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

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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
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