The Oncologist, Vol. 13, No. 2, 98-104, February 2008; doi:10.1634/theoncologist.2007-0190
© 2008 AlphaMed Press
Suberoyl Bis-Hydroxamic Acid Activates Notch-1 Signaling and Induces Apoptosis in Medullary Thyroid Carcinoma Cells
Li Ning,
David Yu Greenblatt,
Muthusamy Kunnimalaiyaan,
Herbert Chen
Endocrine Surgery Research Laboratories, Department of Surgery, University of Wisconsin, and the University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA
Key Words. Suberoyl bis-hydroxamic acid • SBHA • Medullary thyroid carcinoma • Neuroendocrine tumors • Notch-1 • Achaete-scute complex-like 1 • ASCL-1 • Chromogranin A • Apoptosis
Correspondence: Correspondence: Herbert Chen, M.D., H4/750 Clinical Science Center, 600 Highland Avenue, Madison, Wisconsin 53792-7375, USA. Telephone: 608-263-1387; Fax: 608-263-7652; e-mail: chen{at}surgery.wisc.edu; or Muthusamy Kunnimalaiyaan, K4/638 Clinical Science Center, 600 Highland Avenue, Madison, Wisconsin 53792-7375, USA. Telephone: 608-265-3749; Fax: 608-263-8613; e-mail: kunni{at}surgery.wisc.edu
Received October 8, 2007;
accepted for publication December 11, 2007.
Disclosure: No potential conflicts of interest were reported by the authors, planners, reviewers, or staff managers of this article.
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ABSTRACT
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Medullary thyroid carcinoma (MTC) is a neuroendocrine (NE) malignancy that frequently metastasizes and has limited treatments. We recently reported that ectopic expression of Notch-1 in human MTC cells suppresses growth. The objective of this study was to evaluate the ability of suberoyl bis-hydroxamic acid (SBHA) to modulate Notch-1 signaling in MTC cells. At baseline, no active Notch-1 protein was present in MTC cells. Treatment with SBHA resulted in a dose-dependent induction of the Notch-1 intracellular domain, the active form of the protein. Furthermore, with Notch-1 activation there was a concomitant decrease in achaete-scute complex-like 1 (ASCL-1), a downstream target of Notch-1 signaling, as well as the NE tumor marker chromogranin A (CgA). Transfection of Notch-1 small-interfering RNA into MTC cells blocked the effects of SBHA on Notch-1 activation, ASCL-1, and CgA. Importantly, SBHA treatment resulted in a dose-dependent decrease in cell viability. Treated cells had an increase in protein levels of cleaved caspase-3 and poly ADP-ribose polymerase, and changes in the expression of apoptotic mediators including Bcl-XL and Bad, indicating that the growth inhibition was a result of apoptosis. These results demonstrate that SBHA activates Notch-1 signaling, which is associated with the antiproliferative and apoptotic effects in MTC cells. Therefore, Notch-1 activation with SBHA is an attractive new strategy for the treatment of patients with MTC.
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INTRODUCTION
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Medullary thyroid carcinoma (MTC) is a neuroendocrine (NE) cancer that originates from calcitonin-secreting parafollicular C cells in the thyroid. MTC is a relatively rare malignancy, accounting for 3%–5% of all thyroid cancers and causing significant morbidity and mortality [1]. Although surgery is currently the only effective treatment for MTC, approximately 50%–80% of patients already have metastatic disease at the time of initial diagnosis, and thus complete surgical resection is often not possible [2–4]. Therefore, new treatments for patients with advanced MTC are clearly needed.
The Notch-1 signaling pathway controls cell fate in multiple developmental programs and its dysregulation has been implicated in the oncogenesis of several types of cancer [5]. Recent years have seen major advances in the understanding of the dual function of Notch-1 signaling as both an oncogene and a tumor suppressor [6]. We have previously demonstrated the tumor suppressor role of Notch-1 signaling in MTC cell lines [7]. As Notch-1 signaling is minimal or absent in MTC [8], and overexpression of Notch-1 resulted in a reduction in growth and NE markers, activation of Notch-1 signaling is an attractive therapeutic strategy for MTC tumors. However, until now, no known compounds were available to activate the Notch-1 pathway in these tumor cells. Recently, we and others reported that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, activates the Notch-1 pathway in human pancreatic carcinoid and neuroblastoma cells [9, 10].
