WMJ‐8‐B, a novel hydroxamate derivative, induces MDA‐MB‐231 breast cancer cell death via the SHP‐1‐STAT3‐survivin cascade

Abstract

Background and Purpose

Histone deacetylase (HDAC) inhibitors have been demonstrate to have broad‐spectrum anti‐tumour properties and have attracted lots of attention in the field of drug discovery. However, the underlying anti‐tumour mechanisms of HDAC inhibitors remain incompletely understood. In this study, we aimed to characterize the underlying mechanisms through which the novel hydroxamate‐based HDAC inhibitor, WMJ‐8‐B, induces the death of MDA‐MB‐231 breast cancer cells.

Experimental Approach

Effects of WMJ‐8‐B on cell viability, cell cycle distribution, apoptosis and signalling molecules were analysed by the MTT assay, flowcytometric analysis, immunoblotting, reporter assay, chromatin immunoprecipitation analysis and use of siRNAs. A xenograft model was used to determine anti‐tumour effects of WMJ‐8‐B in vivo.

Key Results

WMJ‐8‐B induced survivin reduction, G2/M cell cycle arrest and apoptosis in MDA‐MB‐231 cells. STAT3 phosphorylation, transactivity and its binding to the survivin promoter region were reduced in WMJ‐8‐B‐treated cells. WMJ‐8‐B activated the protein phosphatase SHP‐1 and when SHP‐1 signalling was blocked, the effects of WMJ‐8‐B on STAT3 phosphorylation and survivin levels were abolished. However, WMJ‐8‐B increased the transcription factor Sp1 binding to the p21 promoter region and enhanced p21 levels. Moreover, WMJ‐8‐B induced α‐tubulin acetylation and disrupted microtubule assembly. Inhibition of HDACs was shown to contribute to WMJ‐8‐B′s actions. Furthermore, WMJ‐8‐B suppressed the growth of MDA‐MB‐231 xenografts in mammary fat pads in vivo.

Conclusions and Implications

The SHP‐1‐STAT3‐survivin and Sp1‐p21 cascades are involved in WMJ‐8‐B‐induced MDA‐MB‐231 breast cancer cell death. These results also indicate the potential of WMJ‐8‐B as a lead compound for treatment of breast cancer and warrant its clinical development.

Abbreviations

ChIP

chromatin immunoprecipitation

HAT

histone acetyltransferase

HDAC

histone deacetylase

MTT

3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide

PI

propidium iodide

PTPs

protein tyrosine phosphatases

SHP‐1

src homology 2 (SH2)‐domain containing the tyrosine phosphatase‐1

TNBC

triple‐negative breast cancer

Introduction

Breast cancer is the most prevalent cancer and the leading cause of cancer‐related death among females worldwide (Torre et al., 2015). Surgical resection with chemotherapy, hormone‐based agents or agents that directly target the human EGF receptor 2 (HER2 also known as Neu) are the preferred strategies in the treatment of breast cancer. However, approximately 15% of diagnosed breast cancers are characterized as triple‐negative breast cancer (TNBC), lacking HER2, oestrogen and progesterone receptors (Hudis and Gianni, 2011; Torre et al., 2015). Since TNBC exhibits a relatively higher histological grade and poorer prognosis, developing novel agents or therapeutic strategies is an ongoing urgent need to improve the therapeutic outcomes of TNBC.

In addition to genomic and genetic alterations, epigenetic modifications that occur on histone proteins also contribute to cancer pathologies (Berdasco and Esteller, 2013). Acetylation of histone not only alters the structure of chromatin, but also modulates the access of transcription factors to gene promoters. It is believed that histone deacetylases (HDACs), a group of enzymes that negatively regulate histone acetylation, are main epigenetic regulators in gene expression. To date, 18 HDAC family members have been identified and are divided into four classes. Class I consists of HDAC 1, 2, 3 and 8. Class II HDACs are further grouped into two subgroups, Class IIA (HDAC 4, 5, 7 and 9) and Class IIB (HDAC 6 and 10). Class III consists of sirtuins 1–7, and HDAC11 is the only member of Class IV (Bolden et al., 2006). HDACs not only target histones but also non‐histone nuclear and cytoplasmic proteins, which participate in a great variety of cellular processes (Bolden et al., 2006; Peng and Seto, 2011). It has been reported that the abnormal expression of HDACs and alterations in their acetylation levels are common hallmarks of human cancers (Bolden et al., 2006). There is also growing evidence indicating that HDAC inhibitors are effective at inducing cell cycle arrest and apoptosis in different types of human cancer cell lines, as well as disrupting angiogenesis (West and Johnstone, 2014) and suppressing tumour progression in preclinical models (Bolden et al., 2006; Falkenberg and Johnstone, 2014). Moreover, it appears that the apoptotic and growth‐suppressive activities of HDAC inhibitors are restricted to transformed cells (Marks et al., 2001). HDAC inhibitors thus represent promising anti‐tumour agents. However, the precise mechanisms underlying the anti‐tumour activities of HDAC inhibitors remain incompletely understood.

Survivin, the smallest member of inhibitors of apoptosis protein (IAP) family, is commonly detected during embryonic development while its expression is negligible in most adult normal tissues. However, survivin is found to be highly expressed in most cancers including breast cancers (Ambrosini et al., 1997; Hunter et al., 2007). We recently demonstrated that survivin knockdown leads to cell cycle arrest and cell death in colorectal cancer cells (Hsu et al., 2012; Chang et al., 2013) and hypopharyngeal carcinoma cells (Yen et al., 2016). Despite its pivotal role in regulating cell division and apoptosis (Altieri, 2008), survivin also promotes angiogenesis (Sanhueza et al., 2015), metastasis and chemotherapy resistance (Ambrosini et al., 1997). Therefore, survivin is considered as an independent prognosis factor and a valuable target for therapeutic intervention of cancer (Altieri, 2003; Altieri, 2008; Shirai et al., 2009). Many transcription factors such as Sp1 and STAT3 (Sehara et al., 2013) transactivate survivin expression. In contrast, survivin is transcriptionally repressed by p53, which counteracts the binding of Sp1 to the survivin promoter region (Mirza et al., 2002; Hsu et al., 2011; Chang et al., 2013). Loss of p53 function may lead to deregulated survivin expression. The aberrant expression of survivin is also regulated by various mechanisms such as miRNAs (Huang, Lyu, Wang & Liu, 2015a), Akt (Sierra et al., 2010) or MAP kinase (Carter et al., 2006) pathways.

