Cancer Letters 362 (2015) 83–96

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

17-DMCHAG, a new geldanamycin derivative, inhibits prostate cancer cells through Hsp90 inhibition and survivin downregulation Jifeng Wang a,1, Zhenyu Li b,1, Zhiyuan Lin a,1, Baobing Zhao b, Yang Wang a, Ruixian Peng a, Meifang Wang a, Chunhua Lu b, Guowei Shi a,*, Yuemao Shen b,** a

Department of Urology, The Fifth People’s Hospital of Shanghai, Urology Research Center, Fudan University, Shanghai 200240, China Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan, Shandong 250012, China b

A R T I C L E

I N F O

Article history: Received 19 December 2014 Received in revised form 17 March 2015 Accepted 17 March 2015 Keywords: Geldanamycin derivative Hsp90 AR Survivin Prostate cancer

A B S T R A C T

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the stability of many client proteins, including androgen receptor (AR) and survivin, making Hsp90 an attractive molecular therapeutic target for prostate cancer. Several Hsp90 inhibitors have shown antitumor activity in various preclinical models and in clinical trials. Geldanamycin is a well-known inhibitor of Hsp90, but its associated liver toxicity limited its clinical development. Here, we report a highly effective and low-hepatotoxic geldanamycin derivative that exhibits antitumor activity against human prostate cancer cells. Treatment of cells with 17-DMCHAG (17-(6-(3,4-dimethoxycinnamamido)hexylamino)-17-demethoxy-geldanamycin) dosedependently suppressed the proliferation, reduced colony formation and induced apoptosis of human prostate cancer cell lines. 17-DMCHAG exhibits anti-invasive and anti-migratory activities in prostate cancer cells through down-regulating of transcription factors Zeb1, Snail1, Slug, and mesenchymal marker Vimentin, while up-regulating the epithelial marker of E-cadherin. Furthermore, 17-DMCHAG treatment damaged the Hsp90/AR and Hsp90/survivin complexes and induced the proteasome-dependent degradation of AR and survivin, then inhibited the activity of these two proteins. In vivo, we observed that 17-DMCHAG showed strong antitumor effects in LNCaP and DU-145 cell-xenografted nude mice. Thus, 17-DMCHAG is a potential treatment for prostate cancer. © 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction Prostate cancer is the most common life-threatening carcinoma in the developed world with increasing rates in the developing world [1,2]. Prostate cancer is often present in multiple locations within the prostate and has variable characteristics. Like many other cancers, prostate cancer has considerable tumor heterogeneity, which has been observed between patients from different geographical and ethnic populations, different individuals within these populations, different tumor foci within the same patient, and different cells within the same tumor focus [3]. The high heterogeneity of prostate cancer affects key cancer pathways, drives phenotypic variation, and presents a significant challenge in the treatment of prostate cancer [4–6].

* Corresponding author. Tel.: +86 21 64300477; fax: +86 21 24289026. E-mail address: [email protected] (G. Shi). ** Corresponding author. Tel.: +86 531 88382108; fax: +86 531 88382108. E-mail address: [email protected] (Y. Shen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2015.03.025 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

Androgen deprivation therapy (ADT) that targets androgen signaling remains the standard treatment for patients with advanced prostate cancer. AR is a nuclear receptor that is activated by binding of the androgenic hormone testosterone or dihydrotestosterone in the cytoplasm; subsequently, AR is translocated into the nucleus where it plays a key role in prostate carcinogenesis and progression [7,8]. AR is a client protein of Hsp90, and it relies on Hsp90 for its function in both normal and malignant prostate cells. Hsp90 inhibition has been shown to disrupt the localization of the AR [9,10]. Moreover, Hsp90 is highly expressed in prostate cancer cells compared with normal prostate epithelium, making it a potential selective target for the treatment of prostate cancer [11–13]. Survivin is a member of the inhibitor of apoptosis (IAP) family with multiple functions [14]. It is highly overexpressed in transformed cell lines and in multiple types of adenocarcinomas, including prostate adenocarcinoma [15]. Increasing evidence has suggested that survivin is associated with the progression of prostate cancer, and it is not expressed in normal prostate secretory epithelial cells, implying that survivin plays an important role in the survival strategies of prostate cancer. Thus, suppressing survivin is considered an attractive therapeutic strategy for prostate cancer therapeutics [16,17]. Geldanamycin (GA) was identified as the first natural product inhibitor of Hsp90 that binds to the N-terminal ATPase domain of

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Fig. 1. 17-DMCHAG inhibits proliferation and induces apoptosis in prostate cancer cells. (A) Chemical structure of 17-DMCHAG. (B) The viability of LNCaP, C4-2B, DU145 and PC-3 cells following 24, 48 and 72-h exposure to various concentrations of 17-DMCHAG. The cell survival rate was calculated using the MTT assay. Each value represents the mean ± SD of three independent experiments. (C) Flow cytometric analysis of 17-DMCHAG-induced cell death in LNCaP and DU145 cells after treatment with various 17-DMCHAG concentrations for 24 h. The data shown represent the mean ± SD (n = 3). *P < 0.05 vs the control. (D) Flow cytometric analysis of 17-DMCHAGinduced cell death in LNCaP and DU145 cells after treatment with 1 μM 17-DMCHAG for 24, 48, or 72 h. The data shown represent the mean ± SD (n = 3). *P < 0.05 vs the control. (E) Colony-forming capability was measured by colony formation assay in DU145, LNCap, PC-3 and RWPE-1 cells after treatment with various 17-DMCHAG concentrations for 10 days. (F) Quantitative analysis of the relative colony number for each group is shown on the right. *P < 0.05 vs the control. (G) Detection of apoptosis using Annexin V-FITC staining. After treatment with 1 μM or 2 μM of 17-DMCHAG for 24 h, LNCaP cells were analyzed using a FACS Calibur flow cytometer. (H) Western blot analysis of apoptosis-related proteins after treatment with 17-DMCHAG. DU-145 cells were treated for the indicated times with different concentrations of 17DMCHAG. The expression levels of Bcl-2 and cleaved caspase-3 were determined by Western blotting. β-actin was used for normalization and verification of protein loading. The relative expression levels of proteins were analyzed by Image J software. Intensity values were expressed as fold change compared to control. Data were collected from 3 independent experiments, and the mean ± S.E. (error bars) was calculated, *P < 0.05 vs the control. (I) Cells were pretreated with z-VAD-fmk at 50 μM for 2 h before treatment of 17-DMCHAG at 1 μM for 24 h, the expression levels of cleaved caspase-3 were determined by Western blotting. The relative expression levels of proteins were analyzed by Image J software. Data were collected from 3 independent experiments, and the mean ± S.E. (error bars) was calculated, *P < 0.05 vs the control. (J) Cells were pretreated with z-VAD-fmk at 50 μM for 2 h before treatment of 17-DMCHAG at 1 μM for 24 h, the number of live cells (PI negative cells) was analyzed by flow cytometry. The data shown represent the mean ± SD (n = 3). *P < 0.05 vs the control.

