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

Journal of Steroid Biochemistry & Molecular Biology 132 (2012) 256– 261
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Journal of Steroid Biochemistry and Molecular Biology
jo u r n al hom epage: www.elsevier.com/locate/jsbmb
Anti-proliferative effects of a novel isoflavone derivative in medullary thyroid
carcinoma: An in vitro study
Yona Greenman a,∗ , Meital Grafi-Cohen a , Orly Sharon a , Etty Knoll a , Fortune Kohen b ,
Naftali Stern a , Dalia Somjen a
a Institute of Endocrinology, Metabolism and Hypertension, Tel-Aviv Sourasky Medical Center and the Sackler Faculty of Medicine, Tel-Aviv University, Israel
b Department of Biological Regulation, The Weizmann Institute, Rehovot, Israel
a r t i c l e i n f o
Article history:
Received 16 April 2012
Received in revised form 25 June 2012
Accepted 27 June 2012
Keywords:
Medullary thyroid carcinoma
Phytoestrogen
TT cell line
Estrogen receptor
1. Introduction
a b s t r a c t
Differentiated follicular thyroid cancer is significantly more
prevalent in female patients, which account approximately for 75%
of diagnosed cases [1]. That endogenous estrogen may have a role
in the pathogenesis of these disorders is supported by the increase
in the female:male ratio from 0.8 in pre-pubertal to 5.3 in postpubertal
children, with a gradual decrease in the sex ratio to 2.2
at the peri-menopausal age [2]. The epidemiological observations
led to pathological and in vitro studies that demonstrated expression
of estrogen receptors in normal and malignant thyroid tissues
[3,4] as well as estrogen-mediated promotion of thyroid tumor cell
growth [5] and metastatic phenotype [6].
Abbreviations: cD-tBoc, 5-(25)-pentyl-carbamic acid tert-butyl ester N-t-Boc-7-
(O)-carboxymethyl daidzein; DPN, diaryl-propionitrile; PPT, propylpyrazole triol.
∗ Corresponding author at: Institute of Endocrinology, Metabolism and Hypertension,
Tel.:-Aviv Sourasky Medical Center, 6 Weizmann Street, Tel-Aviv 64239, Israel.
Tel: +972 36973899; fax: +972 36973053.
E-mail address: Greenman@tasmc.health.gov.il (Y. Greenman).
0960-0760/$ – see front matter ©
2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jsbmb.2012.06.006
Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or
recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in
the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line,
might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor
(ER) � mRNA expression in TT cells was more abundant than ER�, with a ratio of 48:1. Estradiol-17�
(E2) increased DNA synthesis in a dose dependent manner. [ 3 H]-thymidine incorporation was also stimulated
by the ER� agonist DPN and the ER� agonist PPT. cD-tBoc inhibited TT cell growth as assessed
by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific
activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise
inhibited. The inhibitory effect of cD-tBoc on [ 3 H]-thymidine incorporation could be blocked by the ER�
antagonist PTHPP but not by the ER� antagonist MPP, suggesting that the antiproliferative effect of cDtBoc
on these cells is mediated through ER�. Furthermore, cD-tBoc potently increased apoptosis and cell
necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect
elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation
of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell
line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.
© 2012 Elsevier Ltd. All rights reserved.
In contrast, the sex-hormone milieu contribution to the pathogenesis
and clinical aspects of medullary thyroid carcinoma (MTC)
is less clear. Thyroid glands from healthy men were reported to
have twice as many C-cells as women in a post-mortem study
[7]. Accordingly, maximum observed calcitonin concentrations are
higher in men, suggesting the need for different normal ranges
for males and females [8,9]. From the epidemiological point of
view, the female:male ratio for this malignancy is reported to vary
between 1 and 1.3 [10,11]. Nevertheless, male sex was consistently
associated with decreased survival in most studies [12,13], reminiscent
of the poor prognosis of differentiated follicular thyroid
cancer in men [13]. Furthermore, male patients had larger tumors
as well as a higher incidence of extrathyroidal extension and distant
metastasis in comparison to their female counterparts [1]. When
adjustments for staging of disease were performed, the influence
of gender on survival lost statistical significance in some instances,
reflecting the fact that male patients had more advanced disease at
diagnosis [14].