Suberoyl bis-hydroxamic acid (SBHA), a relatively new HDAC inhibitor, is thought to exert anticancer effects by relieving inhibition of genes that regulate cell survival, proliferation, and apoptosis [11–13]. Histones are basic proteins that, by complexing with DNA, form nucleosomes leading to the compact structure of chromatin. Modification of histones by acetylation and deacetylation, which affects chromatin structure, plays an important role in the regulation of gene transcription and expression. The dynamic equilibrium between histone acetylation and deacetylation is regulated by histone acetyltransferases and HDAC enzymes [14]. Recent research has also shown that the Notch signaling pathway is regulated by an HDAC corepressor complex that is sensitive to HDAC inhibitors [15]. Therefore, we hypothesized that SBHA may be able to affect the Notch-1 signaling cascade by HDAC inhibition.
The objectives of this study were twofold: first, to evaluate the effects of SBHA on the growth of MTC cells and on the Notch-1 signaling cascade in MTC cells, and second, to investigate the mechanism by which SBHA regulates growth in MTC cells. We found that SBHA effectively induced Notch-1 signaling, suppressed production of NE tumor markers, and inhibited cell proliferation. Furthermore, we demonstrated that the growth inhibition was mediated by apoptosis. These findings suggest that Notch-1 signaling activation with SBHA may have potential therapeutic effects in patients with MTC.
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MATERIALS AND METHODS
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Cell Culture and Reagents
Human MTC cells (TT) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 18% fetal bovine serum (Sigma-Aldrich, St Louis, MO), 100 IU/ml penicillin and 100 µg/ml streptomycin (Life Technologies) in a humidified atmosphere of 5% CO2 in air at 37°C. SBHA (Biomol, Plymouth Meeting, PA) was dissolved in dimethylsulfoxide (DMSO) at a stock concentration 50 mg/ml and stored at –20°C. Fresh dilutions in medium were made for each experiment.
Cellular Proliferation Assay
TT cell proliferation was measured by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay (Sigma-Aldrich), as previously described [16]. Briefly, cells were seeded in quadruplicate on 24-well plates and incubated for 24 hours under standard conditions to allow cell attachment. The cells were then treated with SBHA in concentrations of 0–20 µM and incubated for up to 6 days. The MTT assay was performed by replacing the standard medium with 250 µl of serum-free medium containing MTT (0.5 mg/ml) and incubating at 37°C for 3 hours. After incubation, 750 µl of DMSO (Sigma-Aldrich) was added to each well and mixed thoroughly. The plates were then measured at 540 nm using a spectrophotometer (µQuant; Bio-Tek Instruments, Winooski, VT). Experiments were performed at least twice.
Notch-1 RNA Interference Assays
Small-interfering RNA (siRNA) against Notch-1 and nonspecific siRNA (sc-44226 and sc-37007, Santa Cruz Biotechnology, Santa Cruz, CA) were transfected into TT cells per the manufacturer's instructions. Briefly, TT cells were plated at a density of 30%–60% confluency in six-well plates. On the next day, transfection with siRNA using Lipofectamine 2000 (Invitrogen, San Diego, CA) was carried out. On the following day, the medium was changed with complete medium and the incubation was continued for 2 more days. Cell lysates were prepared and Western blot was done as described below for Notch-1 and other desired proteins.