STAT3 is found to be constitutively active and correlated with increased invasion, metastasis and chemo‐resistance in many human cancers including breast cancer (Dolled‐Filhart et al., 2003; Yu and Jove, 2004; Kortylewski et al., 2005; Gupta et al., 2011). STAT3 activation has also been linked to a poor prognosis in breast cancer patients (Diaz et al., 2006). STAT3 is expressed as inactive monomers in the cytoplasm. STAT3 activation occurs via phosphorylation of tyrosine or serine residues by protein kinases JAKs, c‐Src (Schreiner et al., 2002) or MAPKs (Tkach et al., 2013). These phosphorylations allow STAT3 to form homodimers, translocate to the nucleus and transactivate genes involved in tumourigenesis. In contrast, protein tyrosine phosphatases (PTPs) such as Src homology 2 (SH2)‐domain containing the tyrosine phosphatase‐1 (SHP‐1) (Jung et al., 2015), SHP‐2 (Kim et al., 2003) or PTP 1B (PTP‐1B) (Gu et al., 2003) could dephosphorylate and thereby inactivate STAT3 signalling. There is increasing evidence that SHP‐1 activation suppresses cell proliferation and induces apoptosis in different types of malignant cells (Liu et al., 2014; Fan et al., 2015). Pharmacologically targeting SHP‐1‐STAT3 signalling may be a potential therapeutic strategy for cancer treatment.

Recently, agents derived from the pharmacophore hydroxamate with diverse biological and pharmacological properties have attracted much attention in the field of drug discovery (Bertrand et al., 2013). In addition, most HDAC inhibitors have been hydroxamate derivatives. A number of novel hydroxamate derivatives as HDAC inhibitors have been shown to exhibit broad‐spectrum anti‐tumour activities (Deroanne et al., 2002; Venugopal et al., 2013; Rajak et al., 2014; West and Johnstone, 2014). Vorinostat (suberoylanilide hydroxamate) (Grant et al., 2007) and belinostat (PXD101) (Poole, 2014) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma. Another hydroxamate‐based HDAC inhibitor, panobinostat (LBH589), also exhibits anti‐tumour properties and is currently undergoing clinical trials (Giles et al., 2006). These observations suggest that additional hydroxamate‐based HDAC inhibitors may exhibit anti‐tumour activities capable of therapeutic applications and serve as a basis for developing novel anti‐tumour drugs. In an effort to develop novel anti‐tumour hydroxamate‐based HDAC inhibitors, we synthesized a series of aliphatic hydroxamate derivatives, the WMJ‐8 compounds (Figure 1A), and investigated their anti‐tumour properties. Of these compounds, WMJ‐8‐B was selected for its ability to facilitate MDA‐MB‐231 cell death. The aims of this study were to characterize the underlying mechanisms by which WMJ‐8‐B induces the death of MDA‐MB‐231 human basal TNBC cells.

Figure 1.

WMJ‐8‐B suppressed cell proliferation and affected cell cycle distribution in MDA‐MB‐231 cells. (A) Chemical structures of WMJ‐8‐A, WMJ‐8‐B, WMJ‐8‐C, WMJ‐8‐D, WMJ‐8‐E and WMJ‐8‐F. (B) 4T1, T47D, MCF‐7 and MDA‐MB‐231 cells were treated with vehicle or WMJ‐8‐A~F at 10 μM for 48 h. Cell viability was then determined by an MTT assay. Each column represents the mean ± SEM of six independent experiments performed in triplicate. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. Technical replicates were used to ensure the reliability of singe values for each experiment. (C) MDA‐MB‐231, MCF‐7, MCF‐10A and Hs68 cells were treated with vehicle or WMJ‐8‐B at the indicated concentrations for 24 h. Cell viability was then determined by an MTT assay. Each column represents the mean ± SEM of six independent experiments performed in triplicate. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. (D) Cells were treated with vehicle or WMJ‐8‐B at the indicated concentrations for 24 h. The acetylation status of H3 was then determined by immunoblotting. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. (E) Cells were treated with vehicle or WMJ‐8‐B at the indicated concentrations for 24 h. The percentage of PI‐stained cells in subG1, G0/G1, S and G2/M phases was then analysed by flow cytometric analysis as described in the Methods section. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using one‐way ANOVA, with Tukey's post hoc test. *P < 0.05, compared with the control group.

Methods

Synthesis of WMJ‐8

WMJ‐8 compounds were synthesized as described in the Supporting Information.

Cell culture

MDA‐MB‐231, MCF‐7, T47D, 4T1 and Hs68 cell lines were obtained from the Bioresource Collection and Research Centre (Hsinchu, Taiwan). MCF‐10A and HS578T cell lines were kindly provided by Professor Yuan‐Soon Ho (Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan). MDA‐MB‐468 cell line was kindly provided by Professor Wei‐Chien Huang (Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan). MCF‐10A cells were maintained in DMEM/F12 medium containing 0.5 μg·mL⁻¹ hydrocortisone, 10 μg·mL⁻¹ insulin, 20 ng·mL⁻¹ EGF, 10% FCS, 100 μg·mL⁻¹ streptomycin and 100 U·mL⁻¹ of penicillin G in a humidified 37°C incubator. Other cells were maintained in DMEM (MDA‐MB‐231, Hs68, HS578T, and 4T1), MEM (MCF‐7), RPMI1640 (T47D), DMEM/F12 (MDA‐MB‐468) medium containing 10% FCS, 100 μg·mL⁻¹ streptomycin and 100 U·mL⁻¹ of penicillin G in a humidified 37°C incubator.

MTT (cell viability) assay

The colorimetric MTT assay was employed to determine cell viability as described previously (Chen et al., 2015).

Flowcytometry

Propidium iodide (PI)‐single staining

MDA‐MB‐231 cells were treated with the indicated concentrations of WMJ‐8‐B for 24 h. Cells were washed with PBS twice and fixed in 70% ethanol at 0°C for another 24 h. Cells were washed with phosphate‐citric acid buffer and stained in the dark with staining buffer containing 30 μg·mL⁻¹ PI, 0.1% Triton X‐100 and 100 μg·mL⁻¹ RNase A for 30 min. Flowcytometry was performed using the FACScan and Cellquest program (BD Biosciences, San Jose, CA, USA). The percentage of PI‐stained cells in the subG1 (Apoptosis, Apo), G0/G1, S or G2/M region was analysed using the ModFit programmes (BD Biosciences, San Jose, CA, USA).

Annexin V‐FITC and PI‐double staining

MDA‐MB‐231 cells were treated with the indicated concentrations of WMJ‐8‐B for 24 h. Cells were harvested and immediately stained with annexin V‐FITC (2 μg·mL⁻¹) and PI (50 μg·mL⁻¹) at 37°C for 15 min in the dark. Flowcytometry was performed using the FACScan and Cellquest program (BD Biosciences, San Jose, CA, USA). The FCS Express programme (BD Biosciences, San Jose, CA, USA) was employed to determine the percentage of stained cells in different quadrants. The lower left quadrant (LL, annexin V⁻PI⁻) of each panel represents the viable cells. The lower right quadrant (LR, annexin V⁺PI⁻) represents the early apoptotic cells. The upper right quadrant (UR, annexin V⁺PI⁺) represents advanced apoptotic and necrotic cells.