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Fig. 1. (continued)

Hsp90 to inhibit its chaperone function, and induces massive death of tumor cells via an apoptotic mechanism [18,19]. However, the use of GA as a chemotherapeutic agent has not proceeded because of its associated hepatotoxicity at effective concentrations [20]. Many GA derivatives have been reported, and some derivatives with lower hepatotoxicity have entered into clinical trials, specifically 17allylamino-17-demethoxygeldanamycin (tanespimycin, 17-AAG) and 17-[2-(dimethylamino)ethyl]amino-17-demethoxygeldanamycin (alvespimycin, 17-DMAG) [21,22]. Modification of the 17-position of GA maintains the excellent antitumor activity of GA and endows compounds with reduced hepatotoxicity [23]. We have designed and synthesized more than 200 GA derivatives [23–25]. In the present study, one GA derivative, 17-DMCHAG (as shown in Fig. 1A), was further examined for its in vitro and in vivo anticancer activities. This compound showed highly effective antitumor activity against prostate cancer and low hepatotoxicity in vivo, findings that provide theoretical foundations for targeted therapies for prostate cancer. Materials and methods 17-DMCHAG was dissolved in dimethyl sulfoxide (DMSO) to prepare 10 mmol/L stock solutions that were stored at −20 °C.

Measurement of cell death Cell death induced by compounds was determined by evaluating the plasma membrane integrity by examining the permeability of cells to propidium iodide (PI). Cells were trypsinized, collected by centrifugation, washed once with PBS, and resuspended in PBS containing 5 μg/mL PI. The level of PI incorporation was quantified by flow cytometry using a FACScan flow cytometer (Beckman Coulter EPICS XL). Colony formation assay Cells were cultured in 6-well plates (1000/well) overnight and with new medium replaced in the presence of 17-DMCHAG, and the plates then continued to be incubated at 37 °C with 5% CO2 for 10 days. On the last day, the medium was removed, and after washing with PBS and fixing with methanol, the colonies were stained with crystal violet solution for 3 hr at room temperature. The images were acquired with a scanner and visible colony numbers were counted after washing and air-drying. Assessing apoptosis by Annexin-V/PI staining Cells were seeded in a 6-cm dish one day before treatment with the compounds. After 17-DMCHAG treatment for 24 h, cells were stained with Annexin V and PI following the manufacturer’s protocol (Annexin V FITC Apoptosis Detection Kit; BD Pharmingen, USA). Subsequently, cells were analyzed by flow cytometry and BD CellQuest Pro software using the FL1 channel for FITC and FL3 detector for PI as described elsewhere. Western blot analysis

Antibodies and reagents Anti-β-actin, 3-(4,5)-dimethylthiazol(-2-yl)-3,5-diphenyltetrazolium bromide (MTT), MG132, DAPI (4,6-diamidino-2-phenylindole), Hoechst 33258 and RNaseA were purchased from Sigma-Aldrich (USA). Lipofectamine was purchased from Invitrogen (USA). Annexin V, FITC Apoptosis Detection Kit, z-VAD-fmk, and Matrigel were purchased from BD Pharmingen (USA). Anti-AKT, anti-phospho-AKT (ser 473), anti-Her2, anti-EGFR, anti-CDk4, anti-C-Raf, anti-Survivin, anti-Bcl-2, antivimentin, anti-Zeb1, anti-slug, anti-caspase 3 and anti-PSA were purchased from Cell Signaling Technology (USA). Anti-MMP9, anti-Snai1, anti-AR, anti-NKX3.1, anti-Ecadherin, anti-Hsp90, and anti-Hsp70 were purchased from Santa Cruz Biotechnology (USA). Cells and cell culture The human prostate cancer cell lines LNCaP, DU145, PC-3, C42B and RWPE-1 were purchased from the American Type Culture Collection. Cultures were maintained in a humidified incubator at 37 °C in an atmosphere of 5% CO2. Cytotoxicity assays The cytotoxicity of compounds was measured using the MTT assay as previously described [26].

After treatment with or without compounds for different time courses, cells were harvested and lysed in ice-cold lysis buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 1 mg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride). The lysate was mixed with an equal volume of 2 × loading buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.2 mg/mL bromophenol blue in 0.1 M Tris–HCl (pH 6.8)) and boiled for 10 min immediately. The boiled lysates were subjected to 8–12% SDS–polyacrylamide gels and developed under 100 V and then were transferred to Immobilon-P membranes (Millipore). After blocking the membranes with 5% skim milk in phosphate-buffered saline with 0.1% Tween20 for 1 h, they were incubated overnight with the corresponding primary antibodies in blocking solution at 4 °C. The primary antibodies were detected using either a peroxidase-conjugated ImmunoPure goat anti-rabbit IgG (H + L) or a peroxidaseconjugated ImmunoPure Goat Anti-Mouse IgG (H + L) secondary antibody and enhanced chemiluminescence. Wound healing assay Cells (4 × 105) were seeded in a 6-well plate in complete medium and grown overnight. Next, wounds were created using a sterile 10-μL pipette tip. The cells were then rinsed with PBS and covered with fresh medium with 2% FBS supplemented with 0.2 μM 17-DMCHAG. The control cells were cultured in culture medium without

