A novel HSP90 modulator with selective activity against thyroid cancers in vitro

A novel HSP90 modulator with selective activity against thyroid cancers in vitro Abbas Samadi, PhD,a Peter Loo, BS,a Rithwi Mukerji, MD,a Gemma O’Donnell, PhD,b Xiaqin Tong, MS,b Barbara N. Timmermann, PhD,b and Mark S. Cohen, MD, FACS,a Kansas City and Lawrence, KS

Background. Heat shock protein 90 (HSP90) is a chaperone protein regulating several client proteins involved in thyroid cancer development. The purpose of this study was to mechanistically evaluate a novel, natural product drug with anticancer activity in thyroid cancer cell lines in vitro for future translational applications. Methods. A total of 285 natural plant extracts and compounds were evaluated for anticancer activity by MTS assay. Apoptosis and cell cycle arrest were characterized by annexin V-propidium iodide (PI) flow cytometry. HSP90 and client protein modulation, as well as apoptosis confirmation, as demonstrated by Western blot analysis. Results. Of the 285 compounds and products tested, 45 demonstrated antiproliferative activity in thyroid cancers by MTS assay. BTIMNP_D004 demonstrated the greatest inhibition (IC50 = 155--2,890 nM in thyroid cancers). Activity was cancer-cell selective compared to fibroblasts, with increased potency over 17-AAG in BCPAP, FTC133, and DRO81-1 cells. D004 modulated cell cycle arrest after 18 hours (G1/G0~S and G2/M) with 30% FTC133 cells shifted, 22% BCPAP cells shifted, and 15% SW1736 cells shifted versus controls ( P < .01, P < .01, and P < .05, respectively). A total of 1 mM D004 induced significant apoptosis, with 76% BCPAP cells gated after 18 hours (annexin V-PI staining vs <3% in controls, P < .01; and 80% FTC133 cells vs 4% controls; P < .01). Western blot analysis demonstrated modulation of HSP90 expression levels, with inhibition of HSF-1, AKT, and caspase-3 expression, and cleavage of PARP in both BCPAP and FTC133 cells. Conclusion. BTIMNP_D004 is a novel natural product drug with anticancer activity against thyroid cancers in vitro, and may act through induction of apoptosis, modulation of cell cycle arrest, and modulation of heat shock chaperone proteins including HSP90. These preliminary in vitro data support future preclinical studies for translational applications. (Surgery 2009;146:1196-207.) From the Department of Surgery, University of Kansas Medical Center,a Kansas City; and Department of Medicinal Chemistry, The University of Kansas,b Lawrence KS

THYROID CANCER is the most prevalent endocrine malignancy diagnosed each year and represents 1% of all malignancies worldwide, with papillary cancers representing the majority of cases. In the Presented at the 30th Annual Meeting of the American Association of Endocrine Surgeons, Madison, Wisconsin, May 2--5, 2009. Supported in part by research grants from the National Institutes of Health (P20 RR016443 to B.N.T. and M.S.C.) through the University of Kansas Center for Cancer Experimental Therapeutics (CCET) National Institutes of Health Center of Biomedical Research Excellence (COBRE) Program. Reprint requests: Mark S. Cohen, MD, FACS, Vice Chairman for Research, Department of Surgery, University of Kansas Medical Center, 2035 Sutherland Institute, Mail Stop 2005, 3901Rainbow Boulevard, Kansas City, KS 66160. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2009 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2009.09.028

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United States, thyroid cancers represent the majority of all endocrine malignancies and endocrine cancer deaths, as evidenced by 37,000 new cases diagnosed in 2008 and 1,600 cancer-related deaths in the same year.1 Whereas papillary cancers account for up to 85% of all thyroid malignancies, greater than 80% of these carry an excellent prognosis, with 20-year cause-specific mortality less than 1% after thyroidectomy and frequently radioiodine ablative therapy.2 In contrast, poorly differentiated and recurrent thyroid cancers, medullary cancers, and anaplastic cancers are associated with a much lesser 5- and 10-year disease-specific survival, which is primarily due to a lack of effective systemic therapies.3 Recent discoveries have improved our understanding of the genetic and molecular basis of thyroid cancer, with new therapies and targeted approaches being tested in phase I and II human

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clinical trials worldwide. Papillary thyroid cancers (PTC) have been characterized by alterations of one of several kinases including rearrangements of the RET (RET/PTC) receptor tyrosine kinase (13-43% of cases), point mutations in the BRAF serine/threonine kinase (29--69% of cases), rarely rearrangements of the NRTK1 receptor tyrosine kinase (5--13% of cases), or amplification of the catalytic subunit of phosphatidylinositol-3-kinase (#12% of cases).4-9 Follicular thyroid cancers (FTC), which make up approximately 10--15% of all thyroid cancers, are often associated with Ras oncogene mutations in 40--53% of cases or rearrangements between the PAX8 transcription factor and the peroxisome proliferator-activated receptor (PPAR) in 25--63% of cases.9,10 Medullary thyroid cancers (MTC), which comprise 5--9% of all thyroid malignancies, are familial in 25% of cases (as part of the MEN 2 syndromes) or sporadic in 75% of cases.11 Almost all familial MTC and greater than 50% of sporadic MTC are due to mutations of the transmembrane tyrosine kinase receptor RET proto-oncogene. Recent evidence also points to a high prevalence (#50%) of TP53 mutations in MTC.9 Anaplastic thyroid cancers (ATC), which comprise 1--5% of all thyroid cancers, carry the worst clinical prognosis, with most patients dying of the disease within months of diagnosis. ATC also have mutations in BRAF (10--35% of cases) and Ras proto-oncogenes (2060% of cases), but uniquely have a high prevalence of TP53 mutations (67--88% of cases).12 Heat shock protein 90 (HSP90) is a cellular chaperone protein required for the activation of several eukaryotic protein kinases, including the cyclin-dependent kinase 4. Because multiple oncogenic proteins are substrates for the HSP90--mediated protein folding process, HSP90 has emerged as an exciting target for the development of cancer chemotherapeutics. Examples of client proteins dependent on the HSP90 protein folding machinery include the steroid hormone receptors, AKT, Her2, Raf, Bcr-Abl kinase, MEK, mutant p53, and telomerase.13 Many of these same proteins are part of oncogenic pathways responsible for several different thyroid cancers. Therefore, inhibition of HSP90 results in the simultaneous disruption of multiple signaling nodes and leads to induction of apoptosis. Currently, there are more than 20 clinical trials in progress based on HSP90-targeted drugs, and several reports have attempted to explain the high level of differential selectivity observed for HSP90 inhibitors.14 This combination of attributes makes HSP90 a novel target for the development of new drugs. To date, most clinical and translational

