fig. 10c) [70, 71].

8 Current Medicinal Chemistry, 2015, Vol. 22, No. 1

(a)

Dalenc et al.

!
H2N

HA

H
N

"

(b)
HA
HO

HO

Catalyst

!
HO

$
H
N

HN

5,6!-EC

Dendrogenin A
DDA

Fig. (8). The addition of 5,6-EC to HA. a) HA contains three nitrogen atoms (, and ) that can react via an SN2 reaction.
b) 5,6-EC gives a single product of conjugation with the primary () amine of HA.

HO

HO

HO
HN

HO

N
H
DDA
HA, GID, Cyt

HO
HN

HO

N
H
C15
WA, GID, Cyt

HN

C16
HA, GID, Cyt

N
H

NH2
HO

HO
N

HO
N

NH2

HO

C17
NA, GID, Cyt

HO
HN

HO

NH2

C21
WA, GID, Cyt

HN

H
N

C23
HA, NID, NCyt

NH2
HO

HO

HN

H
N

HO

H2N
DDB
HA, NID, NCyt

HO

HO
HN
N
H
C28
HA, NID, NCyt

NH
H2N

HO

HN

NH

N
H
C29
HA, NID, NCyt

Fig. (9). Chemical structure and biological properties of some of the products of condensation of 5,6-epoxysterols with
biogenic amines. HA: highly active; WA: weakly active; NA: not active; GID: general inducer of cell differentiation; NID:
Selective inducer of cell differentiation into neurons; Cyt: cytotoxic; NCyt: not cytotoxic.

Dendrogenin A is a Tumor Suppressor

Current Medicinal Chemistry, 2015, Vol. 22, No. 1

Fig. (10). Cell differentiation tests. a) U937 can differentiate into dendritic cells. b) P19 cells can differentiate into neurons. c)
P19 cells are pluripotent cells that can differentiate into different cell types.

While 5,6-EC or biogenic amines alone or in association did not induce dendritic outgrowth, some but
not all SA induced dendrite outgrowth at low nanomolar concentrations. The HA adduct on 5,6-EC gave a
compound that was named dendrogenin A (DDA).
DDA induced dendritic outgrowth on both U937 and
P19 cells at low doses and was cytotoxic at M concentrations. This property was observed for all sterols
containing the histamine motif except those in which
the sterol side chain was not present and those in which
a double bond was present between C7-C8. Replacement of HA by diaminobutane (putrescine) (C21, see
Fig. 9) gave a less efficient compound than DDA but it
did stimulate dendrite outgrowth in both cell lines and
induce cytotoxicity when used at higher concentrations.
Replacement of HA by spermidine (C23 and DDB, see
Fig. 9) or spermine (C28 and C29) affected their biological properties. These compounds were not cytotoxic at concentrations up to 1 mM and stimulated dendrite outgrowth only in P19 cells and not in U937 cells,
suggesting a different mechanism of action than that
observed with DDA. Of these, the use of 7dehydrocholesterol as a template for epoxidation and
conjugation gave the most active compound, and induced dendrite outgrowth specifically of P19 cells but
with no cytotoxicity up to 1 mM (DDB, see Fig. 9).
When looking at other cancer cells, DDA was found
to induce a dose-dependent cytotoxicity in all cell lines
tested with IC50 values ranging from 0.4 M in an intestinal carcinoma cell line (SW620) to 5.5 M in an
acute myeloid leukemia cell line (KG1) following a 72

hour treatment. Interestingly, human and mouse cell

lines representative of the most frequent cancers such
as leukemia (U937, NB4, KG1), colorectal cancers
(HCT-8, SW-620), neuroblastoma (SK-N-SH, SHSY5Y, Neuro2A), glioma (U87), lung carcinoma
(A549), melanoma (SKMEL-28, B16F10) and breast
cancer (MCF-7, TSAH2d) were found to be highly sensitive to DDA. None of these cell lines were sensitive to
spermine or spermidine adducts (C21, C23, DDB, C28,
C29, see Fig. 9) up to concentrations of 1 mM [66].
To further investigate the putative stimulation of
cancer cell differentiation in vitro, the effect of DDA
on a BC cell line (MCF-7) and on a melanoma cell line
(SKMEL-28) were studied. Treatment of MCF-7 cells
with DDA induced the arrest of cells in the G0-G1
phase of the cell cycle. DDA modified cell morphology, cells became flattened and had increased granulosity. Oil red O staining of cells revealed the accumulation of neutral lipids containing vesicles. Biochemical
analysis showed that these neutral lipids were triacyl
glycerides (TG). Additionally, DDA induced the expression of milk fat globulin, one of the major proteins
found in milk. Altogether these data showed that by
arresting them DDA induced MCF-7 cell differentiation and activated the lactation function of the original
breast ductal cell (see Fig. 11a). DDA appeared to be at
least 5 times more efficient at inducing MCF-7 cell
differentiation [66] compared to a range of other compounds including Tam, other AEBS ligands such as
tesmilifene or PBPE, and other SERMs such as
raloxifene, bazedoxifen, 4OHTam and clomiphene [6,

10 Current Medicinal Chemistry, 2015, Vol. 22, No. 1

Dalenc et al.