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Fig. 2. 17-DMCHAG suppresses the cell migration and invasion of prostate tumor cells. (A) Effect of wound healing. After DU-145 (a) and C4-2B (b) cells were wounded using a 10-μL micropipette tip, they were incubated with or without 17-DMCHAG (0.2 μM) for 24, 48, or 72 h. The images were taken at 0, 24, 48 and 72 h. The dotted lines show the area where the scratch wound was created. Scale bar is 200 μm. Quantitative data are presented as means ± SD of three independents experiments. *P < 0.05 is interpreted to be significant as compared with control. (B) DU-145 and LNCaP cells were placed in Transwell chambers and incubated with or without 17-DMCHAG (0.2 μM) for 36 h. Representative images of cells stained with crystal violet are shown at 100× magnification (left panel). Quantitative data are presented as means ± SD of three independents experiments (right panel). *P < 0.05 indicates a significant difference compared with the controls. (C) DU-145 and LNCaP cells were placed in Transwell chambers coated with Matrigel and incubated with or without 17-DMCHAG (0.2 μM) for 48 h. Representative images of cells stained with crystal violet are shown at 100× magnification (left panel). Quantitative data are presented as means ± SD of three independents experiments (right panel). *P < 0.05 indicates a significant difference compared with the controls. (D) Western blot analysis of E-cadherin and Vimentin after treatment with 17-DMCHAG. DU-145 cells were treated for the indicated times with different concentrations of 17-DMCHAG. β-actin was used for normalization and verification of protein loading. The relative expression levels of proteins were analyzed by Image J software. Intensity values were expressed as fold change compared to control. (E) DU-145 cells were treated with 1 μM 17-DMCHAG for 12 or 24 h, and E-cadherin and Vimentin mRNA levels were determined by quantitative real-time PCR. The mRNA levels were normalized to that of GAPDH mRNA and are expressed as the mean ± SE. (F) Western blot analysis of Zeb1, MMP9, snail1 and Slug after treatment with 17-DMCHAG in DU-145 cells. β-actin was used for normalization and verification of protein loading. (G) The relative expression levels of Zeb1, MMP9, snail1 and Slug in (F) were analyzed by Image J software. Intensity values were expressed as fold change compared to control. Data were collected from 3 independent experiments, and the mean ± S.E. (error bars) was calculated, *P < 0.05 vs the control.

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Fig. 2. (continued)

17-DMCHAG. Following incubation for 48 h, the wounds were photographed under an inverted microscope.

(5′-TCCCTCGAATGCAACTCTCT-3′) and anti-sense (5′-GCCACATCTCTGCAGTCAAA3′). Each assay was performed in triplicate.

Transwell assay

Small interfering RNA transfection

Transwell assay was performed using Transwell chambers (Corning, New York, NY, USA) with a polycarbonate membrane (8-μm polyester membrane filter pores). The invasion assay used coated Matrigel in the upper chamber, which simulated the characteristics of the extracellular matrix, and the migration assay used Transwell chambers only. Cells were starved for 24 h prior to the experiment, and then 5 × 104 cells were seeded into the upper chamber with 150 μL serum-free medium. A total of 500 μL of medium containing 20% FBS was placed in the lower chamber as chemoattractant. After incubation at 37 °C for 36 or 48 h, the cells adhering to the lower surface of the membrane on the upper chamber were fixed in 4% paraformaldehyde for 30 min, followed by staining with 0.1% crystal violet, and counted in three random shots by microscopy, and photographed. The average numbers of cells in the three random shots were reported. All experiments were performed in triplicate.

For the siRNA transfection, Du-145 and LNCaP cells were plated into 100 mm plate and allowed to grow to 70% confluence. Then the cells were transiently transfected with scrambled or survivin-specific siRNA (Santa Cruz Biotechnologies, Santa Cruz, CA) using Lipofectamine. 36 h post transfection, the cells were subsequently prepared for use in Semiquantitative RT-PCR analysis and Annexin-V assays.

Quantitative real-time PCR Total RNA was extracted from cultured cells after 48 h of treatment using TRIzol reagent (Invitrogen). Two micrograms of total RNA was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). Real-time monitoring of PCR amplification of cDNA was performed using DNA primers and the ABI PRISM 7500 HT Sequence Detection System (Applied Biosystems) with SYBR PCR Master Mix (Applied Biosystems). Target gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in the respective samples as an internal standard, and the comparative cycle threshold (Ct) method was used to calculate relative amount of target mRNAs. Oligonucleotide primers used for PCR amplification of human GAPDH, E-cadherin, Vimentin, Survivin, PSA, NKX3.1 and FKBP5 were as follows: for GAPDH, sense (5′-TCCTGTTCGACAGTCAGCCGCA-3′) and anti-sense (5′-ACCAGGCGCCCAATACGACCA-3′); for E-cadherin, sense (5′GTAACCGATCAGAATGAC-3′) and anti-sense (5′-CGTGGTGGGATTGAAGAT-3′); for Vimentin, sense (5′-CCGACACTCCTACAAGATTTA-3′) and anti-sense (5′CAAAGATTTATTGAAGCAGAA-3′); for Survivin, sense (5′-CACCGCATCTCTACATTC AAG-3′) and anti-sense (5′-CAAGTCTGGCTCGTTCTCAG-3′); for PSA, sense (5′-CACAGCCTGTTTCATCCTGA-3′) and anti-sense (5′-AGGTCCATGACCTTCACAGC3′); for NKX3.1, sense (5′-GGACTGAGTGAGCCTTTTGC-3′) and anti-sense (5′CAGCCAGATTTCTCCTTTGC-3′); for PSA, sense (5′-CACAGCCTGTTTCATCCTGA-3′) and anti-sense (5′-AGGTCCATGACCTTCACAGC-3′); for FKBP5, sense