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research on HSP90 inhibitors has focused on inhibitors such as 17-allylamino-17-demethoxygeldanamycin (17-AAG).14 Some difficulties encountered clinically with this inhibitor include hepatotoxicity, the need to administer the drug as an intravenous infusion, and the low numbers of patients with metastatic thyroid cancer who have responded to this therapy. These issues highlight a critical need for novel and improved chemotherapeutics against this disease. Medicinal plants and their derivatives have become increasingly important in drug discovery for the treatment of human diseases including cancer. The Solanaceae is a plant family produce withanolides, of which the most important and well-described is Withaferin A.15 These compounds exert a number of different effects including antistress immunomodulatory, cardiac, and cytotoxic activities.16 Their roles as anticancer agents in thyroid cancer are as yet undefined. Although research in recent years has generated new targeted therapies in thyroid cancer, a gap remains in the treatment of anaplastic tumors and recurrent papillary cancers not amenable to operative resection. The search for novel small-molecule inhibitors with good efficacy against anaplastic and recurrent PTC with a low toxicity profile is critical to advancing the treatment of these malignancies. The purpose of this study is to evaluate a novel natural product drug for anticancer activity in thyroid cancers in vitro and its potential mechanism of action, including modulation of heat shock chaperone proteins in multiple thyroid cancer cell lines, for future translational applications. MATERIALS AND METHODS Cell culture conditions. Established human thyroid cancer cell lines were grown in selective media. Papillary cancer cells (BCPAP and TPC1; obtained from Rebecca Schweppe, MD, University of Colorado, Denver, CO) and anaplastic cells (SW1736; obtained from Nils-Erik Heldin, MD, PhD, Dept of Genetics and Pathology, Rudbeck Laboratory, Uppsala, Sweden) were cultured in T75 cell culture flasks using Dulbecco’s modified Eagle’s medium ([DMEM] for BCPAP) or Roswell Park Memorial Institute (RPMI) medium 640 (for TPC1 and SW1736) (flasks and media obtained from Sigma Chemicals, St. Louis, MO) with 10% (w/v) fetal bovine serum (FBS; Sigma), and a standard 13 antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA). Follicular cancer cell lines (FTC133, FTC236, and FTC238) were obtained from Peter Goretzki, MD, (Department of Surgery, Lukaskrankenhaus,

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University Dusseldorf, Dusseldorf, Germany) and established from a single patient (FTC236 from a lymph node metastasis and FTC238 from a lung metastasis). These cells were also cultured in T75 cell culture flasks using DMEM/F-12 media (1:1) with glutamine (0.365 g/L) and 10% (w/v) FBS, supplemented with 13 penicillin-streptomycin solution, insulin, and human TSH (all materials obtained from Sigma). MTC (DRO81-1) cells (gifts from G. Juillard, MD, University of California, Los Angeles, CA) were cultured in T75 cell culture flasks using DMEM with glutamine, 10% (w/v) FBS, 13 penicillinstreptomycin solution, and amphotericin B (0.25 mg/mL) (all materials obtained from Sigma). All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO2. D004 stock solution (10 mM) was prepared in dimethyl sulfoxide (DMSO; D004 was purified by Dr Barbara Timmermann, Department of Medicinal Chemistry, University of Kansas, Lawrence, KS). For drug treatment studies, cell media were switched to 2% FBS to decrease fetal serum--related protein effects on Western blot analysis. Cell proliferation assay. Once 75% confluent, cells were trypsinized (0.25% trypsin), counted, and plated in 96-well microtiter plates (Costar, Cambridge, MA, USA) (2 3 103 cells/well) in 100 mL of growth media. After an overnight attachment period, cells were treated with varying concentrations of D004 for 3 days. All studies were performed in triplicate and repeated at least 3 times independently. After the 3-day treatment period, the number of viable cells was determined using a colorimetric assay for cell proliferation (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay; Promega, Madison WI), which measures the bioreduction of the MTS (3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay by dehydrogenase enzymes of metabolically active cells into a water-soluble formazan product in the presence of the electron coupling reagent phenazine methosulfate (PMS). To perform the assay, 20 mL of combined MTS/ PMS solution containing 2 mg/mL MTS and 150 mmol/L PMS in buffer (0.2 g/L KCl, 8.0 g/L NaCl, 0.2 g/L KH2PO4, 1.15 g/L, 133 mg/mL CaCl2-2H2O, 100 mg/mL, MgCl2.6H2O, pH 7.35) was added to each well. After 3 hours of incubation at 37°C in a humidified 5% CO2 atmosphere, absorbance was measured at 490 nm in a microplate reader. Triplicate wells with predetermined cell numbers were subjected to the assay described above in parallel with the test samples to normalize the