Determination of mitochondrial membrane potential

MDA‐MB‐231 cells were treated with the indicated concentrations of WMJ‐8‐B for 24 h. Cells were harvested and immediately stained with 200 nM MitoStatus Red (BD Biosciences, San Jose, CA, USA) at 37°C for 15 min in the dark. Flow cytometry was performed using the FACScan and Cellquest programme (BD Biosciences, San Jose, CA, USA).

Immunoblotting

Cells were harvested in an extraction buffer containing 0.5% NP‐40, 140 mM NaCl, 10 mM Tris (pH 7.0), 0.05 mM pepstatin A, 0.2 mM leupeptin and 2 mM PMSF. Equal amounts of protein samples were subjected to SDS‐PAGE and transferred onto a NC membrane (Pall Corporation, Washington, NY, USA). After being blocked in a 5% non‐fat milk‐containing blocking buffer for 1 h, proteins were identified using specific primary antibodies followed by HRP‐conjugated secondary antibodies. Enhanced chemiluminescence was used to detect immunoreactivity as per the manufacturer's instructions. Quantitative data were obtained using a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP).

Transfection in MDA‐MB‐231 cells

MDA‐MB‐231 cells (5 × 10⁴ cells per well) were transfected with Sp1‐luc, STAT3‐luc or survivin promoter‐luc (survivin‐luc) plus renilla‐luc for the reporter assay or transfected with pcDNA, HDAC4‐Flag or HDAC6‐Flag for immunoblotting using K2 Transfection reagent (Biontex Laboratorys GmbH, München, Germany) according to the manufacturer's instructions.

Reporter assay (Dual‐Glo luciferase assay)

After transfection with reporter constructs plus renilla‐luc, cells with or without treatments were harvested. The luciferase reporter activity was examined using a Dual‐Glo luciferase assay system kit (Promega) according to the manufacturer's instructions and was normalized based on renilla luciferase activity.

Chromatin immunoprecipitation (ChIP) analysis

After different treatments, cells were cross linked with 1% formaldehyde at 37°C for 10 min. Cells were rinsed with ice‐cold PBS and harvested in SDS lysis buffer followed by sonication six times for 15 s each. Cells were centrifuged for 10 min, and supernatants were collected and diluted in ChIP dilution buffer. An aliquot of each sample was used as ‘Input’ in the PCR analysis. The remainder of the soluble chromatin was immunoprecipitated with normal IgG, STAT3 or Sp1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 16 h. Protein A‐Magnetic Beads (Millipore, Billerica, MA, USA) were added and incubated for another 2 h at 4°C with a gentle rotation to collect the immune complexes. The complexes were sequentially washed in the following washing buffers: low salt immune complex washing buffer, high‐salt immune complex washing buffer and LiCl immune complex washing buffer. After being washed with Tris‐EDTA buffer twice, the complexes were eluted twice for 100 μL aliquots of elution buffer each. The cross‐linked chromatin complex was reversed by incubation with 0.2 M NaCl and heating at 65°C for 4 h. DNA was purified using GP™ DNA purification spin columns (Viogene, New Taipei City, Taiwan). PCR was performed using PCR Master Mix (Promega, Madison, WI, USA), according to the manufacturer's protocol. Ten percent of the total purified DNA was used for the PCR in a 50 μL reaction mixture. The 228‐bp survivin promoter fragment between −264 and −37 and 202‐bp p21 promoter fragment between −234 and −33 were amplified using PCR with the following primer pairs: sense: 5′‐ttc ttt gaa agc agt cga gg‐3′ and antisense: 5′‐tca aat ctg gcg gtt aat gg‐3′(for survivin promoter); sense: 5′‐act ggg gga gga ggg aag t‐3′ and antisense: 5′‐gcg gcc ctg ata tac aac c‐3′ (for p21 promoter). This was done with an initial denaturation at 95°C for 5 min, 30 cycles of 30 s at 95°C, 30 s at 56°C and 45 s at 72°C and final extension for another 10 min at 72°C. The PCR products were analysed by 1.5% agarose gel electrophoresis.

Suppression of Shp‐1, STAT3 or survivin expression

Target gene suppression was performed as previously described (Chen et al., 2015). For shp‐1, stat3 or survivin suppression, pre‐designed siRNAs targeting the human shp‐1, stat3 or survivin (BIRC5) and negative control siRNA were purchased from Sigma‐Aldrich (St Louis, MO, USA). The siRNA oligonucleotides were as follows: negative control siRNA, 5′‐gaucauacgugcgaucaga‐3′; shp‐1 siRNA, 5′‐cugaacugcuccgauccca‐3′; stat3 siRNA, 5′‐ggauaacgucauuagcaga‐3′ and survivin siRNA, 5′‐ccucuacuguuuaacaaca‐3′.

Immunoprecipitation

Cells were lysed in a lysis buffer containing 1 mM MgCl₂ and 125 mM NaCl, 1 mM PMSF, 1% Triton X‐100, 10 μg·mL⁻¹ leupeptin, 10 μg·mL⁻¹ aprotinin, 100 μM sodium orthovanadate and 20 mM Tris–HCl, pH 7.5. Cell lysate was centrifuged for 30 min at 4°C; the supernatant was collected and incubated with antibodies against Sp1 with gentle rotation at 4°C for 16 h. Protein A‐Magnetic Beads (Millipore) were added to collect the immune complexes at 4°C for another 2 h. After being washed three times with lysis buffer, the immunoprecipitated complexes were subjected to immunoblotting for assessing Sp1 acetylation status.

Immunofluorescence microscopy

For determination of tubulin distribution, MDA‐MB‐231 cells were seeded on glass cover slips for 24 h. Cells were treated with WMJ‐8‐B, colchicine or paclitaxel for 24 h. After treatment, cells were washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were permeabilized for 30 min in 0.1% Triton X‐100 in PBS, washed twice and incubated with 1% BSA in PBS for another 1 h. To observe tubulin distribution, cells were reacted with rabbit anti‐β tubulin antibody (Cell Signalling, Danvers, MA, USA) (1:100 dilution in PBS) for 16 h at 4°C. After being washed, slides were incubated for 1 h with FITC‐conjugated goat anti‐rabbit IgG. Slides were mounted with DAPI‐containing mounting solution (SlowFad Gold, Thermo Fisher Scientific, Waltham, MA, USA) and then observed under a confocal microscope (Zeiss, LSM 410). Green fluorescence indicated β tubulin, and blue fluorescence represented nuclei.