At present, the only effective treatment modality for MTC is
complete excision of the tumor by total thyroidectomy, and, when
indicated, lymph node neck dissection. Unfortunately, curative
surgery is often unachievable, as over 50% of patients present with

Y. Greenman et al. / Journal of Steroid Biochemistry & Molecular Biology 132 (2012) 256– 261 257
stage III or IV disease at the time of diagnosis [11]. Currently available
treatments for patients with residual or recurrent disease after
primary surgery have low efficacy rates. Chemotherapeutical regimens
may achieve partial remission rates in the range of 10–20%,
that are generally short lived [15]. Therefore cytotoxic chemotherapy
is not recommended as first-line therapy for these patients.
Similarly, the role of adjunctive external beam irradiation to the
neck in MTC is controversial, and its use is considered only in
selected cases [15]. In the absence of established effective systemic
therapies, the development of new drugs for MTC has been an area
of intense research. Compounds that inhibit receptor or intracellular
kinases, and in particular molecules that block RET kinase
activity or downstream signaling pathways have shown promise in
clinical trials [16]. Nevertheless, the low rate of partial responses
and absence of complete responses in the various trials should
encourage the development of different treatment strategies.
In view of the putative role of estrogen in the development of
thyroid neoplasia as implied by the data described herein, targeting
of estrogen receptors could be an attractive strategy for the
management of MTC. Here we explore whether proliferation of
the human MTC TT cell line, may be curbed by novel isoflavone
derivatives generated in our laboratory, which possess potent anticancer
effects in human ovarian cancer cells through interaction
with estrogen receptor (ER) � [17].
2. Materials and methods
2.1. Reagents
All reagents were of analytical grade. Chemicals, steroids and
PPT (ER� agonist) were purchased from Sigma (St. Louis, MI). DPN
(ER� agonist), MPP (anti-ER�) and PTHPP (anti-ER�) were purchased
from Tocris Bioscience (Bristol, UK). The apoptotic inhibitor
Z-VADFMK Me ester was obtained from Axxora (San Diego, CA).
Methyl-[ 3 H]-thymidine (5 Ci/mmol) was obtained from New England
Nuclear (Boston, MA). Synthesis of t-Boc derivatives of carboxy
alkyl isoflavones were prepared as described previously [17] by
Bioline Innovations, Jerusalem, Israel.
2.2. Cell culture
The human medullary carcinoma cell line TT was purchased
from the American Type Culture Collection (ATCC). Cells were cultured
in Ham’s F12K medium with 2 mM l-glutamine adjusted to
contain 1.5 g/L sodium bicarbonate and 10% fetal bovine serum,
grown to sub-confluence and then treated with various hormones
or agents for 24 h as indicated.
2.3. Preparation of total RNA, RT-PCR and RNA quantification
Total RNA from TT cells was extracted using the TRIzol reagent
(Gibco Life Technologies) according to the manufacturer’s instructions.
Extracted RNA (1 �g) was then reverse transcribed using
the RevertAid TM First Strand cDNA synthesis kit from Fermentas
Life Science (St. Leon-Rot, Germany). mRNA expression was
quantified with an ABI 7700 Real Time PCR System using specific
primer probe sets for estrogen receptors (ER) � and � obtained from
Applied Biosystems (Foster City, CA). Each RT-PCR contained 12.5 �l
TaqMan Universal PCR Master Mix, 1.25 �l Assays-on demand
Gene Expression Assay Mix for either ER� (HS00174860M1) or
ER� (HS00230957M1), 2.5 �l nuclease-free water, and 9 �l cDNA.
Endogenous controls (RNAse P) were run in triplicate to assure
repeatability. In this system, cycle threshold (CT) indicates the fractional
cycle number at which the reporter fluorescence generated
by cleavage of the probe passes a fixed threshold above baseline and
�CT represents relative gene expression. The relative difference in
expression of the gene of interest and of the internal reference gene
is represented by ��CT. Changes of gene expression in relation to
the calibrator are represented by 2 −�C T .
2.4. Assessment of DNA synthesis
TT cells were grown until sub-confluence, and then treated with
various hormones or agents for 24 h as indicated. At the end of incubation,
[ 3 H]-thymidine was added for 2 h. Cells were then treated
with 10% ice-cold trichloroacetic acid (TCA) for 5 min and washed
twice with 5% TCA and then with cold ethanol. The cellular layer was
dissolved in 0.3 ml of 0.3 M NaOH, aliquots were taken for counting
radioactivity, and [ 3 H]-thymidine incorporation into DNA was
calculated.