Immunofluorescence LNCaP cells were grown on coverslips and treated with or without 0.2 μM 17-DMCHAG for 12 or 48 h followed by treatment with 1 nmol/L R1881 for 6 h. After treatment, cells were fixed with 4% formaldehyde, permeabilized for 10 min in 0.2% Triton X-100 in PBS, and then incubated for 1 h in blocking buffer (5% BSA in PBS). Next, cells were incubated with AR (1:250) antibody overnight, and then were visualized with Cy3-conjugated Addinipure Goat Anti-Mouse IgG (H + L). Nuclei were stained by incubating cells with 10 μg/mL Hoechst 33258 (Sigma) in PBS and then washing extensively with PBS. Images were obtained by fluorescence microscopy. Immunoprecipitation Samples were incubated overnight with 3 μg of primary antibody at 4 °C, after which 10 μL of Protein A/G Plus agarose beads was added to the mixture, which was then incubated for 6 h at 4 °C. The immunoprecipitated protein complexes were washed three times with lysis buffer. After the supernatant was discarded, the antibody/protein complexes were resuspended in 50 μL of loading buffer and boiled for 10 min to dissociate the immunocomplexes from the beads. The supernatant collected after centrifugation was separated with 10% sodium dodecyl sulfate– polyacrylamide gel electrophoresis and assayed with protein immunoblotting. Serum analysis of aspartate aminotransferase and alanine aminotransferase The in vivo hepatotoxicity of 17-DMCHAG was measured using in vivo toxicity assays as described previously [24]. Briefly, whole blood was centrifuged at 300 × g for 10 min; the supernatant corresponding to the serum was next removed and analyzed. The assays to measure alanine aminotransferase (ALT) and aspartate

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Fig. 3. 17-DMCHAG induces the degradation of HSP90 client proteins and inhibits the AR pathway in LNCaP cells. (A) DU-145 cells were treated for the indicated times with different concentrations of 17-DMCHAG, and proteins regulated by Hsp90 were assessed by Western blotting. β-actin was used for normalization and verification of protein loading.The relative expression levels of proteins were analyzed by Image J software. Intensity values were expressed as fold change compared to control. (B) LNCaP cells were cultured in the presence of 17-DMCHAG or 17-AAG at the indicated concentrations for 24 h, and proteins regulated by Hsp90 or AR were assessed by Western blotting. β-actin was used for normalization and verification of protein loading. (C) The relative expression levels of proteins used in (B) were analyzed by Image J software. Intensity values were expressed as fold change compared to control. (D) LNCaP cells were pretreated with 10 μM MG132 for 4 h in the presence or absence of 1 μM 17-DMAG for an additional 12 h. Whole-cell lysates were subjected to Western blot analysis using antibodies against Hsp90, HER2, EGFR, AR, Raf-1, Akt, CDK4 and β-actin. β-actin was used for normalization and verification of protein loading (left panel). The relative expression levels of proteins were analyzed by Image J software. Intensity values were expressed as fold change compared to control (right panel). (E) LNCaP cells were treated with or without 1 or 2 μM of 17-DMAG for 12 h and then were subjected to immunoprecipitation followed by Western blotting analysis with antibodies against AR and Hsp90. The relative levels of proteins were analyzed by Image J software. Intensity values were expressed as fold change compared to control. (F) LNCaP cells were treated with 1 μM 17-DMCHAG for the indicated times, as well as 0.1 nM R1881 for 12 h, and AR localization was assessed by immunofluorescence staining. Nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). Scale bar is 50 μm. (G) LNCaP cells were treated with 1 μM 17-DMCHAG for 12 or 24 h, and PSA, FKBP5 and NKX3.1 mRNA levels were determined by quantitative real-time PCR. The mRNA levels were normalized to that of GAPDH mRNA and are expressed as the mean ± SE. All of the experiments were repeated at least three times. Data were collected from 3 independent experiments, and the mean ± S.E. (error bars) was calculated, *P < 0.05 vs the control.

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Fig. 3. (continued)

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aminotransferase (AST), as indirect indicators of liver damage, were performed following the requirements of the manufacturer. The results are expressed as units per liter. In vivo antitumor assays Six- to eight-week-old male athymic nude mice (BALB/c-nu) were obtained from Slac Laboratory Animal (Shanghai, China). LNCaP or DU-145 cells (1 × 106 cells per animal) were injected subcutaneously to generate orthotopic xenografts. Next, the mice bearing tumor cells were randomly divided into treatment and control groups (7 mice per group). The drugs were injected via the caudal vein every three days at a dose of 10 mg/kg body weight, whereas the blank control group received an equal volume of 5% glucose injection containing 1% DMSO and 2% lecithin. During treatment, subcutaneous tumors were measured with a vernier caliper every three days, and body weight was monitored regularly. Tumor volume was calculated by the formula (V = ab2/2, where a and b stand for the longest and shortest diameters, respectively). After treatment for 21 d with drugs, the animals were euthanized, and solid tumors were removed. Data points are expressed as average tumor volume ± SE. All of the animal protocols in the present study were approved by the Shanghai Medical Experimental Animal Care Commission.

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Table 1 Cytotoxicity of 17-DMCHAG on prostate cancer cells. Time

Cell line

IC50 (μM)

24 h

RWPE-1 LNCaP Du-145 PC-3 C4-2B RWPE-1 LNCaP Du-145 PC-3 C4-2B RWPE-1 LNCaP Du-145 PC-3 C4-2B

2.322 0.303 0.673 2.032 0.653 1.971 0.135 0.194 0.890 0.114 0.805 0.022 0.089 0.320 0.038

48 h

72 h

Immunohistochemical staining The sections of heart, liver, spleen, lung, kidney, and tumor for immunohistochemistry were prepared from euthanized mice with tumor xenografts as previously described [27]. Statistical analysis Student’s t tests were used to determine the significant differences between the treatment and the control groups, and P ≤ 0.05 was considered to be biologically and statistically significant. All of the experiments were conducted in three independent replications.