Western Blot Analysis
Total cellular proteins were isolated as described, and the protein concentrations were determined with a bicinchoninic assay kit (Pierce, Rockford, IL). Cellular extracts (30–50 µg) were denatured by boiling for 5 minutes and separated by 8% or 10% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) by electroblotting. Membranes were blocked in milk (5% nonfat dry milk and 0.05% Tween 20 in phosphate-buffered saline) and exposed to primary and secondary antibodies as described. The following primary antibody dilutions were used: Notch-1 (1:1,000, Santa Cruz Biotechnology), ASCL-1 (1:1,000; BD Pharmingen, San Diego, CA), glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (1:10,000, Trevigen, Gaithersburg, MD), acetyl-histone H4 (Lys12), Bcl-XL, Bad, caspase-3, cleaved caspase-3, poly (ADP-ribose) polymerase (PARP) (1:1,000, Cell Signaling Technology, Beverly, MA), and chromogranin A (CgA) (1:1,000, Zymed Laboratories, San Diego, CA). Primary antibody incubations were kept overnight at 4°C and then, depending on the antibody, membranes were washed three times for 5 minutes or three times for 10 minutes in wash buffer (0.1% Tween 20 in phosphate-buffered saline). Next, the membranes were incubated with a 1:2,000 dilution of horseradish peroxidase–conjugated anti-mouse secondary antibody (for ASCL-1) (Cell Signaling Technology) or anti-rabbit secondary antibody (for Notch-1, acetyl-histone H4 [Lys12], Bcl-XL, Bad, caspase-3, cleaved caspase-3, PARP, CgA, and G3PDH). Membranes were developed by Immun-Star (Bio-Rad Laboratories, Hercules, CA) for CgA, caspase-3, cleaved caspase-3, PARP, and G3PDH or by Super West Femto chemiluminescence substrate (Pierce) for acetyl-histone H4 (Lys12), Notch-1, ASCL-1, Bcl-XL, and Bad, according to the manufacturers' directions.
Statistical Analysis
Analysis of variance with Bonferroni post hoc testing was performed using a statistical analysis software package (SPSS version 10.0, SPSS, Chicago, IL). A p-value of < 0.05 was considered significant.
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RESULTS
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SBHA Activates the Notch-1 Signaling Cascade in TT Cells
It is known that a functional Notch cascade relies on HDAC activity [15], and we previously reported that overexpression of Notch-1 intracellular domain (NICD) inhibits cell proliferation and alters the NE phenotype of TT cells [8]. Therefore, we wanted to determine whether the HDAC inhibitor SBHA was capable of activating the Notch-1 signaling cascade in TT cells. We first confirmed the HDAC inhibitory effect of SBHA in cells treated with varying concentrations of SBHA. As shown in Figure 1A, treatment with SBHA led to a dose-dependent increase in histone H4 acetylation. Interestingly, acetylation of histone H4 was detected in as low as 10 µM and higher concentrations of SBHA. Next, we analyzed the cell lysates for expression of Notch-1 signaling cascade proteins. At baseline, no detectable amount of NICD protein was present in TT cells (Fig. 1B). However, SBHA treatment resulted in a dose-dependent induction of NICD (Fig. 1B). To confirm that the induced Notch-1 (NICD) was active, we measured levels of ASCL-1, which is a known downstream target of the Notch-1 signaling pathway. SBHA treatment of TT cells resulted in a dose-dependent decrease in ASCL-1 protein (Fig. 1B), which corresponded to the levels of Notch-1 protein. These results demonstrated that SBHA activates the Notch-1 signaling pathway in TT cells.
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Figure 1. SBHA activates Notch-1 signaling in TT cells. Treatment with SBHA led to a dose-dependent increase in histone H4 acetylation (A); SBHA treatment resulted in a dose-dependent induction of NICD, the active form of the Notch-1 protein. With Notch-1 activation, there was a concomitant decrease in ASCL-1, a downstream target of Notch-1 signaling (B). Abbreviations: AH4, acetyl-histone H4; ASCL-1, achaete-scute complex-like 1; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; NICD, Notch-1 intracellular domain; SBHA, suberoyl bis-hydroxamic acid.