2.5. Assessment of cell proliferation
Cells were grown until sub-confluence and then treated with
various compounds for 24 h as indicated. At the end of incubation,
cell proliferation was assayed using the cell proliferation kit
based on XTT colorimetric assay (Biological Industries, Kibbutz Beit
Haemek, Israel).
2.6. Assessment of cell death and detection of apoptosis and
necrosis
Cells were grown until sub-confluence and then treated with
various compounds for 24 h as indicated. At the end of incubation,
photometric enzyme-immunoassay for the quantitative
in vitro determination of cytoplasmic histone-associated-DNAfragments
(mono- and oligo-nucleosomes) after induced cell death
was assayed using Cell Death Detection ELISA plus kit from Roche
kit Molecular Biochemicals. This is a “sandwich” assay constructed
to identify DNA fragments through the use of two antibodies, one
against histones and the second directed against DNA. Cell necrosis
was also assayed at the end of the incubation by measuring lactic
dehydrogense (LDH) activity released to the culture medium from
the cytoplasm of disintegrating cells using a standard commercial
LDH assay (Advia Centaur, Siemens).
2.7. Assessment of creatine kinase specific activity
Cells were grown until sub-confluence, treated for 24 h with the
various hormones as specified, and were then collected and homogenized
in an extraction buffer as previously described. Supernatant
extracts were obtained by centrifugation of homogenates at
14,000 × g for 5 min at 4 ◦ C in an Eppendorf micro centrifuge.
Creatine kinase activity (CK) was assayed by a coupled spectrophotometric
assay (17). Protein was determined by Coomassie blue dye
binding using bovine serum albumin (BSA) as the standard.
2.8. Statistical analysis
Results are expressed as mean ± SEM. Differences between the
mean values obtained from the experimental and the control
groups were evaluated by analysis of variance (ANOVA). A p value
less than 0.05, was considered significant.
3. Results
3.1. Effect of E2 and specific ER˛ and ERˇ agonists and
antagonists on DNA-synthesis and CK activity in the human
medullary thyroid cancer TT cell line
ER� mRNA was significantly more abundant than ER� in
the TT cell line, with a ratio of 48:1 (Fig. 1a). E2 elicited a

258 Y. Greenman et al. / Journal of Steroid Biochemistry & Molecular Biology 132 (2012) 256– 261
Fig. 1. Estradiol-17(, ER� and ER� agonists stimulate DNA synthesis and CK activity in medullary thyroid carcinoma cell line TT. (a) ER� and ER� mRNA expression in
cultured human TT cells; (b) effect of treatment (24 h) with increasing concentrations of estradiol-17� (E2) on DNA synthesis, *p < 0.05; (c) effect of MPP (ER� antagonist)
and PTHPP (ER� antagonist) on the stimulatory effect of E2, DPN (ER� agonist) and PPT (ER� agonist) on DNA synthesis, *p < 0.05; **p < 0.01 for the difference between
hormonal treated and control treated cells; # p < 0.05 for the difference between hormone + antagonist treated and hormonal treated cells; and (d) effects of raloxifene on E2-,
DPN- and PPT-induced changes in CK specific activity, *p < 0.05 for the difference between hormonal treated and control treated cells; # p < 0.05 for the difference between
hormone + Ral treated and hormonal treated cells.
dose-dependent increase in DNA synthesis in TT cells as assessed by
[ 3 H]-thymidine incorporation (Fig. 1b). Stimulation of proliferation
occurred even at low concentrations of E2, which are equivalent to
physiological E2 levels in adult females (0.3–3 nM). The ER� agonist
DPN at low concentration (42 nM) stimulated [ 3 H]-thymidine
incorporation by 68% (similar to the extent of increase in DNA
synthesis achieved with E2 at similar concentration). Incubation
with higher concentrations of DPN had no effect on cell proliferation.