Reverse‐transcription‐quantitative real‐time PCR (RT‐qPCR)

Total RNA was isolated from cells using the RNAspin RNA isolation kit (GE Healthcare, Little Chalfont, UK). The GoScript™ Reverse Transcription System (Promega, Madison, WI, USA) was used for cDNA synthesis according to the manufacturer's instructions. The cDNAs were stored at −20°C until qPCR was performed in the StepOne Real‐Time PCR systems (Applied Biosystems, Grand Island, NY‐USA). Real‐time PCR was performed with the GoTaq qPCR Master Mix (Promega, Madison, WI, USA) and cycling conditions were as follows: hot‐start activation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing/extension at 60°C for 60 s respectively. Primer pairs for the two transcripts of GAPDH and survivin are as follows: GAPDH sense, 5′‐gtc agt ggt gg acct gac ct‐3′; GAPDH anti‐sense, 5′‐agg ggt cta cat ggc aac tg‐3′; survivin sense, 5′‐gcc ttt cct taa agg cca tc‐3′; survivin anti‐sense, 5′‐aac cct tcc cag act cca ct‐3′.

SHP‐1 activity assay

To determine SHP‐1 phosphatase activity, we used a PTP assay system (Promega, Madison, WI, USA) to measure phosphate release as an index of phosphatase activity as previously described (Chen et al., 2015). After different treatments, cells were harvested, and equal amounts (200 μg) of cellular protein were incubated with anti‐SHP‐1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 15 μL protein A‐Magnetic Beads (Millipore, Billerica, MA, USA) at 4°C for 2 h to immunoprecipitate SHP‐1. Immune complexes were collected, washed and incubated with the phosphoprotein substrate (amino acid sequence END (pY)INASL, 100 μM), in protein phosphatase assay buffer [60 mM 2‐mercaptoethanol, 0.1 M NaCl, 20 mM 4‐morpholinepropanesulfonic acid (pH 7.5) and 0.1 mg·mL⁻¹ serum albumin]. Reactions were initiated by the addition of the substrate and carried out for 20 min at 30°C. Appropriate phosphate standard solutions containing free phosphate were also prepared for the standard curve. Reactions were terminated by the addition of the Molybdate Dye solution. The absorbance at 630 nm was measured on a microplate reader. Nonspecific hydrolysis of END (pY)INASL by lysates was assessed in normal IgG immunoprecipitates.

Transwell invasion assay

The cell invasion assays were performed using transwell plates (Corning, NY, USA). The bottom face of the filter in the transwell plate was coated with 0.2% gelatin. The bottom chambers were filled with DMEM containing 10% FBS. MDA‐MB‐231 cells (2 × 10⁴ cells per well) in 200 μL DMEM medium (without FBS) in the absence or presence of WMJ‐8‐B (10 μM) were seeded in the top chambers. Cells were allowed to invade for 24 h. Non‐invaded cells (on the top side of the filter) were scraped with a cotton swab, and invaded cells were fixed and stained with 0.5% toluidine blue in 4% paraformaldehyde. The cells were photographed under an inverted contrast phase light microscope (×40, Nikon, Japan). Stained MDA‐MB‐231 cells that invaded through the membrane were also quantified by dissolving in 33% acetic acid and measuring the absorbance at 570 nm.

Randomization and blinding

In every single experiment, the same cell was used to evaluate the effects of WMJ‐8‐B versus the related control. Formal randomization was therefore not employed. Mice used in the xenograft model were randomly allocated to cages by vivarium staff and randomized into vehicle‐treated or WMJ‐8‐B‐treated group before the treatment. For blinding, we had different people conducting the experiments (operator) and analysing the data (analyst).

Ethics statement

The protocols described below were approved by the Taipei Medical University Laboratory Animal Care and Use Committee (Permit Number: LAC‐2015‐0215). The present study was also carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publication No 85–23, revised 1996).

Mouse xenograft model

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The breast xenograft model with nude_nu/nu mice as described previously (Nakayama et al., 2009; Xie et al., 2009) was employed to determine the anti‐tumour effects of WMJ‐8‐B in vivo. Three‐week old female nude_nu/nu mice with body weight about 19 g were obtained from BioLasco (Taipei, Taiwan) and used for the experiment presented in Figure 9. All the mice were housed (three mice per cage) in clean specific pathogen‐free rooms (standard 12 h light/dark cycle at 22°C) in Laboratory Animal Centre of Taipei Medical University and maintained on standard chow and autoclaved water. All mice were randomly allocated to individually ventilated cages by vivarium staff, upon transfer from BioLASCO into the animal housing room. All mice purchased from BioLASCO were acclimatized in the animal housing room for 7 days prior to starting experiments. MDA‐MB‐231 cells were harvested and resuspended in PBS, and 4 × 10⁶ cells in a volume of 200 μL were injected into the mammary fat pad of each mouse. Once the tumour reached approximately 200 mm³, animals were randomized into the control group (six mice) and the treatment group (six mice), which received WMJ‐8‐B 20 mg·kg⁻¹·day⁻¹. The treatment was administered i.p. once daily for 34 days. Tumours were measured every day by a digital calliper. Tumour volume was calculated using the formula V (mm3) = [ab2] × 0.52, where a is the length and b is the width of the tumour (Chang et al., 2015). The body weights of the nude mice were examined daily during the 34 days of treatment with vehicle or WMJ‐8‐B. At the end of the treatment, animals were killed by carbon dioxide inhalation, and tumours were removed and weighed. The study conforms to the Guide for the Care and Use of Laboratory Animals (NIH publication No 85–23, revised 1996) and was approved by the Taipei Medical University Animal Care and Use Committee.

Figure 9.