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SBHA Decreases Levels of CgA
NE tumors such as MTC frequently cause debilitating symptoms in patients because of excess tumor secretion of various bioactive amines and peptides. CgA is an acidic glycoprotein that is cosecreted with hormones such as calcitonin by MTC cells. Most NE tumors produce CgA, and the protein is thus a useful marker for this class of malignancies [17]. We previously reported that ectopic expression of Notch-1 in TT cells altered the NE phenotype of these cells by suppressing protein expression of CgA [8]. To study whether SBHA had the ability to alter the NE phenotype in MTC, we performed Western blot analysis for the expression of CgA in TT cells. Because the doubling time of TT cells is long and the appearance of a reduction in either growth or markers is seen after as few as 48 hours, we carried out our experiments for a minimum of 48 hours. After a 48-hour treatment of TT cells with varying concentrations of SBHA, levels of CgA decreased in a dose-dependent manner, indicating a change in the NE phenotype of these tumor cells (Fig. 2).
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Figure 2. SBHA decreases the level of CgA in TT cells. TT cells were treated with SBHA (0–20 µM) for 48 hours and cellular extracts were analyzed by Western blotting for expression of CgA. G3PDH was used to confirm equal protein loading. After SBHA treatment, protein levels of CgA decreased in a dose-dependent manner. Abbreviations: CgA, chromogranin A; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; SBHA, suberoyl bis-hydroxamic acid.
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DOWNREGULATION OF NOTCH-1 EXPRESSION BY RNA INTERFERENCE BLOCKS THE EFFECTS OF NE MARKER REDUCTION BY SBHA IN TT CELLS
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To determine whether the Notch-1 signaling cascade is necessary for the effects of SBHA on NE tumor markers, TT cells were transiently transfected with siRNA against Notch-1, nonspecific siRNA, or vehicle (Lipofectamine) alone. On the next day, transfected cells were treated with 15 µM SBHA or DMSO for 2 days and cell lysates were analyzed by Western blot. As shown in Figure 3, no active Notch-1 protein (NICD) was present in DMSO-treated cells transfected with siRNA or vehicle. As expected, the absence of NICD was associated with no effect on ASCL-1 and CgA. On the other hand, SBHA-induced NICD expression in TT cells transfected with nonspecific siRNA or vehicle resulted in a marked decrease in NE tumor markers. As predicted, NICD protein was absent in SBHA-treated cells with siRNA against Notch-1. More importantly, the absence of NICD in these cells resulted in no effect on the levels of ASCL-1 and CgA. These results indicate that SBHA-associated changes in NE tumor markers in TT cells are dependent upon induction of Notch-1.
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Figure 3. Notch-1 RNA interference blocks the effects of SBHA in TT cells. Cells were transfected with nontargeting or targeting Notch-1 siRNA, and then treated with SBHA (15 µM) for 48 hours. With Notch-1 inhibition by targeting siRNA, TT cells did not show a reduction in either ASCL-1 or CgA compared with the control group. Abbreviations: ASCL-1, achaete-scute complex-like 1; DMSO, dimethylsulfoxide; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; NICD, Notch-1 intracellular domain; NS, nonspecific siRNA; SBHA, suberoyl bis-hydroxamic acid; siRNA, small-interfering RNA.
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SBHA Inhibits TT Cell Proliferation
After confirming that SBHA activates Notch-1 signaling and alters the NE phenotype in MTC cells in a Notch-1–dependent manner, we used the MTT assay to examine the effects of SBHA on cellular proliferation. Treatment of TT cells for up to 6 days with SBHA (0–20 µM) resulted in the inhibition of cell growth in a dose-dependent manner (Fig. 4). At 6 days of 15 µM of SBHA treatment, growth was inhibited by 50% relative to control cells. Moreover, the dose-dependent growth-inhibitory effect was seen as early as 48 hours after 10 µM of SBHA treatment.
