Anti-proliferative effects of a novel isoflavone derivative in medullary ...

260 Y. Greenman et al. / Journal of Steroid Biochemistry & Molecular Biology 132 (2012) 256– 261
Fig. 4. cD-tBoc induces thyroid cancer TT cell death through the induction of apoptosis and necrosis. (a) Effect of cD-tBoc on apoptotic cell death in cultured TT cell lines
as assessed by the detection of histone–DNA fragments, p < 0.001; (b) effect of the general apoptosis inhibitor ZV on the inhibitory effect of cD-tBoc on DNA synthesis in
cultured human TT cells, *p < 0.05 for the difference between cD-tBoc treated and control treated cells and # p < 0.05 for the difference of cD-tBoc treated + ZV compared to
cD-tBoc treated alone; (c) effect of ZV on cD-tBoc inhibition of CK activity, *p < 0.05 for the difference between cD-tBoc treated and control treated cells and # p < 0.05 for the
difference of cD-tBoc treated + ZV compared to cD-tBoc treated alone; and (d) effect of cD-tBoc on LDH activity, reflecting cell necrosis in cultured human TT cells, *p < 0.05
for the difference between cD-tBoc treated and control treated cells.
already achieved at the lowest concentration tested (0.0312 �M)
(Fig. 4a). Co-incubation with the antiapoptotic agent ZV (Z-VAD-
FMK) reversed the growth inhibitory effect elicited by cD-tBoc
as measured by thymidine incorporation (Fig. 4b), and CK activity
(Fig. 4c), indicating that cD-tBoc-induced apoptosis is a major
mechanism through which this compound retards growth in the
TT cell line. Finally, cD-tBoc increased TT cell release of LDH by
67% (Fig. 4d), reflecting cell necrosis and thus indicating that this
is another pathway by which this compound inhibits cell proliferation.
4. Discussion
An increase in calcitonin secretion by normal thyroid C-cells as
a result of estrogen stimulation has been demonstrated in vitro
[18] as well as in animal models [19] and humans [20], thus suggesting
the presence of estrogen receptors in these cells. Normal
[21] and hyperplastic C-cells [22] have been shown by immunocytochemistry
to express exclusively ER�. This predominant ER�
expression has been described initially for medullary thyroid carcinoma
samples as well [23], but subsequently ER� has been detected
in most MTC samples by immunostaining using different antibodies
[23], or by RT-PCR [4]. In fact, as measured by quantitative
real time RT-PCR, MTC samples had higher expression levels of
ER� mRNA than ER� mRNA, in equivalent levels to those found
in normal thyroid tissue [4]. Although estrogen receptor expression
has been clearly demonstrated previously in TT cells [24], here
we show that ER� is the predominant mRNA isoform expressed in
these cells. Furthermore, our results indicate that both specific ER�
and ER� agonists stimulate DNA synthesis and CK activity, indicating
increased TT cell proliferation. This effect was receptor specific
as MPP, an ER� antagonist, blocked the effects of the ER�-specific
PPT agonist, whereas PTHPP, an ER� antagonist, blocked the proliferative
effects of the ER�-specific DPN agonist. Hence, despite
the more pronounced mRNA expression of ER� in TT cells, both
receptor isoforms significantly mediated E2 induced proliferation
in these cells.
Our results are in contrast with those of Cho et al., who reported
opposite effects of ER� (stimulatory) and ER� (inhibitory) on cell
proliferation and apoptosis of TT cells [24]. A possible explanation
to this discrepancy it that the TT cell line strain used by
these authors had no endogenous ER receptor expression and that
proliferation studies were performed following infection of these
cells with adenoviral vectors carrying the either the human ER�
or the ER� receptor, thus creating a more artificial experimental
paradigm.
Another important finding of the present study is that a synthetic
derivative of the isoflavone daidzein, cD-tBoc, potently
inhibited TT-cell proliferation, through the induction of both cell
apoptosis and necrosis. The exact mechanisms by which this compound
exerts its anti-tumoral effects have not been clarified yet
although our results point to a predominant mediating effect of
ER�. This hypothesis is supported by our data showing abrogation
of the antiproliferative effects if cD-tBoc by a specific ER� antagonist
but not by an ER� antagonist. Furthermore, cD-tBoc inhibited
TT-cell proliferation induced by an ER� agonist but not by and
ER� agonist. Finally, daidzein, the parent compound of cD-tBoc
has been shown to have a greater affinity for ER� [25]. Our findings
support recent data published by our group suggesting that
cD-tBoc inhibition of proliferation of the follicular thyroid carcinoma
cell line WRO and of the anaplastic thyroid carcinoma cell
line ARO is mediated by ER� [26]. Nevertheless, in TT cell lines,