Fig. (11). DDA-induced cancer cell re-differentiation into normal cells. a) DDA induced lactation in BC cells of ductal origin. b) DDA induced melanoma cell differentiation into melanocytes. c) DDA induced the differentiation of cancer cells of
neuronal origin into neurons.

11, 51, 72]. DDA was then tested on the human melanoma cell line SKMEL-28 and these cells were observed to morphologically change from spindle to dendritic morphology upon DDA treatment. Ultrastructure
analysis by electron microscopy revealed the presence
of melanosomes in the dendrites. Biochemical analysis
revealed that DDA also activated melanin biosynthesis
in these cells. Cell cycle analyses showed that DDA
had arrested cells in the G0-G1 phase of the cell cycle
[66]. These characteristics are typical of melanocytes
[73, 74], evidencing a stimulation of melanoma cell redifferentiation into melanocytes by DDA (see Fig.
11b). When tested on cancer cells of neuronal origin,
DDA displayed a similar differentiation profile as that
determined for DDB, by stimulating neurite outgrowth
(see Fig. 11c). Interestingly, DDA also induced the selective differentiation of P19 cells into neurons [66].
To complete these studies, DDA was tested on normal cells from the brain. Neuronal progenitor cells
from mice were collected. They normally spontaneously produce neurospheres in vitro. It was found that
DDA stimulated the proliferation and expansion of
these neurospheres at a low dose (10 nM), as observed
for EGF and FGF2 treatment. In addition to stimulating
proliferation, DDA stimulated dendrite outgrowth and
the appearance of -tubulin-positive cells, strongly
suggesting their differentiation into neurons (see Fig.
12) [75]. Altogether these data showed that DDA had
very interesting pharmacological properties and de-

served further in vivo studies for its anticancer and neurodegenerative disease applications.
4.2. In vivo Anticancer and Chemopreventive
Activities of DDA
The impact of DDA was then studied in vivo on tumors implanted into normal immunocompetent mice.
In the first set of experiments TSAH2d cells were implanted into the mammary fat pad of BALB/cJ mice.
TSAH2d cells are breast adenocarcinoma cells (ER(+))
obtained from BALB/cJ mice and are syngeneic for
this strain of mouse [17].
In a chemopreventive setting, DDA was injected at
the same time as the tumor cells were implanted. Mice
were treated every day over a 30 day period by subcutaneous peri-tumoral injection of DDA. DDA was
found to be very effective at controlling tumor development and, importantly, on improving mouse survival. Dose escalation was limited due to drug solubility, but a strong efficacy of DDA was observed even at
extremely low doses (3.7g/kg). Similar effects of Tam
were seen at 5.6 mg/kg, which corresponded to a 1500
fold greater concentration than that of the injected dose
of DDA. Importantly, although Tam controlled tumor
development it did not improve mouse survival, in contrast to DDA. A similar efficacy was obtained for the
treatment of established breast tumors implanted 10
days before DDA treatment (tumors detected by man-

Dendrogenin A is a Tumor Suppressor

Current Medicinal Chemistry, 2015, Vol. 22, No. 1

11

Fig. (12). DDA induced the differentiation of normal progenitor cells. DDA stimulated the multiplication of neurospheres
and activated the differentiation of progenitor cells into neurons.

ual palpation), showing that DDA also displayed anticancer properties on BC cells [17]. DDA was next
tested on the very aggressive mouse melanoma cell line
B16F10 implanted in immunocompetent syngeneic
C57/Bl6 mice and it was found that DDA was extremely efficient at delaying tumor development and
improving mouse survival. Again, it was extremely
efficient even at low doses [17], suggesting that DDA
is a good candidate for melanoma treatment and prevention. Analyses of the phenotypic characteristics of
BC and melanoma tumors from DDA-treated animals
showed that DDA induced the same differentiation
characteristics as those observed in vitro. Additionally,
the use of immunocompetent mice made it possible to
check tumor infiltration by immune cells after DDA
treatment. It was found that DDA strongly stimulated T
lymphocytes and dendritic cells, suggesting that the
immune system contributed to the anticancer activities
of DDA.

chemically-deafened guinea pigs [77]. It was found that

DDA and DDB were extremely potent in maintaining
the electrical responsiveness to a similar extent as that
shown using neurotrophic peptides [78]. The effects of
DDA and DDB were observed both after immediate
administration and one week after animals had been
deafened. In contrast to that found with neurotrophic
peptides, a repopulation of the cochlea with SGN was
not observed. This suggests that DDA may stimulate
dendritic contacts and would explain the recovery of
auditory nerve excitability. These data established that
DDA and DDB constitute a new class of drug with
strong potencies for improving cochlear implant efficacy and treating neuropathy/synaptopathy-related hearing loss [78]. These drugs add a promising new pharmacological perspective to existing strategies [76].