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Figure 4. SBHA suppresses growth of TT cells. TT cells were treated with SBHA (0–20 µM) for up to 6 days, and cell viability was measured with the MTT assay. The results indicate that SBHA led to the inhibition of cell growth in both a dose-dependent and time-dependent manner in TT cells. Abbreviations: MTT, methylthiazolyldiphenyl-tetrazolium bromide; OD, optical density; SBHA, suberoyl bis-hydroxamic acid; siRNA, small-interfering RNA.
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SBHA Regulates the Expression of Apoptotic Mediators and Induces Caspase-Dependent Apoptosis in TT Cells
Next, we explored the mechanism of SBHA-induced growth inhibition in TT cells. We carried out Western blot analysis for apoptotic mediators after SBHA treatment for 2 days. PARP is a well-known marker of apoptosis and caspase-3 is the final executioner of both the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways [18]. As shown in Figure 5, Notch-1 activation with SBHA resulted in cleavage of both PARP and caspase-3, indicating that the observed inhibition of TT cell growth was a result of apoptosis. To confirm SBHA-induced apoptosis, we evaluated the expression of the antiapoptotic protein Bcl-XL and the proapoptotic protein Bad in TT cells after treatment with SBHA for 48 hours. As shown in Figure 5, Bcl-XL expression was markedly downregulated after incubation with SBHA. In contrast, the expression level of Bad was increased after the addition of SBHA.
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Figure 5. SBHA regulates the expression of apoptotic mediators and induces apoptosis. SBHA treatment resulted in increased caspase-3 and PARP cleavage, indicating activation of apoptotic pathways. After SBHA treatment, the antiapoptotic protein Bcl-XL was markedly downregulated. In contrast, the expression level of Bad, a proapoptotic protein, was upregulated. Abbreviations: G3PDH, glyceraldehyde 3-phosphate dehydrogenase; PARP, cleaved caspase-3, poly (ADP-ribose) polymerase; SBHA, suberoyl bis-hydroxamic acid; siRNA, small-interfering RNA.
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DISCUSSION
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Notch-1 is a multifunctional protein that regulates cellular differentiation, development, proliferation, and survival in a variety of contexts [19]. Both the Notch-1 receptor and its ligands are transmembrane proteins with large extracellular domains. Binding of ligand and two proteolytic cleavages in the Notch-1 receptor results in the release of NICD. Once released, NICD translocates to the nucleus and regulates various target genes such as hairy enhancer of split (HES)-1 [20–25], thereby modulating the development and growth of cells. Notch-1 has been shown to play an essential role in the embryologic development of NE cells in the lung and gastrointestinal tract [19–22, 26]. We have previously shown that the Notch-1/HES-1/ASCL-1 signaling pathway plays an important role in regulating the differentiation and growth of NE tumors [8, 27, 28].
Notch-1 signaling is absent in human MTC tumor tissue and MTC cell lines. Furthermore, MTC tumors and cells express high levels of ASCL-1 and CgA [8]. Activation of doxycycline-inducible Notch-1 in MTC cells led to a dose-dependent increase in Notch-1 protein and concomitant reduction in NE tumor markers [8]. We also observed that activation of Notch-1 significantly reduced the growth of MTC cells, and the reduction in growth was dependent on the level of Notch-1 protein [8]. These observations clearly supported the hypothesis that Notch-1 is acting as a tumor suppressor in MTC.
Recently, we and others reported that the HDAC inhibitor VPA activates Notch-1 signaling in human carcinoid cancer and neuroblastoma cell lines [9, 10]. Therefore, we hypothesized that similar effects might occur with SBHA, another HDAC inhibitor. In the current study, we demonstrated that SBHA activates the Notch-1 signaling cascade in a dose-dependent manner in MTC cells. Activation of Notch-1 with SBHA resulted in decreased levels of ASCL-1 and CgA, which supports our earlier findings that Notch-1 signaling inhibits NE tumor markers in MTC cells. Importantly, expression of ASCL-1 and CgA did not decrease in MTC cells treated with SBHA if Notch-1 signaling was blocked by Notch-1–specific siRNA. These results indicated that Notch-1 signaling cascade activation is required for NE tumor marker reduction. Furthermore, the antitumor effects of SBHA were confirmed by cell viability analyses, which showed that SBHA inhibits cell proliferation in a dose-dependent manner.