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absorbance readings. This procedure also provided internal confirmation that the assay was linear over the range of absorbance and cell numbers measured. Data were plotted as a function of the percent viability from controls (cell viability) versus drug concentration (x-axis). The concentration of drug at which 50% of cells were inhibited from growth (IC50 level) was determined as the point of inflection on a standard absorbance-concentration curve. Drugs demonstrating activity on MTS assay were then confirmed using a trypan blue dye exclusion method. A total of 30,000 cells were incubated overnight in 60-mm glass petri dishes and then treated with indicated concentrations of D004 for 24 hours. Cells were collected, trypsinzed, and counted by the viability method of trypan blue exclusion using standard protocols. Viable cells demonstrated exclusion of trypan blue as a percentage of total cells. Plant library screens. A screening analysis for anticancer activity was performed on a subset of samples that demonstrated antioxidant properties, which were obtained from the medicinal plant library at the University of Kansas (laboratory of B.N.T.). This subset included 285 extracts or purely derived compounds. Activity was measured using the MTS assay (run in triplicate); positive activity was defined for those natural product compounds and extracts that demonstrated IC50 values less than 20 mM in at least 2 different thyroid cancer cell lines. MTS was chosen as an initial assay screen to quantitatively compare the effect of drug extract compounds on cell viability. Because MTS is quantitative and uses an inexpensive absorbance assay, it could easily be used to screen a greater number of compounds obtained from the library. The assay can also compare potency of drug compounds on cell growth in a number of different cancer cell lines including normal cells. In addition to MTS, we used a trypan blue viability screen to confirm changes in cell viability with drug treatment. With this method, viable cells do not take up the trypan blue and the cells can be counted and compared quantitatively with control levels. A threshold of 20 mM concentration was chosen based on in vitro data from other clinically applicable drug compounds, such as 17-AAG; these data suggested that concentrations greater than 20 mM would not be achievable in a clinical application. Therefore, compounds with activity below this threshold were felt to be of more interest for further analysis. Western blot analysis. Thyroid cancer cell lines described above were grown (80% confluence) in 100-mm plates. At the end of treatment, cells were washed twice in cold phosphate-buffered saline

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Fig 1. Antiproliferative effects of D004 and 17-AAG on thyroid cancer cell lines by MTS assay in vitro. The viability of D004--treated (A) or 17-AAG--treated (B) thyroid cancer cells (expressed for each cell line as a percentage of its respective control condition viability with error bars) after 72-hour treatment in papillary (BCPAP and TPC1), anaplastic (SW1736), follicular (FTC133, FTC236, and FTC238), and medullary thyroid cancer (DRO81-1) cells, as well as normal lung fibroblast MRC-5 cells (dotted line). IC50 and IC70 values for each cell line treated are listed in the Table. MTS data were confirmed by trypan blue viability exclusion and total cell counting with a hemocytometer. Follicular thyroid cancer cell FTC133, anaplastic thyroid cancer cell SW1736, and papillary thyroid cancer cells TPC1 were treated with indicated concentrations of D004 for 24 hours, and the total cell number was counted by a hemocytometer (A and B). Results are presented as mean ± SD of 4 to 6 independent experiments. Each cell line shows a decrease in cell number and viability with increasing drug concentration. (C) Using the trypan blue viability method, 30,000 cells were incubated overnight in 60-mm glass petri dishes and then treated with the indicated concentrations of D004 for 24 hours. Cells were collected, trypsinzed, and counted. Viable cells demonstrated exclusion of trypan blue as a percentage of total cells.

(PBS) and treated with 0.25% trypsin to remove adherent cells from the culture plate. The cells were then collected by centrifugation (750g for 5 minutes) and washed once with cold PBS. The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 6.8, 150 mM NaCl, 1% [v/v] Nonidet P-40 [NP-40], 0.5% [w/v] sodium deoxycholate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 mM sodium

pyrophosphate, 0.1% [w/v, sodium dodecyl sulfate [SDS]) supplemented with 1 3 antiprotease solution (Calbiochem, San Diego, CA). After lysis on ice for 20 minutes, protein samples were collected from the supernatant after centrifugation of the samples at 12,000g for 20 minutes, and protein levels in whole cell lysates were determined using the bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford,

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Table. IC50 levels in thyroid cancer cell lines Cell type BCPAP (papillary thyroid cancer) TPC1 (papillary thyroid cancer) SW1736 (anaplastic thyroid cancer) FTC133 (follicular thyroid cancer) FTC236 (follicular thyroid cancer) FTC238 (follicular thyroid cancer) DRO81-1 (medullary thyroid cancer) MRC-5 (normal lung fibroblast cells)

IC50* D004 (nM) 155 2,890 2,540 2,150 745 770 689 5,750

± ± ± ± ± ± ± ±

15 35 20 25 30 25 30 35

IC50 17-AAG (nM) >20,000 1,750 ± 25 1,470 ± 35 >20,000 315 ± 20 180 ± 15 2,410 ± 30

IC70 D004 (nM) 205 3,180 3,210 2,825 2,450 2,080 1,005 7,450

± ± ± ± ± ± ± ±

10 35 40 40 48 35 15 50

IC70 17-AAG (nM) >20,000 1,840 ± 30 2,050 ± 35 >20,000 >20,000 340 ± 15 >20,000

The cell proliferation of D004-treated and 17-AAG treated cells were measured by MTS assay. *IC50 level is expressed for each cell line in nanomolar concentration (average of 3 experiments for each condition ± standard deviation) and is defined as the concentration of D004 required to reduce cell proliferation/viability by 50% compared to controls.