4.3. DDA Protects Against Chemically-Induced

Deafness In vivo

The observation that DDA displayed very potent

properties at nanomolar concentrations suggested that
DDA could exist as a cholesterol metabolite in mammalian tissues. Thus, an analytical methodology was
set up to purify, detect, characterize and quantify DDA
in tissue extracts. It was found that synthetic DDA displayed a specific MS/MS fragmentation profile which
would make it easy to find in tissues if it existed, and a
deuterated form of DDA was used for isotope dilution
quantification. Analysis of tissue extracts showed that
DDA was present at a concentration of 46 ng/g in brain
tissue, and its presence was also established in the
liver, blood, spleen and lungs. DDA was also found in
mouse, human and bovine tissues, suggesting that

Among neurodegenerative diseases, deafness represents one of the major neurosensory deficits occurring
around the world [76]. Hearing impairment involves a
progressive degeneration of structures within the inner
ear including the auditory nerve and spiral ganglion
neurons (SGN). Based on in vitro data, DDA and DDB
were tested in vivo on deafened guinea pigs to determine whether they could restore auditory function. It
had been previously established that neurotrophic factor
instillation of the cochlea could restore auditory nerve
excitability through the stimulation of SGN number on

5. DDA IS A METABOLITE
5.1. DDA is Present in Healthy Mammalian Tissues

12 Current Medicinal Chemistry, 2015, Vol. 22, No. 1

DDA is common to mammals and therefore that other

mammalian tissues may also contain DDA. It was next
determined that heat or proteolytic enzymes inactivated
DDA production and that the transformation of 5,6EC and HA into DDA was possible only in the presence of fresh tissue extracts [17]. Thus, DDA is
thought to be produced in tissues by an as-yet unidentified enzyme. DDA is the first SA identified to date in
mammals. Other SA derivatives of cholesterol have
been identified in plants (tomatidine, see Fig. 13) and
fishes (squalamine, Fig. 13) [79]. Tomatidine is a cholesterol derivative generated through seven enzymatic
steps, including C22 and C26 hydroxylations and a
C26 transamination [80-82]. In squalamine, a spermidine molecule is incorporated onto the C3 of the
steroidal backbone of cholesterol [83].
5.2. DDA is not Produced in Cancer Cells
Researchers then investigated whether DDA was
present in normal mammary epithelial cells and normal
melanocytes grown in vitro. In each case, DDA was
found to be present at 8.2 and 5.6 ng/mg protein respectively. However, DDA was not detectable in BC or
melanoma cell lines representative of different cancer
subtypes, suggesting a deregulation of DDA metabolism in breast and skin cancers. DDA was also not detectable in cell lines of other different types of cancer
including leukemia, glioma, neuroblastoma, colon, in-

Dalenc et al.

testinal and lung cancers [17]. DDA content was analyzed in tumor biopsies from patients with breast cancers and compared to DDA levels measured in normal
matched tissues. It was found that while DDA was present in normal breast tissue its concentration drastically
decreased in tumors. Altogether these data established
that DDA metabolism is decreased in cancers, suggesting a deregulation of DDA metabolism during carcinogenesis.
CONCLUSION
Natural compounds extracted from plants, microorganisms and marine organisms are the main source of
natural chemopreventive and anticancer agents [84-90].
The human metabolome has not been fully explored so
far to determine whether pharmocologically active metabolites against degenerative diseases can be identified. The discovery of DDA occurred 100 years after
the seminal discovery of all-trans retinoic acid (ATRA)
by Elmer McCollum as the first natural compound metabolized by mammalian cells from retinol [91]. He
later also discovered vitamin D [92]. ATRA and vitamin D are potent inducers of cancer cell differentiation
that are currently used in the treatment of myeloid leukemia and are currently being evaluated for other cancers [93-95]. The discovery of dendrogenins has now
opened up new and very exciting fields of research and
defining the mechanisms of action of dendrogenins

Fig. (13). Steroidal alkaloid metabolites of cholesterol are present in different plants, fishes and mammals. Tomatidine is
found in green tomato roots, squalamine in fishes and dendrogenin A in mammals.