Results 17-DMCHAG inhibited proliferation and induced apoptosis in prostate cancer cells To evaluate the in vitro antitumor effects of 17-DMCHAG, the cytotoxicities of 17-DMCHAG against the proliferation of a Homo sapiens prostate normal cell line (RWPE-1) and four human prostate cancer cell lines (LNCaP, C42B, DU-145 and PC-3) at 24 h, 48 h and 72 h were evaluated by MTT assay (Table 1). In the cell lines tested, 17DMCHAG inhibited cell growth in a dose-dependent manner. In the prostate cancer cell lines, 17-DMCHAG displayed potent cytotoxicity with IC50 values ranging from 0.022 to 0.320 μM (LNCaP: 0.022 μM; C4-2B: 0.375 μM; DU-145: 0.895 μM; PC-3: 0.320 μM) at 72 h (Fig. 1B). However, 17-DMCHAG showed lower cytotoxicity against RWPE-1 cells in which the IC50 value was 0.805 μM (Fig. 1B). These data indicated that 17-DMCHAG demonstrated high activity against androgen-dependent (LNCaP) and androgenindependent (C42B, DU-145 and PC-3) prostate cancer cells but lower cytotoxicity against normal prostate cells (RWPE-1).

Membrane integrity, which was assessed by propidium iodide (PI) staining, showed that 17-DMCHAG induced cell death of LNCaP and DU-145 in a dose-dependent manner (Fig. 1C). As expected, the ratio of dead cells also increased with increasing exposure time (Fig. 1D), suggesting that cell death is the main contributor to the antiproliferative activity of 17-DMCHAG. Consistently, colony formation assay showed that the numbers of colonies formed in the cells of LNCaP, DU-145 and PC-3 treated with 17-DMCHAG significantly decreased compared with that of the control group (Fig. 1E and F). Apoptosis is one of the important ways that chemotherapeutic drugs kill tumor cells. Flow cytometry and Western blot analyses were applied to explore whether the antiproliferative activity of 17DMCHAG is related to apoptosis. The Annexin V-PI assay revealed that the fraction of cells undergoing apoptosis was significantly increased in a dose-dependent manner with 17-DMCHAG compared with control in LNCaP cells (Fig. 1G) and Du-145 cells (data not shown). In addition, 17-DMCHAG induced caspase-3 activation as shown by increased cleaved caspase-3, together with the decreased expression level of apoptosis-related protein Bcl-2 (Fig. 1H). Pre-treatment of the cells with the caspase inhibitor z-VAD-fmk, which broadly inhibits caspase cleavage, inhibited 17-DMCHAGinduced caspase-3 cleavage (Fig. 1I) and significantly reduced 17-DMCHAG-induced cell death (Fig. 1J). These results suggest that 17-DMCHAG induced apoptosis in a caspase-3-dependent manner. 17-DMCHAG suppressed the migration of prostate cancer cells Recently, accumulating evidence has indicated that epithelialto-mesenchymal transition (EMT) is a critical component of prostate

Fig. 4. 17-DMCHAG suppresses the expression of survivin through damaging the Hsp90/Survivin chaperone complex. (A) Western blot analysis of survivin after treatment with 17-DMCHAG. DU-145 cells were treated for the indicated times with different concentrations of 17-DMCHAG, and then whole-cell lysates were subjected to Western blot analysis using antibodies against survivin and β-actin. β-actin was used for normalization and verification of protein loading. The relative expression level of Survivin was analyzed by Image J software. Intensity values were expressed as fold change compared to control. (B) DU145 cells were pretreated with 10 μM MG132 for 4 h in the presence or absence of 1 μM 17-DMAG for an additional 12 h. Whole-cell lysates were subjected to Western blot analysis using antibodies against survivin and β-actin. β-actin was used for normalization and verification of protein loading. The relative expression level of survivin was analyzed by Image J software. Intensity values were expressed as fold change compared to control. (C) DU-145 cells were treated with or without 1 or 2 μM of 17-DMAG for 12 h and then were subjected to immunoprecipitation followed by Western blotting analysis with antibodies against survivin and Hsp90. The relative levels of proteins were analyzed by Image J software. Intensity values were expressed as fold change compared to control. (D) Western blot analysis of survivin after treatment with 17-DMCHAG, GA or 17-AAG. DU-145 cells were treated with different concentrations of 17-DMCHAG, GA or 17-AAG for 24 h and then whole-cell lysates were subjected to Western blot analysis using antibodies against survivin and β-actin. β-actin was used for normalization and verification of protein loading. The relative expression level of Survivin was analyzed by Image J software. Intensity values were expressed as fold change compared to control. (E) Knockdown of survivin potentiates 17-DMCHAG-induced apoptosis. LNCaP and DU-145 cells were transfected with either scrambled or survivin-specific siRNA. After 48 h, cells were treated with 17-DMCHAG (2 μM) for 24 h. Semiquantitative RT-PCR analysis shows mRNA expression of survivin in DU-145 cells transfected with survivin–siRNA (left panel). Scrambled siRNA was used as a control in parallel. GAPDH was used as an internal control. This is a representative example with mean densitometric values from triplicate blots. *P < 0.05 vs control siRNA transfectants. Apoptosis was evaluated by green fluorescent protein– annexin V + propidium iodide (right panel). Bars represent the mean ± standard deviation of three independent experiments. *P < 0.05 for 17-DMCHAG. Scrambled or survivin– siRNA combined treatment with 17-DMCHAG vs control. Data were collected from 3 independent experiments, and the mean ±S.E. (error bars) was calculated, *P < 0.05 vs the control.