WMJ‐8‐B suppressed the growth of MDA‐MB‐231 tumour xenografts in vivo. (A) Cells were starved in DMEM medium without FBS for 24 h; cells were then seeded in the top chamber in the absence or presence of WMJ‐8‐B (10 μM) using serum (10% FBS) as chemo‐attractant. After 24 h, invaded cells through the gelatin‐coated membrane were stained and quantified. Each column represents the mean ± SEM of seven independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the vehicle‐treated control group; ^# P < 0.05, compared with the group treated with WMJ‐8‐B alone. (B) Nude mice bearing xenografts of MDA‐MB‐231 breast cancer cells were treated with WMJ‐8‐B, 20 mg·kg⁻¹·day⁻¹ i.p., for 34 days. The control group received vehicle only. Tumour volumes were calculated as described in the Methods section. Values represents the mean ± SEM, n = 6. (C) After 34 days of treatment, mice were killed and tumours were dissected and weighed. Each column represents the mean ± SEM. Statistically significant differences were determined using the Student's t‐test. ^* P < 0.05 as compared with the vehicle‐treated control group, n = 6. Protein lysates obtained from five randomly selected xenograft tumours were subjected to immunoblotting for assessing survivin, cleaved caspase 3 (D) and STAT3‐p (E) levels. Values represent the mean ± SEM of five tumours from each group performed in duplicate. Statistically significant differences were determined using Student's t‐test. ^* P < 0.05 as compared with the vehicle‐treated control group. Technical replicates were used to ensure the reliability of single values for each experiment.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are expressed as mean ± SEM (n ≥ 5), where ‘n’ refers to independent values and not replicates. To control for unwanted sources of variation and to reveal relevant trends, normalization was performed to compare the differences after the treatment. For the MTT assay, the viability of vehicle‐treated cells (control group) was considered to be 100% and the viability of the cells treated with the WMJ‐8 compounds was expressed as a percentage of the control. For immunoblotting, protein expression levels (e.g. survivin and p21) were normalized to that of α‐tubulin. The levels of protein modification [e.g. STAT3 phosphorylation or histone 3 (H3) acetylation] were normalized to that of un‐modified protein (e.g. STAT3 or H3). The status of protein modification or protein expression levels in WMJ‐8‐B‐treated cells are expressed as fold changes over that of the vehicle‐treated cells, whose expression was set to 1 (100%). The SEM was normalized appropriately. The status of protein modification or protein expression levels are expressed by normalization that generates control values with no variance (SEM = 0) to reduce the effect of variation from different exposure of blotting, and such data were not subjected to parametric statistical analysis. Statistical analysis was performed using SigmaPlot 10 (Build 10.0.0.54; Systat Software, San Jose, CA, USA). Statistical comparisons between two groups were evaluated by Student's unpaired t‐test for parametric analysis or Mann–Whitney test for non‐parametric analysis. Statistical comparisons among more than two groups were evaluated by one‐way ANOVA with Tukey's post hoc test for parametric analysis or Kruskal–Wallis test followed by Dunn's multiple comparison for non‐parametric analysis. Post hoc tests were run only if F achieved P < 0.05, and there was no significant inhomogeneity. A P value smaller than 0.05 was defined as statistically significant.

Reagents

MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) was from Sigma‐Aldrich (St Louis, MO, USA). DMEM, MEM or RPMI 1640 medium, TrypLE™, FBS and all cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). Mithramycin A, colchicine and paclitaxel were bought from Calbiochem (San Diego, CA, USA). Z‐Val‐Ala‐Asp (OMe)‐FMK (Z‐VAD‐FMK) was purchased from MedChem Express (Monmouth Junction, NJ, USA). U0126 and the histone acetyltransferase (HAT) inhibitor, anacardic acid (AA), were purchased from SelleckChem (Houston, TX, USA). Antibodies against normal IgG, p21, SHP‐1 and Sp1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against survivin, caspase 3 active form, PARP, ERK1/2, ERK1/2 phosphorylated at Thr²⁰²/Tyr²⁰⁴, STAT3, STAT5, STAT3 phosphorylated at Tyr⁷⁰⁵, STAT3 phosphorylated at Ser⁷²⁷ and STAT5 phosphorylated at Tyr⁶⁹⁴ were purchased from Cell Signalling (Danvers, MA, USA). Anti‐mouse and anti‐rabbit IgG conjugated HRP antibodies, as well as antibodies against α‐tubulin and DDDDK (Flag), were obtained from GeneTex Inc (Irvine, CA, USA). K2 Transfection reagent was purchased from Biontex Laboratorys GmbH (München, Germany). HDAC4‐Flag (Addgene plasmid 13821) and HDAC6‐Flag (Addgene plasmid 13823) constructs were provided by Dr Eric Verdin (Department of Medicine, University of California, San Francisco, USA). Survivin promoter luciferase construct (Survivin‐luc) was purchased from Health Research Inc. (Roswell Park Cancer Institute Division, NY, USA). Renilla‐luc and the Dual‐Glo luciferase assay system were purchased from Promega (Madison, WI, USA). All materials for immunoblotting were purchased from Bio‐Rad (Hercules, CA, USA). The enhanced chemiluminescence detection kit was from Millipore (Billerica, MA, USA). All other chemicals were obtained from Sigma‐Aldrich (St Louis, MO, USA).

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY (Alexander et al., 2015a,b).

Results

WMJ‐8‐B induced cell cycle arrest and apoptosis in MDA‐MB‐231 cells

The MTT assay was used to evaluate the effects of WMJ‐8 compounds (WMJ‐8‐A~F) (Figure 1A) on cell viability in mouse (4T1) or human (T47D, MCF‐7 and MDA‐MB‐231) breast cancer cell lines. As shown in Figure 1B, WMJ‐8‐A, WMJ‐8‐B, WMJ‐8‐C, WMJ‐8‐D or WMJ‐8‐E at 10 μM significantly decreased cell viability in 4T1, MCF‐7 and MDA‐MB‐231 cells, but not in T47D cells, after 48 h exposure. However, cell viability was not altered in the presence of 10 μM WMJ‐8‐F in these cell lines (Figure 1B). In the following experiments, we selected WMJ‐8‐B to investigate its underlying mechanisms at inducing MDA‐MB‐231 cell death. The results from the MTT assay further showed that WMJ‐8‐B concentration‐dependently decreased cell viability in MDA‐MB‐231 cells, after 24 h exposure (Figure 1C). In contrast, the suppressive effects of WMJ‐8‐B on cell viability were less pronounced in MCF‐7 breast cancer cells and MCF‐10A human mammary epithelial cells. Moreover, WMJ‐8‐B did not affect cell viability in non‐tumour Hs68 fibroblasts (Figure 1C). To assess whether WMJ‐8‐B is able to inhibit HDACs, we examined the effects of WMJ‐8‐B on H3 acetylation. As shown in Figure 1D, WMJ‐8‐B significantly increased H3 acetylation, suggesting that WMJ‐8‐B is capable of inhibiting HDACs in MDA‐MB‐231 cells. We next determined whether WMJ‐8‐B affects cell cycle progression or induces apoptosis using flowcytometry with PI labelling. As shown in Figure 1E, the percentage of PI‐stained cells in the S region was decreased after the 24 h treatment with WMJ‐8‐B. These effects were accompanied by a concomitant increase in the percentage of PI‐stained cells in the sub‐G1 (apoptosis, Apo) and G2/M regions (Figure 1E). To detect apoptosis, flowcytometry with PI and annexin V‐FITC double labelling was also employed. Treatment of cells with WMJ‐8‐B for 24 h significantly increased the percentage of early (lower right quadrant, LR, annexin V⁺PI⁻ cells) and advanced (upper right quadrant, UR, annexin V⁺PI⁺ cells) apoptotic cells (Figure 2A). WMJ‐8‐B at 10 μM, for 24 h, also significantly reduced the mitochondria membrane potential (Figure 2B). Z‐VAD‐FMK (100 μM), a broad‐spectrum caspase inhibitor, significantly restored WMJ‐8‐B‐decreased cell viability (Figure 2C). In addition, WMJ‐8‐B increased the cleaved (active) form of caspase 3 (Figure 2D) and the selective caspase 3 substrate, PARP (Figure 2E), as determined by immunoblotting. Together, these findings suggest that WMJ‐8‐B induces G2/M cell cycle arrest and apoptosis in MDA‐MB‐231 breast cancer cells.