In contrast, incubation of TT cells with a low concentration
of the ER� agonist PPT (39 nM) had no effect, but at higher concentrations
(390 nM) this agent caused a significant stimulation of
[ 3 H]-thymidine incorporation (data not shown). Consequently, all
subsequent experiments as described below were performed using
DPN concentration of 42 nM and PPT concentration of 390 nM. The
stimulatory effect of E2 (30 nM) on DNA synthesis was attenuated
by co-incubation of either the ER� specific antagonist MPP
(150 nM) or the ER� specific antagonist PTHPP (150 nM), indicating
that the estrogen induced proliferation in these cells is mediated by
both ER� and ER� (Fig. 1c). Furthermore, the stimulatory effect of
the ER� agonist DPN on cell proliferation was more robustly inhibited
by co-incubation with the ER� antagonist PTHPP, than with
MPP. Accordingly, ER� mediated DNA synthesis by PPT was mostly
inhibited by the specific ER� antagonist MPP (Fig. 1c). Nuclear
receptor-dependent estrogenic activity, as measured by creatine
kinase specific activity (CK activity), was significantly stimulated
by E2 (30 nM), DPN (42 nM) and PPT (390 nM, Fig. 1d). Incubation
with the selective estrogen receptor modulator (SERM) Raloxifene
(Ral, 3 �M) that has tissue specific stimulatory or inhibitory activities,
induced CK activity in TT cells (Fig. 1d). On the other hand, Ral
blocked E 2 and PPT but not DPN stimulation of CK activity, reflecting
its predominant antagonism of ER� over ER� (Fig. 1d). These
results indicate that despite the more pronounced mRNA expression
of ER� in TT cells, both receptor isoforms significantly mediate
E2 induced proliferation in these cells.
3.2. Effect of cD-tBoc on human TT cell line growth and survival
in vitro
cD-tBoc significantly inhibited DNA synthesis in cultured TT
cells in a dose dependent manner ranging from 0.0312 to 3.120 �M
(Fig. 2a). Similar inhibitory effects were observed when cell proliferation
was assessed by the XTT assay, with maximal suppression of
proliferation (70%) achieved at cD-tBoc concentration of 0.312 �M
(not shown). Furthermore, cD-tBoc (3.12 �M) completely abrogated
the proliferative effect of E2, as well as of the ER� agonist
DPN, but not of the ER� agonist MPP (Fig. 2b). Concordant with
this finding, the cD-tBoc inhibitory effect on [ 3 H]-thymidine incorporation
could be blocked by PTHPP (anti-ER�) but not with MPP
(anti-ER�), suggesting that the antiproliferative effect of cD-tBoc
on these cells is mediated through ER� (Fig. 2c). Finally, basal CK
activity, as well as E2 and DPN (but no PPT) stimulated CK activity
was suppressed by co-treatment with cD-tBoc (Fig. 2d). Fig. 3
depicts actual photographs of control and treated TT cells responding
to this compound. Shown photographs were obtained following
24 h of culture with vehicle (Fig. 3a) or with cD-tBoc at 3 (Fig. 3b),
30 (Fig. 3c) and 300 nM (Fig. 3d).
3.3. cD-tBoc induces thyroid cancer cell death through the
induction of apoptosis and necrosis
cD-tBoc potently increased apoptosis (1350–1750% stimulation
of histone–DNA fragments), with the maximal effect being

Y. Greenman et al. / Journal of Steroid Biochemistry & Molecular Biology 132 (2012) 256– 261 259
Fig. 2. cD-tBoc inhibits basal as well as E2 and ER� agonist stimulated DNA synthesis in TT cells. (a) Effect of cD-tBoc on basal DNA synthesis in cultured human TT cells,
*p < 0.05 for the difference between hormonal treated and control treated cells; (b) effect of cD-tBoc on E2, DPN and PPT stimulated DNA synthesis in cultured human TT cells
(% of control, *p < 0.05; **p < 0.01); (c) effect of co-treatment with ER� antagonist (MPP) or ER� antagonist (PTHPP) on cD-tBoc induced inhibition of DNA synthesis, *p < 0.05;
**p < 0.01 for the difference between cD-tBoc treated and control treated cells; # p < 0.05 for the difference between cD-tBoc + antagonist treated and cD-tBoc treated cells;
and (d) effect of cD-tBoc on E2, DPN and PPT induced CK activity in cultured human TT cells (% of control), *p < 0.05; # p < 0.05 for the difference between cD-tBoc + antagonist
treated and cD-tBoc treated cells.
Fig. 3. Effect of different doses of cD-tBoc on the morphology of cultured human TT cells (×20). (a) No treatment; (b) cD-tBoc 0.0312 �M; (c) cD-tBoc 0.312 �M; and (d)
cD-tBoc 3.12 �M.