IL). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto nitrocellulose membranes (Hybond; Amersham, Piscataway, NJ). Membranes were blocked with PBS supplemented with 3% (w/v) nonfat dry milk (Bio-Rad Laboratories, Hercules, CA), and probed with the appropriate dilution of primary antibody overnight at 4°C. The blots were washed 3 3 in PBS buffer with Tween-20 for 10 minutes, and then incubated with 1:10,000 dilution of horseradish peroxidase (HRP)conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS-Tween 20 at room temperature for 1 hour. After washing 3 3 in PBS-Tween 20 for 10 minutes, the proteins were visualized by enhanced chemiluminescence (Amersham) was captured on Kodak XAR-5 film (Eastman Kodak, Rochester, NY). Where indicated, the blots were reprobed with antibody against betaactin (Santa Cruz Biotechnology) to ensure equal loading and transfer of proteins. The primary antibodies to phospho(Thr202/ Tyr204)-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-AKT and AKT, procaspase-3, and cleaved poly(adenosine diphosphate-ribose) polymerase (PARP) were obtained from Cell Signaling Technologies (Beverly, MA). Antibodies against HSF-1, HSP90a, HSP90ß, and HSP72, were purchased from Stressgen (Ann Arbor, MI); and secondary antibodies against ERK1/2 and phospho-ERK were obtained from Santa Cruz Biotechnology. Cell cycle arrest analysis. Thyroid cancer cells at 80% confluence were grown overnight and treated with 1 mM D004. After trypsinization, cells (1 3 106 cells) were washed with 0.9% NaCl and fixed with 70% cold ethanol for 30 minutes at room temperature. Cells were collected by centrifugation (700g for 5 minutes) and stained with propidium iodide (PI) (50 mg/mL in PBS for 30 minutes. Cells then were treated with DNAse-free RNAse (1 mg/mL) for 30 minutes and analyzed by flow cytometry.

Apoptosis analysis. Preliminary studies to evaluate for induction of apoptosis were also performed including annexin V-PI costaining with flow cytometry. An analysis of phosphatidylserine (PS) on the outer leaflet of apoptotic cell membranes was performed using annexin V-fluorescein isothiocyanate (FITC) and annexin V-PI to distinguish between apoptotic and necrotic cells. Cells were cultured to monolayer confluence in 100-mm petri dishes for 24 hours then rinsed twice with PBS and trypsinzed and cultured in 6-well plates (1 3 105 cells/mL) for 24 hours. After treatment with D004, media were removed, and cells were washed with PBS. Cells were trypsinized, washed with PBS, and suspended in 1 3 annexin V binding buffer at a concentration of 1 3 106 cells/mL. Cells were transferred to a culture tube, and annexin-V and PI (BD Pharmingen, San Diego, CA) were added; after gentle vortex, the cells were incubated for 20 minutes at room temperature in the dark. After adding 400 mL of 1 3 annexin V binding buffer to each tube, cells were analyzed by flow cytometry (BD LSR II Flow Cytometer; Becton Dickinson, San Diego, CA). Statistics. All result data points were run in triplicate and expressed as a mean ± SEM. Raw data were analyzed by the Student unpaired t test and the Fisher exact test using statistical software (Prism 5.0; GraphPad Software, La Jolla, CA). Significance was defined as a P value < .05. RESULTS Inhibition of thyroid cancer proliferation in vitro. Of the 285 natural product compounds and extracts tested, 45 (16%) met criteria for activity, with IC50 values in several cancer cell lines below 20 mM drug concentration. However, of this group of extracts and pure compounds with activity, only 1 compound---BTIMNP_D004 (D004)--demonstrated IC50 levels in the nanomolar concentration range. Using MTS assays, the in vitro

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Fig 2. D004 treatment alters cell cycle arrest mechanisms. BCPAP, FTC133, and SW1736 cells at 80% confluence were grown overnight and treated with 1 mM D004. After trypsinization, cells were washed with saline and fixed with 70% cold ethanol for 30 minutes at room temperature. Cells were collected by centrifugation and stained with propidium iodide (50 mg/mL in phosphate-buffered saline for 30 minutes). Cells then were treated with DNAse-free RNAse (1 mg/mL) for 30 minutes and analyzed by flow cytometry. Propidium iodide--staining flow cytometry histograms are depicted on the left (controls) and the middle (treated group) for each cell type. The bar graphs demonstrate quantitative percentages of cells in the different cycles of the cell. A total of 1 uM D004 induced a shift in cell cycle arrest (normally arrested in G0/G1), with an increase in the percentage of cells in the G2-M and S phases as follows: 30% cells shifted out of G0/ G1 into G2/M in FTC133 cells (P < .01 compared to controls), 22% shifted in BCPAP cells (P < .01 compared to controls), and 15% shifted in SW1736 cells (P < .05 compared to controls). Of note, changes in the sub-G0 population are suggestive of DNA fragmentation that can represent early apoptosis.

activity of D004 was evaluated against a diverse panel of 7 thyroid cancer cell lines (including papillary, follicular, medullary, and anaplastic cells) and normal lung fibroblasts (Fig 1, A). In these cell lines, we observed that D004 inhibited cell proliferation in all the cells tested; however, it was at least 2-fold more potently selective against thyroid cancer cells (IC50 range, 155 nM--2.89 mM) compared to the normal MRC-5 fibroblasts (IC50 = 5.75 mM; Table). For comparative purposes, MTS data were also generated in the same cell lines for 17-allylamino17-demethoxygeldanamycin (17-AAG), a wellknown HSP90 inhibitor used in clinical trials (MTS cell viability dose-response curves for 17AAG are shown in Fig 1, B). Although D004 and 17-AAG demonstrated inhibition of thyroid cancer cell growth in vitro, there were several differences in the cell-specific response observed between the 2 compounds. Most notably, D004 had significantly increased potency compared to 17-AAG when exposed to BCPAP papillary cancer