Figure 2.

WMJ‐8‐B induced cell apoptosis in MDA‐MB‐231 cells. (A) Cells were treated with vehicle or WMJ‐8‐B at the indicated concentrations for 24 h. Cells were then stained with annexin V‐FITC and PI for 15 min. The percentage of apoptotic cells was then analysed by flow cytometric analysis. Compiled results represent the mean ± SEM of five independent experiments are shown at the bottom of the chart. Statistically significant differences were determined using one‐way ANOVA, with Tukey's post hoc test. *P < 0.05, compared with the control group. (B) Cells were treated with vehicle or WMJ‐8‐B at 10 μM for 24 h. Mitochondria membrane potential was then analysed by flow cytometric analysis as described in the Methods section. Results shown are representative of five independent experiments. Statistically significant differences were determined using Student's test. *P < 0.05, compared with the control group. (C) Cells were treated with vehicle or Z‐VAD‐FMK (100 μM) for 1 h followed by the treatment with WMJ‐8‐B (10 μM) for another 24 h. Cell viability was then determined by an MTT assay. Each column represents the mean ± SEM of six independent experiments performed in duplicate. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the vehicle‐treated control group; #P < 0.05, compared with the group treated with WMJ‐8‐B alone. Cells were treated with WMJ‐8‐B at the indicated concentrations for 24 h. The extent of cleaved caspase 3 (D) and PARP (E) was then determined by immunoblotting. Each column represents the mean ± SEM of seven independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group.

WMJ‐8‐B induced α‐tubulin acetylation and disrupted microtubule assembly in MDA‐MB‐231 cells

The integrity of microtubules, the cellular dynamic structures, is essential for proper chromosome segregation during mitosis. Interference with the assembly or disassembly of α‐ and β‐tubulin into microtubules may lead to G2/M cell cycle arrest and cell death (Jordan and Wilson, 2004). As described above, WMJ‐8‐B caused G2/M cell cycle arrest in MDA‐MB‐231 cells, we thus explored whether WMJ‐8‐B affects tubulin distribution. The effects of paclitaxel and colchicine, two microtubule‐targeting agents, on tubulin distribution in MDA‐MB‐231 cells were also examined. Immunofluorescence microscopy using specific antibodies against β‐tubulin revealed that WMJ‐8‐B, similar to the microtubule‐disturbing agent colchicine, caused cellular microtubule depolymerization. In contrast, the microtubule‐stabilizing drug paclitaxel induced microtubule polymerization in MDA‐MB‐231 cells (Figure 3A). To confirm that WMJ‐8‐B is capable of disrupting microtubule assembly, cell extracts containing soluble (monomeric) or polymeric tubulin were prepared from MDA‐MB‐231 cells treated with WMJ‐8‐B, colchicine or paclitaxel for 24 h. Results from immunoblotting analysis showed that WMJ‐8‐B or colchicine decreased the fraction of polymerized tubulin (in precipitate, pellets) (Figure 3B). As expected, the polymeric form of tubulin was markedly increased in cells treated with paclitaxel (Figure 3B). It is reported that post‐translational modification of tubulin such as acetylation may interfere with microtubule assembly (Haggarty et al., 2003). We thus examined whether WMJ‐8‐B induces α‐tubulin acetylation in MDA‐MB‐231 cells. As shown in Figure 3C, treatment of cells with WMJ‐8‐B for 6 h markedly enhanced α‐tubulin acetylation in a concentration‐dependent manner. Transfection of cells with flag‐tagged HDAC4 (HDAC4‐Flag) or flag‐tagged HDAC6 (HDAC6‐Flag), two class II HDACs, significantly reduced WMJ‐8‐B‐induced α‐tubulin acetylation (Figure 3D). These results support a causal role of selective HDAC inhibition in WMJ‐8‐B‐induced α‐tubulin acetylation and subsequent disruption of microtubule assembly in MDA‐MB‐231 cells.

Figure 3.

WMJ‐8‐B induced α‐tubulin acetylation and disrupted microtubule assembly in MDA‐MB‐231 cells. (A) Cells were treated with the vehicle, WMJ‐8‐B (10 μM), paclitaxel (1 μM) or colchicine (5 μM) for 24 h. Tubulin distribution was determined by immunofluorescence analysis as described in the Methods section. Results shown are representative of five independent experiments. (B) Cells were treated as described in (A). After treatment, the fraction of polymerized tubulin was determined by immunoblotting. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the vehicle‐treated control group. (C) Cells were treated with WMJ‐8‐B at the indicated concentrations for 6 h. The acetylation status of α‐tubulin was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. (D) Cells were transiently transfected for 48 h with pcDNA, HDAC4‐flag or HDAC6‐flag. After transfection, cells were treated with WMJ‐8‐B (2.5 μM) for another 6 h. The acetylation status of α‐tubulin and flag‐tagged HDAC4 and HDAC6 were determined by immunoblotting. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the pcDNA transfection (mock transfection) group treated with vehicle; #P < 0.05, compared with the pcDNA transfection group treated with WMJ‐8‐B.

Survivin reduction contributes to WMJ‐8‐B‐induced MDA‐MB‐231 cell death

Survivin plays a pivotal role in regulating not only apoptosis but also cell cycle progression (Altieri, 2008). Elevated expression of survivin is found in various types of cancers including breast cancer (Ambrosini et al., 1997; Hunter et al., 2007). To determine whether a reduction in survivin induces MDA‐MB‐231 cell death, a survivin siRNA oligonucleotide was employed. As shown in Figure 4A, transfection with survivin siRNA markedly reduced basal levels of survivin in MDA‐MB‐231 cells. This reduction of survivin mimicked the enhancing effects of WMJ‐8‐B on G2/M cell cycle arrest and apoptosis (Figure 4B). Therefore, we examined whether WMJ‐8‐B had any effects on survivin expression. As shown in Figure 4C, WMJ‐8‐B significantly reduced survivin protein levels in MDA‐MB‐231 cells in a concentration‐dependent manner. However, this inhibitory effect of WMJ‐8‐B on survivin was less pronounced in MCF‐7 cells (Figure 4C). WMJ‐8‐B induced a significant decrease in survivin mRNA in MDA‐MB‐231 cells (Figure 4D). Results from the reporter assay with human survivin‐promoter reporter construct (−3569/+1, survivin‐luc) further showed that WMJ‐8‐B reduced survivin‐promoter luciferase activity in MDA‐MB‐231 cells (Figure 4E). These results suggest that a reduction in survivin contributes to WMJ‐8‐B‐induced MDA‐MB‐231 cell death.