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,

Y. Greenman et al. / Journal of Steroid Biochemistry & Molecular Biology 132 (2012) 256– 261 261
inhibition of proliferation was achieved through pathways leading
to both apoptosis and necrosis, while in WRO and ARO cell
lines, cell death occurred through apoptosis only [26]. Clearly further
work is needed to dissect the pathways through which this
novel compound exerts its antitumoral effects.
In conclusion, our results suggest that estrogens are involved
in proliferation of the human MTC cell line TT, and that this property
can be utilized to design promising anti-cancer drugs for the
management of medullary thyroid carcinoma.
Conflict of interest
All authors declare that there are no actual or potential conflicts
of interest, including financial, personal or other relationships
with other people or organizations that could have inappropriately
influenced this work.
Role of the funding source
This work did not receive financial support by any specific sponsor
or grant.
References
[1] A. Machens, S. Hauptmann, H. Dralle, Disparities between male and female
patients with thyroid cancers: sex differences or gender divide? Clinical
Endocrinology 65 (2006) 500–505.
[2] I. Dos Santos Silva, A.J. Swerdlow, Sex differences in the risks of hormonedependent
cancers, American Journal of Epidemiology 138 (1993) 10–28.
[3] H. Inoue, K. Oshimo, H. Miki, M. Kawano, Y. Monden, Immunohistochemical
study of estrogen receptors and the responsiveness to estrogen in papillary
thyroid carcinoma, Cancer 72 (1993) 1364–1368.
[4] C. Egawa, Y. Miyoshi, K. Iwao, E. Shiba, S. Noguchi, Quantitative analysis of
estrogen receptor-alpha and -beta messenger RNA expression in normal and
malignant thyroid tissues by real-time polymerase chain reaction, Oncology 61
(2001) 293–298.
[5] D. Manole, B. Schildknecht, B. Gosnell, E. Adams, M. Derwahl, Estrogen
promotes growth of human thyroid tumor cells by different molecular mechanisms,
Journal of Clinical Endocrinology and Metabolism 89 (2001) 1072–1077.
[6] S. Rajoria, R. Suriano, A. Shanmugam, Y.L. Wilson, S.P. Schantz, J. Geliebter, R.K.
Tiwari, Metastatic phenotype is regulated by estrogen in thyroid cells, Thyroid
20 (2010) 33–41.
[7] S. Guyetant, M.C. Rousselet, M. Durigon, D. Chappard, B. Franc, O. Guerin, J.P.
Saint-Andre, Sex-related C cell hyperplasia in the normal human thyroid: a
quantitative autopsy study, Journal of Clinical Endocrinology and Metabolism
82 (1997) 42–47.
[8] M. d’Herbomez, P. Caron, C. Bauters, C.D. Cao, J.L. Schlienger, R. Sapin, L. Baldet,
B. Carnaille, J.L. Wémeau, French Group GTE (Groupe des Tumeurs Endocrines):
reference range of serum calcitonin levels in humans: influence of calcitonin
assays, sex, age, and cigarette smoking, European Journal of Endocrinology/European
Federation of Endocrine Societies 157 (2007) 749–755.
[9] A. Machens, F. Hoffman, C. Sekulla, H. Dralle, Importance of gender-specific
calcitonin thresholds in screening for occult sporadic medullary thyroid cancer,
Endocrine Related Cancer 16 (2009) 1291–1298.
[10] E. Kebebew, P.H. Ituarte, A.E. Siperstein, Q.Y. Duh, O.H. Clark, Medullary thyroid
carcinoma: clinical characteristics, treatment, prognostic factors, and a
comparison of staging systems, Cancer 88 (2000) 1139–1148.
[11] E. Modigliani, R. Cohen, J.M. Campos, B. Conte-Devolx, B. Maes, A. Boneu, M.
Schlumberger, J.C. Bigorgne, P. Dumontier, L. Leclerc, B. Corcuff, I. Guilhem, the
GETC Study Group, Prognostic factors for survival and for biochemical cure in
medullary thyroid carcinoma: results in 899 patients, Clinical Endocrinology
48 (1998) 265–273.