The observed effects of SBHA in MTC cells may be a result of its activity as an HDAC inhibitor. Because the regulation of the Notch signal transduction pathway has been associated with HDAC activity, SBHA might affect the Notch-1 signaling cascade at different levels simultaneously. It is known that modification of histones by acetylation and deacetylation affects chromatin structure and plays an important role in the regulation of gene transcription and expression. Therefore, we suspect that the induction of Notch-1 by SBHA in MTC cells may be at the transcriptional level. Recent results have shown that Notch-1 expression regulates cell death through apoptosis [29–31]. In this study, we observed that levels of active caspase-3, an important effector of apoptotic cell death, increased in MTC cells treated with SBHA. Proper functioning of active caspase-3 was subsequently demonstrated by the detection of one of its cleaved substrates, PARP. Furthermore, we found that SBHA downregulated the antiapoptotic protein Bcl-XL. In contrast, it induced upregulation of the proapoptotic protein Bad. These observations suggest that, after Notch-1 signaling activation, SBHA inhibits cell growth through apoptosis in MTC cells. Another hydroxamic acid HDAC inhibitor, suberoylanilide hydroxamic acid, has been reported to induce caspase-independent apoptosis, whereas apoptosis induced by SBHA was dependent on activation of caspases [32]. The apoptotic pathway induced by SBHA may therefore be novel and complementary to other apoptosis-inducing agents. We have also recently shown that treatment of human carcinoid cancer cell lines with the HDAC inhibitor VPA resulted in induction of p21 and p27 and downregulation of cyclin D1, indicating cell-cycle arrest [32]. It is apparent that HDAC inhibitors such as SBHA and VPA have important antiproliferative effects in a variety of NE tumors, but that the primary mechanism of this growth suppression, either cell-cycle arrest or apoptosis, varies by the type of NE cancer.
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CONCLUSION
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In summary, our results demonstrate that the HDAC inhibitor SBHA has antiproliferative and proapoptotic effects in MTC cells. These effects are mediated by induction of the Notch-1 signaling cascade. These findings suggest that Notch-1 activation with HDAC inhibitors such as SBHA may present a promising new form of targeted therapy for the treatment of patients with MTC.
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ACKNOWLEDGMENTS
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The authors acknowledge financial support from American Cancer Society Research Scholars Grant 05–08301TBE; National Institutes of Health Grants DK064735, DK066169, and CA109053; American College of Surgeons George H.A. Clowes Jr. Memorial Research Career Development Award; Vilas Foundation Research Grant; Carcinoid Cancer Foundation Research Grant; Association for Academic Surgery Karl Storz Endoscopy Research Grant; Doctors Cancer Foundation Award; and the Society of Surgical Oncology Clinical Investigator Award.
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FOOTNOTES
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Conception/design: Li Ning, David Yu Greenblatt, Muthusamy Kunnimalaiyaan, Herbert Chen
Financial support: Muthusamy Kunnimalaiyaan, Herbert Chen
Administrative support: Muthusamy Kunnimalaiyaan, Herbert Chen
Provision of study materials or patients: Muthusamy Kunnimalaiyaan, Herbert Chen
Collection/assembly of data: Li Ning, David Yu Greenblatt, Muthusamy Kunnimalaiyaan, Herbert Chen
Data analysis and interpretation: Li Ning, David Yu Greenblatt, Muthusamy Kunnimalaiyaan, Herbert Chen
Manuscript writing: Li Ning, David Yu Greenblatt, Muthusamy Kunnimalaiyaan, Herbert Chen
Final approval of manuscript: Li Ning, David Yu Greenblatt, Muthusamy Kunnimalaiyaan, Herbert Chen
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Abstract
Introduction