cells (IC50D004 = 155 nM; IC5017AAG > 20,000 nM; P < .001), FTC133 follicular cancer cells (IC50D004 = 2.15 mM; IC5017AAG > 20 mM; P < .01), and DRO 81-1 medullary cancer cells (IC50D004 = 0.69 mM; IC5017AAG = 2.41 mM; P < .05). 17-AAG had lower IC50 levels (1.7- to 4-fold difference) in SW1736, FTC236, FTC238, and TPC1 cells. Interestingly, D004 was able to achieve IC70 levels (70% cell growth inhibition) in all thyroid cancer cell lines tested (with a range of 205 nM in BCPAP cells to 3.21 mM in SW1736 cells), whereas 17-AAG did not reach this threshold in 4 cell lines (FTC133, FTC236, BCPAP, and DRO811) at concentrations as high as 20 mM. IC50 and IC70 levels for each cell line tested are listed in the Table. To confirm the results observed with MTS assay, TPC1, FTC133, and SW1736 cells were treated with trypan blue and counted for viability. With increasing concentrations of D004 treatment, all 3 cell lines demonstrated a dose-dependent decrease in cell viability, both by hemocytometer

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Fig 3. D004 induces apoptotic cell death in thyroid cancer cells in vitro. (A) Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay was performed as follows: follicular (FTC133) and papillary (BCPAP) thyroid cancer cells were incubated with either 1% dimethyl sulfoxide (DMSO) or D004 (1 mM) for 18 hours. Cells were then incubated with annexin V-FITC and PI and analyzed by flow cytometry. The number of events analyzed for each condition was 10,000. A total of 3 independent experiments were performed, and the percentage of annexin-positive cells in each treatment protocol was determined. Both cell lines demonstrated a shift to the lower right quadrant with treatment, indicating gating of cells toward apoptotic cell death. In BCPAP cells, this gating occurred in 76% of cells compared to 3% in controls (P < .01); in FTC133 cells, however, this gating occurred in 80% of cells compared to 4% in controls (P < .01). In addition, apoptosis was evaluated by annexin V/PI assay in DRO81-1 medullary thyroid cancer cells to compare similar concentrations of D004 with 17-AAG treatment. D004 demonstrated a greater level of apoptotic cells with treatment compared to 17-AAG throughout the dose range tested. Apoptosis was confirmed in BCPAP and FTC133 cells by Western blot analysis (B). D004 induced a decrease in the level of inactive caspase-3 and cleaved its substrate poly(adenosine diphosphate-ribose) polymerase (PARP) after 24 hours of exposure to drug. BCPAP cells demonstrated PARP cleavage at a concentration of D004 as low as 100 nM. These data support the hypothesis that D004 treatment induces apoptosis in thyroid cancer cells in vitro.

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Fig 4. D004 modulates heat shock protein expression in thyroid cancer cells. Follicular (FTC133) and papillary (BCPAP) thyroid cancer cells were treated with 1% dimethyl sulfoxide (DMSO) (as control) or D004 at concentrations from 0.1 to 3 mM for 24 hours. Lysates were prepared and subjected to Western blotting with the indicated antibodies (left). Protein expression densitometry was then normalized to beta-actin levels and compared (results shown in the bar graphs, right) Both cell lines demonstrated a dose-dependent decrease in HSF-1 expression levels, with BCPAP showing a decrease in HSF-1 protein levels after exposure to only 250 nM D004. HSP90a expression levels decreased in BCPAP cells with drug treatment, but increased in FTC133 cells. HSP70 levels increased in both cell lines in a dose-dependent manner. HSP90b levels initially decreased in FTC133 cells, but then returned to normal levels at greater concentrations. These data suggest that D004 is modulating heat shock protein expression.

counting and trypan blue exclusion percentage (Fig 1, C). Induction of cell cycle arrest and apoptosis. Treatment of papillary (BCPAP), follicular (FTC133), and anaplastic (SW1736) thyroid cancer cells with 1 mM of D004 induced a shift in cell cycle arrest (normally arrested in G0/G1)with an increase in the percentage of cells in the G2/M, and S phases (30% cells shifted out of G0/G1 into G2/M in FTC133 cells (P < .01 compared to controls), 22% shifted in BCPAP cells (P < .01 compared to controls), and 15% shifted in SW1736 cells (P < .05 compared to controls). Figure 2 shows PI staining of cells for cell cycle analysis by flow cytometry. Next, drug-induced apoptosis was evaluated by 2 methods using annexin V-PI costaining and flow

cytometry analysis as an initial method followed by confirmation with demonstration of caspase-3 activation and PARP cleavage by Western blot analysis (Fig 3, A and B). D004 greatly increased the population of BCPAP (76 vs 3% controls; P < .01) and FTC133 (80 vs 4% controls; P < .01) in cells stained with annexin V and PI after 24 hours of treatment with 3 mM D004. Of note, less than 3% of cells died by necrosis. In MTC cells (DRO81-1), D004 demonstrated significantly increased levels of apoptotic cells by annexin V-PI staining on flow cytometry over a variety of doses, with the maximal difference noted at 3 mM (23% D004--treated cells vs 5% of 17AAG--treated cells gated to apoptosis; P < .01) compared to treatment with 17AAG at the same concentrations (Fig 3, A, bottom). Confirmation of apoptosis by Western blot analysis demonstrated