Figure 4.

WMJ‐8‐B reduced survivin expression in MDA‐MB‐231 cells. (A) MDA‐MB‐231 cells were transfected with negative control siRNA or survivin siRNA for 48 h. Survivin and α‐tubulin levels were determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the vehicle‐treated control group. (B) Cells were transfected as described in (A). After transfection, Flow‐cytometric analysis was used to determine cell cycle distribution. Each column represents the mean ± SEM of eight independent experiments. Statistically significant differences were determined using Student's t‐test. *P < 0.05, compared with the negative control siRNA group. (C) MCF‐7 and MDA‐MB‐231 cells were treated with vehicle or WMJ‐8‐B at indicated concentrations for 24 h. Protein level of survivin was determined by immunoblotting. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. (D) MDA‐MB‐231 cells were treated with vehicle or WMJ‐8‐B at 10 μM for 6 h. The survivin mRNA level was determined by an RT‐qPCR as described in the Methods section. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the control group. (E) Cells were transiently transfected with survivin promoter reporter construct (−3569/+1, survivin‐luc) and renilla‐luc for 24 h followed by the treatment with WMJ‐8‐B at 10 μM for another 24 h. The reporter assay was performed as described in the Methods section. Each column represents the mean ± SEM of six independent experiments performed in duplicate. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the control group.

STAT3 contributes to the reduction in surviving induced by WMJ‐8‐B in MDA‐MB‐231 cells

The constitutively activated transcription factor STAT3 is found in approximately half of tumour‐derived cell lines and primary breast tumours (Berishaj et al., 2007; Banerjee and Resat, 2016). The effects of WMJ‐8‐B on phosphorylation of STAT3 at Tyr⁷⁰⁵, which is a key step in STAT3 activation, were thus examined. As shown in Figure 5A, WMJ‐8‐B treatment was associated with a time‐dependent decrease in STAT3 Tyr⁷⁰⁵ phosphorylation. In addition, STAT3 Ser⁷²⁷ phosphorylation also modulates STAT3 transcriptional activity, and it may occur in response to various stimuli (Aziz et al., 2010). Treatment of cells with WMJ‐8‐B only slightly, but not significantly, increased STAT3 Ser⁷²⁷ phosphorylation (Supporting Information Fig. S1). In addition to STAT3, STAT5 is also constitutively activated in various types of human cancers including breast cancer (Yu and Jove, 2004). Results from immunoblotting analysis demonstrated that WMJ‐8‐B also decreased STAT5 Tyr⁶⁹⁴ phosphorylation, which is a key step in STAT5 activation, in MDA‐MB‐231 cells (Figure 5B). It is reported that STAT3 plays an important role in transactivating survivin expression (Sehara et al., 2013). Therefore, we aimed to investigate whether STAT3 contributes to the reduced survivin in WMJ‐8‐B‐stimulated MDA‐MB‐231 cells. Cells treated with WMJ‐8‐B for 24 h had a significant decrease in STAT3‐luciferase activity as determined by the luciferase reporter assay (Figure 5C). A ChIP analysis was employed to determine whether the recruitment of STAT3 to the survivin promoter region is altered in the presence of WMJ‐8‐B. Primers encompassing the survivin promoter region (−264 to −37) containing putative STAT3 binding sites were used. As shown in Figure 5D, STAT3 binding to the survivin promoter region was reduced in MDA‐MB‐231 cells treated with WMJ‐8‐B. Moreover, transfection of cells with STAT3 siRNA significantly reduced the basal level of survivin in MDA‐MB‐231 cells (Figure 5E). This indicates that STAT3 might account for the WMJ‐8‐B‐induced survivin reduction. These findings suggest that the STAT3‐mediated survivin reduction is involved, at least in part, in the facilitatory effect of WMJ‐8‐B on cell death.

Figure 5.

STAT3 contributes to WMJ‐8‐B‐induced decrease in survivin in MDA‐MB‐231 cells. (A) Cells were treated with vehicle or WMJ‐8‐B at 10 μM for the indicated periods. The phosphorylation status of STAT3 Tyr⁷⁰⁵ was determined by immunoblotting. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. (B) Cells were treated with vehicle or WMJ‐8‐B at 10 μM for the indicated periods. The phosphorylation status of STAT5 Tyr⁶⁹⁴ was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. (C) Cells were transiently transfected with STAT3‐luc and renilla‐luc for 24 h followed by the treatment with WMJ‐8‐B at 10 μM for another 24 h. A reporter assay was performed. Each column represents the mean ± SEM of seven independent experiments performed in duplicate. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the control group. (D) Cells were treated with vehicle or WMJ‐8‐B at 10 μM for the indicated periods. A ChIP assay was performed as described in the Methods section. Typical traces representative of five independent experiments with similar results are shown. (E) Cells were transfected with negative control siRNA or STAT3 siRNA for 48 h. The protein levels of survivin, STAT3 and α‐tubulin were determined by immunoblotting. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the negative control siRNA group.

SHP‐1 contributes to WMJ‐8‐B‐induced STAT3 dephosphorylation

We next investigated the underlying mechanism by which WMJ‐8‐B reduces STAT3 Tyr⁷⁰⁵ phosphorylation. It is conceivable that WMJ‐8‐B may activate a PTP that dephosphorylates STAT3 resulting in a reduction in survivin. A recent study showed that tyrosine phosphatase SHP‐1‐dependent STAT3 inactivation plays a pivotal role in inducing cancer cell death (Liu et al., 2013; Fan et al., 2015). We thus determined whether SHP‐1 contributes to the ability of WMJ‐8‐B to reduce STAT3 Tyr⁷⁰⁵ phosphorylation and survivin levels in MDA‐MB‐231 cells. As shown in Figure 6A, the SHP‐1 inhibitor NSC87877 significantly restored the WMJ‐8‐B‐decreased STAT3 Tyr⁷⁰⁵ phosphorylation and also reduced its negative impact on survivin (Figure 6B). To confirm more specifically that the inhibitory actions of WMJ‐8‐B on STAT3 Tyr⁷⁰⁵ phosphorylation and survivin expression were mediated by SHP‐1, a SHP‐1 siRNA oligonucleotide was used. As shown in Figure 6C, SHP‐1 siRNA restored WMJ‐8‐B‐decreased STAT3 Tyr⁷⁰⁵ phosphorylation and prevented the WMJ‐8‐B‐induced reduction in survivin (Figure 6D). Moreover, siRNA experiments revealed that SHP‐1 siRNA markedly reduced SHP‐1 levels in the absence or presence of WMJ‐8‐B. WMJ‐8‐B exposure also led to an increase in SHP‐1 phosphatase activity (Figure 6E). In contrast, NSC87877 significantly suppressed WMJ‐8‐B‐increased SHP‐1 phosphatase activity (Figure 6E). As WMJ‐8‐B was shown to exhibit HDAC inhibitory properties, we next examined whether alterations in protein acetylation levels contribute to SHP‐1 activation in the presence of WMJ‐8‐B. As shown in Figure 6F, anacardic acid (AA), a HAT inhibitor, significantly reduced SHP‐1 activity in WMJ‐8‐B‐stimulated MDA‐MB‐231 cells. Together, these results suggest that the inhibition of HDACs is causally related to WMJ‐8‐B‐induced SHP‐1 activation and subsequent cellular events in MDA‐MB‐231 cells.