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cancer progression, facilitating the development of lethal metastatic androgen-independent prostate cancer [28,29]. Thus, inhibition of EMT is considered a potent target for prostate therapy [30]. To this end, the wound healing assay and Transwell migration assay were performed to study the effect of 17-DMCHAG on cancer cell migration. 17-DMCHAG significantly inhibited cell migration compared with the control group (Fig. 2A and B). The inhibitory effect of 17-DMCHAG on cell invasion was confirmed by the Transwell invasion assay, in which the number of invaded cells was significantly reduced after treatment with 17-DMCHAG for 48 h (Fig. 2C). The expression of genes related to cell invasion was further examined. Western blotting (Fig. 2D and E) and real-time PCR analyses (Fig. 2D) showed that the epithelial marker E-cadherin was upregulated, while the expressions of mesenchymal marker Vimentin protein level were significantly decreased following treatment with 17-DMCHAG. It was reported that EMT-inducing transcription factors such as snail, Slug and Zeb1 could inhibit the expression of E-cadherin, but increase the expression of vimentin via activation of promoters of the target genes [31]. Then we examined the protein levels of the Zeb1, snail1 and Slug with 17-DMCHAG treatment. 17DMCHAG (0.5 μM) treatment decreased the protein levels of the Zeb1, snail1 and Slug in DU-145 cells compared to the control group (Fig. 2F and G). In addition, the secretion of MMP-9 was also repressed by 17-DMCHAG in DU-145 cells (Fig. 2F and G). Thus, these findings indicated that 17-DMCHAG could inhibit prostate cancer cell migration and invasion through regulation of EMT. 17-DMCHAG downregulated Hsp90 client proteins and inhibited AR signaling in prostate cancer LNCaP cells GA has been identified as an Hsp90 inhibitor by facilitating the degradation of Hsp90 client proteins required for tumor growth. To determine whether 17-DMCHAG can also regulate Hsp90, the client proteins of Hsp90 were examined, including Her2, EGFR, C-raf, AKT, p-AKT, and CDK4. Western blot analysis demonstrated that these well-known client proteins were significantly downregulated by treatment with 17-DMCHAG in a dose-dependent manner (Fig. 3A–C). Furthermore, degradation of these client proteins was completely blocked by treatment with the proteasome inhibitor MG132 (Fig. 3D), indicating that 17-DMCHAG-induced Hsp90 client protein degradation was proteasome-dependent. AR has been reported to be a client protein of Hsp90. Therefore, we next evaluated the effects of 17-DMCHAG on AR signaling in LNCaP cells. As shown in Fig. 3C, 17-DMCHAG induced AR (including androgen-regulated genes, PSA and KNX3.1) downregulation in LNCaP cells, and it maybe more potent than 17-AAG. Coimmunoprecipitation assay was performed to determine the effect of 17-DMCHAG on the association of Hsp90 and AR. With increasing concentration of 17-DMCHAG, the protein level of pull-down AR with HSP90 antibody significantly decreased, and vice versa (Fig. 3E), indicating that the Hsp90/AR chaperone complex was damaged by 17-DMCHAG. Furthermore, the degradation of AR was

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completely blocked by treatment with the proteasome inhibitor MG132 (Fig. 3D). In addition, 17-DMCHAG significantly inhibited the nuclear translocation of AR induced by R1881. The localization of AR had shifted from predominantly nuclear to cytoplasmic after treatment with 17DMCHAG (Fig. 3F), which is consistent with previous studies that 17AAG inhibited AR nuclear localization in androgen-sensitive prostate cancer cells in vitro. To further confirm the effect of 17DMCHAG on AR function, the levels of mRNAs for several wellcharacterized androgen-regulated genes were measured in LNCaP cells. Quantitative real-time PCR indicated that 17-DMCHAG significantly decreased the mRNA levels of AR target genes PSA, NKX3.1 and FKBP5 (Fig. 3G). These data suggest that 17-DMCHAG treatment inhibited AR funtion as a result of the disruption of the association of AR and Hsp90, which induced the proteasome-dependent degradation of AR and blocked the transcriptional activity of AR. 17-DMCHAG downregulated the survivin protein level through damaging the Hsp90/survivin chaperone complex Survivin is a member of the IAP family with multiple functions [32,33]. Survivin is highly overexpressed in transformed cell lines and in multiple types of adenocarcinomas, including prostate adenocarcinoma. Thus, survivin is becoming a drug target for novel cancer therapeutics [33]. Interestingly, we found that the protein level of survivin was significantly decreased after treatment with 17-DMCHAG for 24 h (Fig. 4A), which was completely blocked by treatment with the proteasome inhibitor MG132 (Fig. 4B). As survivin has been reported to be a client protein of Hsp90 [34], it may undergo proteasome-dependent degradation as a result of the disruption of the association of survivin and Hsp90. To this end, we performed co-immunoprecipitation assay with HSP90 and survivin antibodies separately. With increasing concentration of 17-DMCHAG, the protein levels of bound survivin or HSP90 decreased (Fig. 4C), which is consistent with other well known HSP90 inhibitors such as GA and 17-AAG (data not shown). However, 17-DMCHAG, not GA and 17-AAG, downregulated the protein level of survivin (Fig. 4D), which suggests that downregulation of survivin induced by 17DMCHAG is not absolutely due to the disruption of HSP90. To determine the role of survivin in 17-DMCHAG-induced apoptosis in prostate cancer cells, we knocked down survivin with siRNA. As we expected, knockdown of survivin induced cell death that is comparative to that of 17-DMCHAG treatment only. The cells transfected with suvivin siRNA did not present aggravated apoptosis after with treatment of 17-DMCHAG (Fig. 4E). These data indicate that the induction of apoptosis by 17-DMCHAG is due, at least in part, to the downregulation of survivin. 17-DMCHAG is a promising antitumor agent with low hepatotoxicity Aspartate aminotransferase (AST) and alanine-aminotransferase (ALT) are the most commonly used biomarkers for the assessment