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that 24 hours of treatment with as little as 1 mM of D004 induced activation of caspase-3 (evidenced by a decrease in inactive caspase-3 levels) in both BCPAP and FTC133 cells, with BCPAP cells showing caspase-3 activation at only 250 nM of drug exposure. Both cell lines also exhibited a dose-dependent cleavage of PARP, with BCPAP cells demonstrating PARP cleavage with only 100 nM of drug exposure (Fig 3, B). Modulation of HSP90 and client proteins. To determine if D004 was indeed a modulator of heat shock chaperone protein expression, including HSP90 expression, Western blot analysis was performed on thyroid cancer cell lines (papillary [BCPAP] and follicular [FTC133]) after treatment with a range of D004 concentrations over 24 hours (Fig 4). The heat shock proteins (HSP) evaluated included the most common isoforms of HSP90 (alpha and beta), as well as HSP70 and HSF-1, which is a transcription factor leading to downstream modulation of HSP levels. Again, both BCPAP papillary cancer cells and FTC133 follicular cancer cells demonstrated a dose-dependent decrease in HSF-1 levels after D004 treatment. In BCPAP cells, HSF-1 expression decreased after exposure to only 100 nM of drug. Normalizing densitometry ratios to beta-actin levels, HSP90a expression levels in BCPAP cells decreased by 50% after treatment with 3 mM D004. Conversely, in FTC133 cells, increasing drug concentration led to slightly increased levels of HSP90a expression. In both cell lines, HSP70 levels increased with drug treatment in a somewhat dose-dependent manner, with maximal expression at D004 concentrations between 0.5 and 2 mM. Finally, in FTC133 cells, HSP90b expression levels seem to be decreased at lower drug concentration levels, but return to normal expression with increasing drug doses, paralleling to an extent the increases observed with HSP70 expression. These data suggest that D004 may have a role in heat shock chaperone protein modulation, including modulation of HSP90 (Fig 4). With evidence of modulation of heat shock chaperones on Western blot analysis, the next experiment evaluated the effect of D004 on protein levels of HSP90 clients. Because it is well known that HSP90 client proteins, including AKT and ERK, are involved in prosurvival cancer pathways, we used Western blot analysis to determine the effect of D004 treatment on the expression levels of these HSP90 client proteins, specifically AKT and ERK1/2 (Fig 5). D004 downregulates total but, more importantly, phospho-AKT expression in a dose-dependent manner in FTC133 cells, with a 75% decrease in protein expression

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Fig 5. Modulation of HSP90 client protein expression (D004 modulates AKT and ERK expression levels in thyroid cancer cells). Follicular (FTC133) and papillary (BCPAP) thyroid cancer cells were treated with 1% dimethyl sulfoxide (DMSO) (as control) and D004 at concentrations from 0.1 to 3 mM for 24 hours. Lysates were prepared and subjected to Western blotting with the indicated antibodies. Protein expression densitometry was normalized for beta-actin levels (the factor difference from control is in blue above each band in the blots). D004 decreased phospho-AKT levels in FTC133 cells in a dose-dependent manner, but had no effect on phospho-ERK or total-ERK levels. In contrast, BCPAP cells demonstrated a dose-dependent decrease in phosphorylated ERK levels after 24 hours of D004 treatment.

levels after exposure to only 100 nM D004. The FTC133 cell line showed no change in phosphoERK and total ERK expression levels over the range of D004 doses tested. Papillary (BCPAP) cells, however, demonstrated a dose-dependent decrease in phospho-ERK expression after 100 nM of drug exposure, with almost complete inhibition of expression at 3 mM of D004 without significant change in total ERK expression. DISCUSSION As a molecular chaperone, HSP90 is responsible for properly folding many of the proteins directly associated with malignant progression; thus, inhibition of the HSP90 protein folding machinery results in a combinatorial attack on numerous

Surgery Volume 146, Number 6 pathways.17 Recent studies have demonstrated that HSP90 inhibitors accumulate in tumor cells more efficiently than in normal tissue, leading to high differential selectivities.18,19 Currently, 26 clinical trials targeting HSP90 are in progress, and these studies have demonstrated that HSP90 can be inhibited at doses that are well-tolerated by patients.20-23 Consequently, HSP90 has emerged as a promising therapeutic target for the treatment of cancer, because inhibition of HSP90 results in the simultaneous degradation of multiple anticancer targets.11-14,23,24 This study provides preliminary in vitro data related to the drug BTIMNP_D004, a natural product with anolide that demonstrates selective anticancer activity against thyroid cancer cells in vitro compared to normal fibroblast cells, with anywhere from 2- to 30-fold selectivity depending on the cell type (IC50 levels in BCPAP cells of 155 nM compared to 5.7 mM in MRC-5 lung fibroblasts). In comparing this drug with 17-AAG (a HSP90 inhibitor used clinically) in terms of their respective abilities to decrease thyroid cancer cell growth and viability in vitro as measured by MTS assay, D004 demonstrated significantly increased antiproliferative potency in BCPAP (papillary cancer cells), FTC133 (follicular cancer cells), and DRO81-1 (MTC cells). Although its IC50 levels were not as potent as 17-AAG in TPC1, SW1736, and FTC-238 cells, D004 inhibited greater than 70% of cancer cell growth (IC70) in several of the cell lines at nanomolar and low micromolar doses, whereas 17-AAG could not reach this level of growth inhibition even at 20 mM concentrations. With this preliminary assay data suggesting some anticancer activity specific to thyroid cells, further studies were conducted to explore the mechanism of action by which this cancer cell growth inhibition was occurring. The first set of experiments analyzed the effect of this drug to determine whether it induced apoptosis or modulated cell cycle arrest, or whether cells were dying simply from toxicity and necrosis. In papillary (BCPAP), follicular (FTC133), and anaplastic (SW1736) thyroid cancer cells, D004 shifted these cancer cells out of their normal G0/G1 cell cycle arrest and into S phase and the G2/M checkpoint. In these cell lines, approximately 20 to 30% of arrested cells were allowed to progress through the cell cycle. Allowing cells to come out of arrest and move through the cell cycle could be a potential mechanism by which this drug leads to cancer cell death. This effect was initially demonstrated and presented by our laboratory in NPA and DRO cells (data not shown). However, a recent

