M. Jurášek et al. / Steroids xxx (2014) xxx–xxx
3
tion coupled with electron transport chain (ETC) [9], thus yielding
oxidative stress response elements, reactive species generation
(
(
ROS), and initiating apoptosis [10]. Oxidative phosphorylation
OXPHOS) is a process in which both nuclear and mitochondrially
encoded enzymes are required. Complex of a hormone and its
receptor binds to HREs of the mitochondrial DNA and induce tran-
scription of mitochondrial OXPHOS genes, thus suggesting the pos-
sibility of a direct mechanism for induction of these genes by
hormones [11–14].
Another nandrolone–bodipy conjugate, compound 7, appeared
to be localized on the cell plasma membrane of LNCaP, PC-3,
MCF-7, and HeLa cells. Thus, colocalization with CellMask™ Deep
Red was performed, see Fig. 3. Except for that, we have observed
fluorescence emission of all compounds (4–7) also in small vesicles
resembling lipid droplets, where pure bodipy specifically localized
(
bodipy control localized in lipid droplets-derived vesicles, data
not shown). This localization of the nandrolone–bodipy conjugates
might be caused by insufficient binding capacity of the specific
binding sites inside the cells together with possible excess of
applied conjugates. However, this nonspecific localization in lipid
droplets does not interfere with the functional studies of nandro-
lone–bodipy.
The specificity of nandrolone–bodipy derivative 4 (0.25
localization was studied by competitive assay (displacement
experiments) using a 50-fold (12.5 M)) and 100-fold (25 M))
lM)
l
l
direct (physical) and indirect (functional) associations derived from genomic
context, high-throughput experiments, co-expression, and literature mining
excess of nonfluorescent nandrolone, and other related steroids:
metandrolone, boldenone, trenbolone, and testosterone. Chemical
structures of the steroids are depicted in Fig. S28 in Supplementary
information. Compound 4 was chosen for this competitive assay as
the most effectively and the most rapidly localized one. All the
tested parent nonfluorescent steroids displaced large amount of
nandrolone–bodipy 4 (decreased fluorescence intensity in ER) from
its endoplasmic reticulum localization site within 1 h of incubation
with 50-fold and 100-fold excess of the steroids. The displacement
was monitored by the decrease of fluorescence intensity of the
same (living) cells before and after treatment with the competitors,
see Figs. 4 and S29 in Supplementary information. The most pro-
nounced decrease of fluorescence was observed for pure nandro-
lone, thus confirming functionality of its bodipy derivative 4.
According to our expectations, fluorescent signal from the lipid
droplets was not diminished by incubation with nonfluorescent
steroids, because in lipid droplets, there is no binding specificity
for steroids.
(
required confidence score 0.4). AR – androgen receptor; CYP19A1 – cytochrome
P450, family 19, subfamily A, polypeptide 1; PGR progesterone receptor;
ENSG00000235307 bromodomain-containing protein (protein RING3)
–
–
2
(
(
O27.1.1); FSH – bromodomain-containing protein 2 (protein RING3) (O27.1.1)
801 aa); IGFBP3 – insulin-like growth factor binding protein 3; NUMB – numb
homolog (Drosophila); FOS – FBJ murine osteosarcoma viral oncogene homolog;
NR3C2 – nuclear receptor subfamily 3, group C, member 2; CYP17A1 – cytochrome
P450, family 17, subfamily A, polypeptide 1.
experiments with ER-Tracker™ Red and mitochondria-specific red-
emitting dye. From Fig. 2, it is very likely that compound 4 was pri-
marily localized in endoplasmic reticulum (ER) of the tested cell
lines. Nevertheless, the overlap of the fluorescent signals of com-
pound 4 and ER-Tracker™ Red was not absolute, there was a
remaining fluorescent signal of nandrolone–bodipy outside of ER.
We have found also partial colocalization of this signal with mito-
chondrial sensors (data not shown). Even though that most of the
steroidogenic enzymes reside in ER, notably, some of them can be
also found in mitochondria of mammalian cells [4], which is in
agreement with our findings. It has been recently described [7,8]
that mitochondrial DNA contains regions sequentially similar to
nuclear hormone response elements (HREs). Mitochondria provide
more than 90% of the cell energy supply by oxidative phosphoryla-
2.3. Cytotoxicity assays
Effect on cell proliferation of the prepared fluorescent nandro-
lone–bodipy derivatives was examined in two prostatic cancer cell
lines, LNCaP and PC-3; one breast cancer cell line (MCF-7) and in
Fig. 2. Fluorescence microscopy images of nandrolone–bodipy conjugate 4 colocalized with ER-Tracker™ Red in human prostatic cancer cells (LNCaP) in vitro. (A) compound
was applied in 0.25 M concentration for 3 h; (B) ER-Tracker™ Red in 70 nM concentration for 30 min; (C) merge. From figure C, the colocalization of both compounds is
4
l
apparent. nandrolone–bodipy 4 localizes in endoplasmic reticulum.