Fig. 5. 17-DMCHAG shows low hepatotoxicity and significantly delays LNCaP/DU-145 tumor growth. (A) In vivo hepatotoxicity evaluation of 17-DMCHAG in mice. To assess the damage of 17-DMCHAG to the hepatic parenchyma, the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using the commercially available ALT Assay Kit and AST Assay Kit, respectively, based on a spectrophotometric method. The blood serum samples were treated according to the manufacturer’s instruction. The ALT and AST activities are expressed as U/L. All of the data are expressed as the mean ± standard deviation (SD). (B) 17-DMCHAG inhibits the tumor growth of LNCaP xenografts in mice. (a) Photographs of dissected LNCaP tumor tissues with or without 17-DMCHAG treatment. (b) The mean tumor volume of mice treated was compared ± SE (n = 8). *P < 0.05 vs the control. (c) The PSA, FKBP5 and NKX3.1 mRNA levels of tumor from LNCaP xenografts mice were determined by quantitative real-time PCR. The mRNA levels were normalized to that of GAPDH mRNA and are expressed as the mean ± SE. All of the experiments were repeated at least three times. (C) 17-DMCHAG inhibits the tumor growth of DU-145 xenografts in mice. Upper panel: Photographs of tumor-bearing mice with or without 17-DMCHAG treatment. Lower panel: Photographs of dissected DU-145 tumor tissues with or without 17-DMCHAG treatment. The mean (D) or individual (E, F) tumor volume of treated mice was compared ± SE (n = 7). P < 0.05. (G) Tumor progression was estimated as the relative tumor volume (RTV) calculated from the formula: RTV = (V21/V9) where V21 is the mean tumor volume at day 21 and V9 is the mean tumor volume at day 9. P < 0.05. (H) The mean tumor body weight of treated mice was compared ± SE (n = 7). P < 0.05. (I) Representative H&E-stained sections of the heart, liver, spleen, lung, and kidney from the mice after treatment (original magnification: ×200). Scale bar is 100 μm. (J) Different tumor sections from DU-145 xenografts mice were subjected to H&E-stained and immunohistological analysis with antibodies against Ki-67, Survivin, AKT, EGFR (original magnification: ×200).

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of hepatotoxicity because the damaged hepatocytes can leak large amounts of these enzymes into the bloodstream, leading to increased serum ALT and AST [35]. To examine the hepatotoxicity of 17-DMCHAG, the levels of AST and ALT were analyzed using an activity assay, revealing that 17-DMCHAG treatment had no effect on the activities of AST and ALT compared with those of the control group (197.2 ± 11.2 U/L and 60.7 ± 6.9 U/L, respectively) (Fig. 5A). It was noted that the AST and ALT levels in the 17-DMCHAG-treated group were obviously lower than those of the 17-AAG-treated group (237.4 ± 29.4 U/L and 66.6 ± 8.1 U/L, respectively) (Fig. 5A). These results suggested that 17-DMCHAG is a promising antitumor agent with low hepatotoxicity. Based on the potent inhibitory in vitro effects of 17-DMCHAG on prostate tumor cell lines and lower hepatotoxicity, the in vivo antitumor activity was evaluated on PCa xenografts with LNCaP cells. The 17-DMCHAG-treated group exhibited a significant decrease in the tumor volume compared with the control group after 21 d (78.76 and 395.67 mm3, respectively) (Fig. 5B a, b). Quantitative realtime PCR analyses demonstrated that the PSA, FKBP5 and NKX3.1 mRNA levels of tumors from LNCaP xenografts were decreased after treated with 17-DMCHAG (Fig. 5B c) that is consistent with AR inhibition in vitro. In addition, we tested whether 17-DMCHAG suppressed the tumor growth of AR-negative xenografts. DU-145 cells were subcutaneously inoculated into male nude mice and were subjected to similar experiments with half the mice receiving injection of solvent or 17-DMCHAG. All of the animals treated with 17-DMCHAG (n = 7) exhibited a significant decrease in the tumor volume compared with control mice starting on day 12 (144.07 and 382.92 mm3, respectively) and after 21 d (487.83 and 1525.23 mm3, respectively; Fig. 5C and D). When each animal was considered individually, the incidence of mice progressing with a tumor volume of 500 mm3 or greater was significantly diminished by day 15 in 17-DMCHAGtreated animals (0/7) compared with controls (7/7) (Fig. 5E and F). The rate of tumor progression at days 9 and 21 was also significantly decreased in the treatment group compared with control mice (1321.49 mm3 for control mice vs 398.66 mm3 for treated mice; P < 0.001) (Fig. 5G). No obvious side effect and body weight loss were observed (Fig. 5H). Immunohistochemical analysis indicated decreased Ki67 expression after treatment with 17-DMCHAG (Fig. 5I) in the tumor cells in vivo. Inhibition of tumor progression by 17DMCHAG may result from decreased proliferation (reduced Ki67, the proliferation marker). The client proteins of Hsp90, such as AKT and EGFR, were decreased in tumors from LNCaP xenografts after treatment with 17-DMCHAG. The level of survivin was also decreased compared to the control group. These data demonstrate that 17-DMCHAG inhibited HSP90 and downregulated survivin. More importantly, there were no damage to organs such as the heart, liver, spleen, lung and kidney (Fig. 5J). The results showed that 17DMCHAG was successful in suppressing the tumor growth of DU145 xenografts similar to LNCaP xenografts (Fig. 5C). These findings suggest that 17-DMCHAG suppresses prostate tumor growth in vivo without side effects. Discussion Prostate cancer is the most common cancer that is the leading cause of male mortality worldwide [1,36]. Therapy with cytotoxic LHRH analogs remains the standard first-line treatment for advanced prostate cancer; however, a large percentage of PCa patients will eventually progress to castration-resistant disease within 18– 24 months and fail to respond to ADT and die within 2–3 years [37,38]. There are limited therapeutic options available for castrationresistant prostate cancer (CRPC); only the first-line chemotherapy drug docetaxel could improve prostate cancer patient survival by months. With advances in understanding the molecular mechanisms