Dendrogenin A is a Tumor Suppressor

remains a major project. The identification of their biosynthetic enzymes is also a major challenge since no
similar enzymatic conjugation of epoxides with histamine or other biogenic amines has been reported to
date. These two thema are currently under investigation
in our team. 5,6-EC are known as autoxidation products of cholesterol, which makes people think that they
can only be artefactually produced [57], however the
discovery of DDA has established that a metabolic
pathway indeed exists and is involved in the transformation of 5,6-EC. Interestingly, 5,6-EC is more than
just an autoxidation product of cholesterol since an asyet-unidentified cytochrome P450 has been reported to
catalyse the stereoselective biosynthesis of 5,6-EC in
bovine adrenals [96]. These data strongly suggest that
the 5,6 double bond of cholesterol is a template for its
steroselective transformation to produce compounds
such as DDA. The tumor suppressive, chemopreventive
and neuroprotective properties of DDA make this compound a very promising one for the complementation
therapy of degenerative diseases in which its biosynthesis is already known to be altered.
LIST OF CHEMICAL NAMES AND ABBREVIATIONS
4-hydroxytamoxifen

Current Medicinal Chemistry, 2015, Vol. 22, No. 1

D8D7I

13

= EBP, 3-hydroxysteroid-8,7isomerase

Dendrogenin A = DDA, 5-hydroxy-6-[2-(1Himidazol-4-yl)-ethylamino]cholestan-3-ol

Desmosterol

= Cholest-5,24-dien-3-ol

DHCR24

= 3-hydroxysteroid-24-reductase

DHCR7

= 3-hydroxysteroid-7-reductase

DPM

= Diphenylmethane

ER

= Estrogen receptor

HA

= Histamine

Lanosterol

= Lanosta-8,24-dien-3-ol

Lazofoxifene

= (5R,6S)-6-phenyl-5-[4-(2pyrrolidin-1-yletho xy)phenyl]5,6,7,8-tetrahydronaphthalen-2-ol

MBOPE

= [4-(2-morpholin-4-ylethoxy)phenyl]-phenylmethanone

PBPE

= 1-[2-[4-(phenylmethyl)phenoxy]
ethyl]-pyrrolidine

Raloxifene

= Keoxifen, [6-hydroxy-2-(4hydroxyphenyl)-1-benzothiophen3-yl]-[4-(2-pipe ridin-1-ylethoxy)phenyl]methanone

SA

= Steroidal alkaloid

SC5D

= 3-hydroxysteroid-5-desaturase

SERM

= Selective estrogen receptor modulator

Tamoxifen

= ICI 46,474; nolvadex; (Z)-2-[4-(1,

2-diphenylbut-1-enyl)phenoxy]N,N-dimethylethanamine

5,6-EC

= 4OHTam, afimoxifene, (Z)-4-(1(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenylbut-1-enyl) phenol

= 5,6-epoxycholesterol

5,6-EC

= 5,6-epoxycholes terol

7-dehydro
cholesterol

= Cholest-5,7-dien-3-ol

AEBS

= antiestrogen binding site

Azido-MBPE

= 4-[2-(2-azido-4-benzylphenoxy)ethyl]morpholine

Tesmilifene

Bazedoxifene

= 1-{4-[2-(azepan-1-yl)ethoxy]benzyl}-2-(4-hydroxyphenyl)-3methyl-1H-indol-5-ol

= DPPE, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine

Triparanol

BC

= Breast cancer

Boxidine

= (1-[2-[[4'-(Trifluoromethyl)-4biphenylyl]oxy] ethyl]pyrrolidine)

= Mer-29, metasqualene, 2-(4-chlo

rophenyl)-1-[4-[2-(diethylamino)ethoxy]phenyl]-1-(4-methylphenyl)ethanol

Zymostenol

= 5-Cholest-8-ene-3-ol

ChEH

= Cholesterol-5,6-epoxide hydrolase

Zymosterol

= 5-Cholesta-8,24-dien-3-ol

Cholesterol

= Cholest-5-en-3-ol

Clomiphene

= 2-[4-[(E)-2-chloro-1,2diphenylethenyl]phenoxy]-N, Ndiethylethanamine

CONFLICT OF INTEREST
Dendrogenins are developed for clinical applications by the company AFFICHEM, of which MP and
SSP are founders.

14 Current Medicinal Chemistry, 2015, Vol. 22, No. 1

Dalenc et al.

ACKNOWLEDGEMENTS
All authors contributed to the writing of this manuscript. This work was founded by the institute National de la sant et de la recherch mdicale, the University of Toulouse III, and the Institut Claudius Regaud. This work was supported by the Agence Nationale pour la Recherche (ANR): ANR 11-RPIB-015-02
DAML, ANR-11-PHUC-0001, the DIRECCTE
Midi-Pyrnes and the Communaut Urbaine de Toulouse mtropole : Onco San Tech (projet DEMODA
RMN13001BBA) and by the Labex Trail (Universit
Bordeaux).

[13]

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