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study25 indicated that those cell lines and many others described in the literature were genetically homologous with a melanoma cell line rather than a thyroid cancer cell line; as a result, the data presented in this study are focused only on genetically confirmed thyroid cancer cell lines. Additionally, an increase noted in the sub-G0 population in both cell lines when treated with D004 suggested that the cells may be undergoing DNA fragmentation, which is a biochemical hallmark of apoptosis. Apoptotic mechanisms were then evaluated to confirm this effect. Annexin V-PI staining with flow cytometry analyses demonstrated that there was a significant shift (as high as 76 to 80% with drug treatment) of cells gated toward apoptotic rather than necrotic cell death (Fig 3, B). This effect was confirmed by demonstrating dose-dependent decreases in inactive caspase-3 levels (indicating activation of caspase-3) on Western blot analysis, as well as observation of cleavage of PARP at concentrations of 100 nM D004 in BCPAP cells and 1 mM in FTC133 cells, respectively. In addition to inducing well-differentiated thyroid cancer cells to undergo apoptosis after drug treatment, D004 also induced gating of cells toward apoptotic cell death in DRO 81-1 medullary thyroid cancer cells, which was demonstrated by annexin V-PI staining with flow cytometry analysis. This observation was compared using equivalent doses of 17-AAG and D004 and was found to be a more potent inducer of apoptosis in this cell line over the range of doses tested. In addition to effects on cell cycle arrest and induction of apoptosis in thyroid cancer cells, the last set of experiments performed in this study explored the potential role of D004 in the modulation of chaperone HSP expression and client protein expression levels. Western blot analysis data in BCPAP and FTC133 cells demonstrated that D004 decreased HSF-1 expression and increased HSP70 expression levels in a dose-dependent manner. BCPAP cells treated with drug also showed decreased HSP90a expression levels, with a 50% decrease in expression at a concentration of 3 mM D004. HSP90ß levels were observed to decrease slightly at lower concentrations of drug in FTC133 cells, and these expression levels returned to normal levels at higher concentrations. Although these data on HSP90 in FTC133 are not robust or conclusive, the changes in HSP90ß at higher concentrations appear to parallel increases in expression of HSP70 observed in this cell line with treatment. HSP70 induction has been described to play a role in replenishing

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depleted HSP90 in the cell. This is only 1 potential explanation of the results observed with HSP90ß, and further experiments are needed to repeat and more carefully elucidate the role of D004 on HSP90 modulation and its mechanism of action. Because modulation of HSP expression was observed with D004 treatment, the last experiment performed in this study evaluated the protein expression levels of AKT and ERK (client proteins of HSP90) in BCPAP and FTC133 cells. After normalizing protein expression levels for betaactin levels, BCPAP cells demonstrated a dosedependent decrease in phospho-ERK expression with treatment, even at a drug concentration as low as 100 nM, with almost complete inhibition at 3 mM D004 concentration. FTC133 cells demonstrated a dose-dependent decrease in both phospho-AKT and total AKT levels with D004 treatment but, as expected (because this cell does not have a mutated B-raf gene or an abnormal MAP-kinase pathway), did not show any changes in total or phospho-ERK expression levels. BCPAP cells, conversely, have a homozygous V600E B-raf mutation, and the inhibition of ERK with drug treatment suggests the possibility that this drug, as reported in vitro with 17-AAG, is likely inhibiting the MAPkinase pathway in this cell. Future studies will be necessary to determine whether this inhibition is due to modulation of HSP90 activity and its client proteins or other mechanistic pathways. Overall, these data support the hypothesis that D004, a novel natural product drug compound, has anticancer activity in thyroid cancer cells in vitro. Preliminary data also suggest a possible mechanism of action involving modulation of cell cycle arrest, induction of apoptosis, and modulation of HSP expression including HSP90. Because there is thyroid cancer cell--specific selectivity and potency compared to 17-AAG, this drug may provide a novel avenue of research for targeted drug therapy in thyroid cancers resistant or minimally responsive to 17-AAG and other HSP90 inhibitors. To our knowledge, this report is the first to relate the activity and some of the cellular mechanisms associated with this drug in thyroid cancer cells. Further in vitro and possibly in vivo studies are warranted to more completely define the role of this drug as a potential novel therapy for thyroid cancer. The authors would like to acknowledge Fiemu Nwariaku, MD (UT Southwestern, Dallas, TX), and Rebecca Schweppe, MD (University of Colorado, Denver, CO), for providing us with their thyroid cancer cell lines. We are also grateful to Joyce Slusser, MD, PhD (Flow Cytometry Core Director, University of Kansas Medical

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Center), for assistance with the flow cytometry studies, as well as John Robertson, MD (Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS), for providing the caspase-3 antibody.