[12] S. Schröder, W. Böcker, H. Baisch, C.G. Bürk, H. Arps, I. Meiners, H. Kastendieck,
P.U. Heitz, Klöppel, Prognostic factors in medullary thyroid carcinomas. Survival
in relation to age, sex, stage, histology, immunocytochemistry, and DNA
content, Cancer 61 (1988) 806–816.
[13] N. Bhattacharyya, A population-based analysis of survival factors in differentiated
and medullary thyroid carcinoma, Otolaryngology-Head and Neck Surgery
128 (2003) 115–123.
[14] S. Roman, R. Lin, J.A. Sosa, Prognosis of medullary thyroid carcinoma: demographic,
clinical and pathological predictors of survival in 1252 cases, Cancer
107 (2006) 2134–2142.
[15] American Thyroid Association Guidelines Task ForceR.T. Kloos, C. Eng, D.B.
Evans, G.L. Francis, R.F. Gagel, H. Gharib, J.F. Moley, F. Pacini, M.D. Ringel, M.
Schlumberger, S.A. Wells Jr., Medullary thyroid cancer: management guidelines
of the American Thyroid Association, Thyroid 19 (2009) 565–612.
[16] M. Cakir, A.B. Grossman, Medullary thyroid cancer: molecular biology and novel
molecular therapies, Neuroendocrinology 90 (2009) 323–348.
[17] F. Kohen, B. Gayer, T. Kulik, V. Frydman, N. Nevo, S. Katzburg, R. Limor, O. Sharon,
N. Stern, D. Somjen, Synthesis and evaluation of the antiproliferative activities
of derivatives of carboxyalkyl isoflavones linked to N-t-Boc-hexylenediamine,
Journal of Medicinal Chemistry 50 (2007) 6405–6410.
[18] G.A. Williams, S.C. Kukreja, E.N. Bowser, G.K. Hargis, C.P. Greenberg, W.J. Henderson,
Prolonged effect of estradiol on calcitonin secretion, Bone and Mineral
1 (1986) 415–420.
[19] T. Naveh-many, G. Almogi, N. Livni, J. Silver, Estrogen receptors and biologic
response in rat parathyroid tissue and C cells, Journal of Clinical Investigation
90 (1992) 2434–2438.
[20] M.I. Whitehead, G. Lane, P.T. Townsend, G. Abeyasekera, C.J. Hillyard, J.C.
Stevenson, Effects in postmenopausal women of natural and synthetic estrogens
on calcitonin and calcium-regulating hormone secretion. Relevance to
postmenopausal osteoporosis, Acta Obstetricia et Gynecologica Scandinavica
Supplement 106 (1981) 27–32.
[21] A.H. Taylor, F. Al-Azzawi, Immunolocalisation of oestrogen receptor
beta in human tissues, Journal of Molecular Endocrinology 24 (2000)
145–155.
[22] C. Bléchet, P. Lecomte, L. De Calan, P. Beutter, S.S. Guyétant, Expression of sex
steroid hormone receptors in C cell hyperplasia and medullary thyroid carcinoma,
Virchows Archiv 450 (2007) 433–439.
[23] A.M. Cho, M.K. Lee, K.H. Nam, W.Y. Chung, C.S. Park, J.H. Lee, T. Noh, W.I. Yang,
Y. Rhee, S.K. Lim, H.C. Lee, E.J. Lee, Expression and role of estrogen receptor
a and b in medullary thyroid carcinoma: different roles in cancer growth and
apoptosis, Journal of Endocrinology 195 (2007) 255–263.
[24] K. Yang, C.E. Pearson, N.A. Samaan, Estrogen receptor and hormone responsiveness
of medullary thyroid carcinoma cells in continuous culture, Cancer
Research 48 (1988) 2760–2763.
[25] G.G.J.M. Kuiper, J.G. Lemmen, B. Carlsson, J.C. Corton, S.H. Safe, P.T. van der Saag,
B. van der Burg, J.Å. Gustafsson, Interaction of estrogenic chemicals and phytoestrogens
with estrogen receptor �, Endocrinology 139 (1998) 4252–4263.
[26] D. Somjen, M. Grafi-Cohen, S. Katzburg, G. Weisinger, E. Izkhakov, N. Nevo,
O. Sharon, Z. Kraiem, F. Kohen, N. Stern, Anti-thyroid cancer properties of a
novel isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-
Boc-hexylenediamine in vitro and in vivo, Journal of Steroid Biochemistry and
Molecular Biology 126 (2011) 95–103.