of the tumorigenesis and progression of PCa, many approaches are currently being evaluated to improve the treatment of PCa [39,40]. Many potential targets have been identified for therapeutic intervention and have allowed the rational design and development of novel agents that target these molecules. In the present study, 17DMCHAG presents potent antitumor activity in vitro and in vivo against both types of PCa cells. Inhibition of Hsp90 function has been shown to cause degradation of multiple cancer-related proteins, crippling the tumorigenic and metastatic potential of tumors regardless of their tissue or cellular origin. Several Hsp90 inhibitors have potent antitumor activity in various preclinical models and are in clinical trials, including lung, gastric, breast, colon, ovarian, skin, bone, and blood cancers [41–43]. As an androgen receptor co-activator and chaperone protein, Hsp90 plays a significant role in preventing apoptosis and facilitating prostate cancer survival; most importantly, Hsp90 is a regulator of EMT in prostate cancer. Thus, Hsp90 has garnered interest as a treatment target in prostate cancer [44,45]. A significant proportion of the work on Hsp90 inhibitors has been devoted to developing analogs of geldanamycin, including 17AAG and 17-DMAG [20,46,47]. However, there seems to be no further development of 17-AAG and 17-DMAG due to the combined effects of the drug and metastatic liver disease. Based on the druggability and liver toxicity, we designed and synthesized a new geldanamycin derivative, 17-DMCHAG. Our data demonstrate that 17-DMCHAG showed high activity against androgen-dependent (LNCaP) and androgen-independent (C42B, DU-145 and PC-3) prostate cancer cells with lower cytotoxicity against normal prostate cells (RWPE-1) (Fig. 1B). More importantly, 17-DMCHAG showed low hepatotoxicity in vivo (Fig. 5A) and no side effects (Fig. 5C,H,J), most likely because the aroyl group used was 3,4-dimethoxycinnamic acid [48], which was reported to be associated with liver-protective activity, and acquired the pharmacologically combinational effects based on the hybridization principle [49]. As an Hsp90 inhibitor, 17-DMCHAG downregulated the protein level of client proteins such as Her2, EGFR, AKT, C-Raf and CDK4. AR is a nuclear receptor that is activated by binding of the androgenic hormone testosterone or dihydrotestosterone in the cytoplasm; subsequently, AR is translocated into the nucleus where it plays a key role in prostate carcinogenesis and progression. As a client protein of Hsp90, the protein level of AR was downregulated by 17DMCHAG; next, its nuclear translocation was blocked, and its transcription activity was inhibited. Subsequently, the mRNA levels of the AR target genes PSA, NKX3.1, and FKBP5 were decreased. In addition, 17-DMCHAG could induce apoptosis of LNCaP cells. Thus, 17-DMCHAG could suppress the proliferation of androgen-dependent prostate cancer cells. In recent years, accumulating evidence has indicated that EMT is a critical component of prostate cancer progression, facilitating the development of lethal metastatic androgen-independent prostate cancer. Thus, inhibition of EMT is considered a potent target for prostate therapy [50–52]. Our research showed that 17-DMCHAG treatment resulted in down-regulation of mesenchymal marker Vimentin and the EMT Marker Zeb1, snail and slug proteins while up-regulation of the epithelial marker E-cadherin (Fig. 2). This finding indicated that 17-DMCHAG treatment targeted EMT markers, thus inhibiting the epithelial-to-mesenchymal transition of androgenindependent prostate cancer cells. Our results indicated that 17DMCHAG may be a potential therapeutic agent for treatment of prostate cancer metastasis. Survivin, a strong apoptosis inhibitor, is highly overexpressed in transformed cell lines and in multiple types of adenocarcinomas, including prostate adenocarcinoma. Increasing evidence has shown that survivin is not expressed in normal prostate secretory epithelial cells but is strongly expressed in prostate cancer cells, suggesting that survivin plays an important role in the survival strategies of

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HRPC, and downregulation of survivin could be considered an attractive therapeutic strategy for prostate cancer [53,54]. Smallmolecule like YM155 that suppresses survivin expression has been proven to be effective in suppressing prostate cancer tumor growth and phase I and II trials have been completed for its safety and efficacy in patients [55,56]. In the present study, our results showed that treatment of cells with 17-DMCHAG resulted in significant induction of apoptosis associated with the downregulation of survivin in prostate cancer cells. The mechanism by which 17-DMCHAG inhibits survivin involves disruption of the Hsp90/survivin chaperone complex and downregulation of survivin. It was reported that survivin specifically binds to members of the caspase family of proteins and inhibits the activity of caspase-3 to block apoptosis. Our data also demonstrated that 17-DMCHAG induced apoptosis of prostate cancer cells in a caspase-3-dependent manner (Fig. 1H). Take together, we conclude that 17-DMCHAG disrupts the association of Hsp90 and survivin that induced decrease of survivin followed by caspase-3 activation and final apoptosis. To test whether 17-DMCHAG shows any antitumor effects in vivo, LNCaP (androgen dependent) and DU-145 (androgen independent) were xenografted into nude mice, and tumor-bearing animals were treated by vein injection with 17-DMCHAG (10 mg/kg/3 d) after the development of palpable tumors. We observed that 17-DMCHAG showed the strongest antitumor effect compared with the control. Based on these results, 17-DMCHAG appears to have tumor suppressive effects on both androgen-dependent and androgenindependent prostate cancer cells in vivo with low hepatotoxicity and no side effects, suggesting that 17-DMCHAG treatments might be a potential treatment for prostate cancer. In conclusion, our research indicated that the expression of survivin and AR, two client proteins of Hsp90 and important molecules of tumor cell survival and metastasis, was significantly decreased in prostate cancer cells treated with 17-DMCHAG compared with control cells, ultimately causing cell growth inhibition and induction of apoptosis. Importantly, our observations from in vivo preclinical animal model studies revealed that 17-DMCHAG showed strong antitumor effects on LNCaP and DU-145 cellxenografted nude mice with low hepatotoxicity, suggesting that 17DMCHAG might provide a potential treatment approach for prostate cancer. Further, in-depth testing of 17-DMCHAG as a mechanismbased anticancer drug for prostate cancer is needed. Acknowledgements This work was supported by the 973 Program (2010CB833802), NSFC Projects (81302214, 81373304, 91313303), Program for Changjiang Scholars and Innovative Research Team in University (IRT13028), Shanghai Nature Science Foundation of Shanghai Science and Technology Committee (13ZR1432700), Shanghai Key Medical Specialty Program (ZK2012A22), and Shanghai Minhang Health Bureau Foundation (No. 2012MW01).

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[27]

[28]

Conflict of interest

[29]

The authors declare that they have no competing interests. [30]

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