REFERENCES 1. American Cancer Society. Cancer Facts & Figures 2008. Atlanta, GA: American Cancer Society; 2008: 5. Available from URL: www.cancer.org. 2. Randolph GW, Thompson GB, Branovan DI, Tuttle RM. Treatment of thyroid cancer: 2007---a basic review. Int J Radiat Oncol Biol Phys 2007;69(2 Suppl):S92-9. 3. Sanders EM Jr, LiVolsi VA, Brierley J, Shin J, Randolph GW. An evidence-based review of poorly differentiated thyroid cancer. World J Surg 2007;31:934-45. 4. Santoro M, Carlomagno F. Drug insight: small-molecule inhibitors of protein kinases in the treatment of thyroid cancer. Nat Clin Pract Endocrinol Metab 2006;2:42-52. 5. Xing M. BRAF mutation in thyroid cancer. Endocr Relat Cancer 2005;12:245-62. 6. Groussin L, Fagin JA. Significance of BRAF mutations in papillary thyroid carcinoma: prognostic and therapeutic implications. Nat Clin Pract Endocrinol Metab 2006;2:180-1. 7. Hou P, Liu D, Shan Y, et al. Genetic alterations and their relationship in the phosphatidylinositol 3-kinase/Akt pathway in thyroid cancer. Clin Cancer Res 2007;13:1161-70. 8. Pierotti MA, Greco A. Oncogenic rearrangements of the NTRK1/NGF receptor. Cancer Lett 2006;232:90-8. 9. Santoro M, Fusco A. New drugs in thyroid cancer. Arq Bras Endocrinol Metabol 2007;51:857-61. 10. Kondo T, Ezzat S, Asa SL. Pathogenetic mechanisms in thyroid follicular-cell neoplasia. Nat Rev Cancer 2006;6:292-306. 11. Schlumberger M, Carlomagno F, Baudin E, Bidart JM, Santoro M. New therapeutic approaches to treat medullary thyroid carcinoma. Nat Clin Pract Endocrinol Metab 2008;4:22-32. 12. Garcia-Rostan G, Costa AM, Pereira-Castro I, et al. Mutation of the PIK3CA gene in anaplastic thyroid cancer. Cancer Res 2005;65:10199-207. 13. Blagg BS, Kerr TD. Hsp90 inhibitors: small molecules that transform the Hsp90 protein folding machinery into a catalyst for protein degradation. Med Res Rev 2006;26:310-38. 14. Messaoudi S, Peyrat JF, Brion JD, Alami M. Recent advances in Hsp90 inhibitors as antitumor agents. Anticancer Agents Med Chem 2008;8:761-82. 15. Yokota Y, Bargagna-Mohan P, Ravindranath PP, Kim KB, Mohan R. Development of withaferin A analogs as probes of angiogenesis. Bioorg Med Chem Lett 2006;16:2603-7. 16. Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern Med Rev 2000;5:334-46. 17. Yu XM, Shen G, Neckers L, et al. Hsp90 inhibitors identified from a library of novobiocin analogues. J Am Chem Soc 2005;127:12778-9. 18. Neckers L. Heat shock protein 90: the cancer chaperone. J Biosci 2007;32:517-30. 19. Neckers L, Kern A, Tsutsumi S. Hsp90 inhibitors disrupt mitochondrial homeostasis in cancer cells. Chem Biol 2007;14: 1204-6. 20. Chaudhury S, Welch TR, Blagg BS. Hsp90 as a target for drug development. ChemMedChem 2006;1:1331-40. 21. Tsutsumi S, Neckers L. Extracellular heat shock protein 90: a role for a molecular chaperone in cell motility and cancer metastasis. Cancer Sci 2007;98:1536-9.

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22. Chiosis G, Huezo H, Rosen N, et al. 17AAG: low target binding affinity and potent cell activity---finding an explanation. Mol Cancer Ther 2003;2:123-9. 23. Banerji U, Judson I, Workman P. The clinical applications of heat shock protein inhibitors in cancer---present and future. Curr Cancer Drug Targets 2003;3:385-90. 24. Sausville EA, Tomaszewski JE, Ivy P. Clinical development of 17-allylamino, 17-demethoxygeldanamycin. Curr Cancer Drug Targets 2003;3:377-83. 25. Schweppe RE, Klopper JP, Korch C, et al. Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab 2008;93:4331-41.

DISCUSSION Dr Herbert Chen (Madison, WI): When you looked at your thyroid cancer cell lines, specifically in looking at the follicular and the medullary, did you see any change in differentiation status of the follicular thyroid cancer cell lines? And then related in the medullary line, did you see any changes in calcitonin expression? Dr Mark S. Cohen (Kansas City, KS): In the medullary line, we did not measure calcitonin from the in vitro cultures. That’s something we plan to do because, obviously, that would indicate a functional inhibition as well as what we see in culture. With regard to the follicular line, we did not observe any change in differentiation. Dr Orlo H. Clark (San Francisco, CA): How did you grow your cells, in what media? And did you try stimulating with thyroid-stimulating hormone (TSH) or epidermal growth factor (EGF) or anything else to see if it changed any of the results of your apoptosis? Dr Mark S. Cohen (Kansas City, KS): The media is very important, we found, because if you do your exper-

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iments in media with 10% fetal serum, you may observe that the effects of your drug on expression of prosurvival and signaling proteins may be significantly affected and even blunted due to the quantity of proteins and antibodies in the serum. For our experiments, we actually did a 2% fetal serum media to negate this effect. With regard to your second question, we typically don’t add supplements such as TSH or EGF to the media for the very reason that it has the potential to change effects or possibly even protect cells from apoptosis. Basically we try to use a very simple media with 2% fetal serum to avoid confounding effects from additives. Discussant from the floor: This seems to generate a lot of tumors; does it also affect the normal cells? It seems very nonselective in a sense. And I’m wondering if you would address that. Dr Mark S. Cohen (Kansas City, KS): That black line in the graph I presented is our dose-response curve for normal lung fibroblasts, and it showed significantly less toxicity, a 2- to 30-fold difference compared to thyroid cancer cells, where it showed pretty significant effects. Discussant from the floor: With regard to the dosage, if you do have to give this to people, is it something that’s tolerable? Is this something that can be achieved in clinical trials? Dr Mark S. Cohen (Kansas City, KS): Well, with IC50 levels in the nanomolar range, given the molecular weight of this small molecule, this would project an easily achievable therapeutic dose in animals, including humans. To verify this, we plan to perform extensive pharmacokinetic analyses in animal models and, from preliminary animal data not presented today, we believe we can achieve therapeutic levels with very small doses, as low as 2--5 mg of drug per kilogram body weight per day. That would be consistent with an achievable level clinically when translating to a human model.