Figure 6.

SHP‐1 contributes to WMJ‐8‐B‐induced STAT3 dephosphorylation. Cells were pretreated with the SHP‐1 inhibitor NSC 87877 at the indicated concentrations for 30 min. After treatment, cells were treated with WMJ‐8‐B at 10 μM for another 2 h. The extent of STAT3 Tyr⁷⁰⁵ phosphorylation (A) and survivin protein level (B) was determined by immunoblotting. Each column represents the mean ± SEM of seven independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the vehicle‐treated control group; #P < 0.05, compared with the group treated with WMJ‐8‐B alone. Cells were transfected with negative control siRNA or SHP1 siRNA for 48 h. The extent of STAT3 Tyr⁷⁰⁵ phosphorylation (C) and survivin protein levels (D) was determined by immunoblotting. Each column represents the mean ± SEM of five (for STAT3 Tyr⁷⁰⁵ phosphorylation) or six (for survivin level) independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the negative control siRNA‐transfected control group (mock transfection); #P < 0.05, compared with the negative control siRNA transfected group in the presence of WMJ‐8‐B. Cells were treated with vehicle, NSC 87877 (10 μM) (E) or AA (10 μM) (F) for 30 min followed by the treatment with WMJ‐8‐B (10 μM) for another 10 min. Cells were harvested for the SHP‐1 phosphatase activity assay as described in the Methods section. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the vehicle‐treated control group; #P < 0.05, compared with the group treated with WMJ‐8‐B alone. NSC: NSC87877, SHP‐1 inhibitor; AA: anacardic acid, HAT inhibitor.

Sp1 mediates WMJ‐8‐B‐induced p21^cip/Waf1 expression in MDA‐MB‐231 cells

The survivin promoter that up‐regulates the expression of survivin can be activated not only by STAT3 but also by the transcription factor Sp1 (Mityaev et al., 2008). In MDA‐MB‐231 cells, mithramycin A, a Sp1 inhibitor, significantly decreased the basal level of survivin (Figure 7A). We thus determined whether Sp1 transactivity is altered in the presence of WMJ‐8‐B. In contrast to the inhibitory effects of WMJ‐8‐B on STAT3 transactivity, WMJ‐8‐B significantly increased Sp1‐luciferase activity in MDA‐MB‐231 cells (Figure 7B). It is likely that the inhibitory effects of WMJ‐8‐B on survivin expression may not involve Sp1. Next, the effect of WMJ‐8‐B on p21^cip/Waf1 levels was examined because Sp1 also plays an important role in the induction of the cell cycle inhibitor p21^cip/Waf1. As shown in Figure 7C, WMJ‐8‐B significantly increased p21^cip/Waf1 expression in MDA‐MB‐231 cells. In contrast, WMJ‐8‐B‐increased p21^cip/Waf1 induction was markedly reduced in the presence of mithramycin A (Figure 7C). To further confirm whether Sp1 is recruited to the endogenous p21^cip/Waf1 promoter region in response to WMJ‐8‐B, a ChIP analysis was employed. Primers encompassing the p21^cip/Waf1 promoter region (−234 to −33) containing putative Sp1 binding sites were used. As shown in Figure 7D, the binding of Sp1 to the p21^cip/Waf1 promoter region (−234 to −33) increased after WMJ‐8‐B exposure. Sp1 has been shown to enhance transcription through various mechanisms including its modification such as acetylation (Chang and Hung, 2012). We thus explored whether Sp1 acetylation is altered in MDA‐MB‐231 cells after WMJ‐8‐B exposure. As shown in Figure 7E, WMJ‐8‐B caused an increase in Sp1 acetylation. The results from the ChIP analysis demonstrated that AA significantly inhibited WMJ‐8‐B‐induced Sp1 binding to the p21^cip/Waf1 promoter region (Figure 7F). Moreover, WMJ‐8‐B‐induced p21^cip/Waf1 induction was also reduced in the presence of AA (Figure 7G).However, ERK signalling also contributes to Sp1 activation (Milanini‐Mongiat et al., 2002). To explore whether ERK signalling contributes to WMJ‐8‐B′s actions, a pharmacological ERK signalling inhibitor, U0126, was used. As shown in Supporting Information Fig. S2a, U0126 significantly inhibited WMJ‐8‐B‐induced p21^cip/Waf1 induction. In contrast, WMJ‐8‐B‐induced survivin reduction was not altered in the presence of U0126 (Supporting Information Fig. S2a). The enhancing effects of WMJ‐8‐B on Sp1‐luciferase activities (Supporting Information Fig. S2b) and Sp1 binding to the p21^cip/Waf1 promoter region (Supporting Information Fig. S2c) were also reduced in the presence of U0126. Furthermore, WMJ‐8‐B caused an increase in ERK1/2 phosphorylation in MDA‐MB‐231 cells (Supporting Information Fig. S2d). Together, these results suggest that an inhibitory effect on HDACs and an increase in ERK signalling may contribute to Sp1 activation and p21^cip/Waf1 induction in WMJ‐8‐B‐stimulated MDA‐MB‐231 cells.

Figure 7.

Sp1 mediates WMJ‐8‐B‐induced increased p21 expression in MDA‐MB‐231 cells. (A) Cells were treated with vehicle or mithramycin A (0.3 μM) for 30 min followed by the treatment with WMJ‐8‐B (10 μM) for another 24 h. Survivin protein level was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the vehicle‐treated control group. (B) Cells were transiently transfected with Sp1‐luc and renilla‐luc for 24 h. After transfection, cells were treated with WMJ‐8‐B (10 μM) for another 24 h. A reporter assay was performed. Each column represents the mean ± SEM of seven independent experiments performed in duplicate. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the control group. (C) After treatment as described in (A), the protein level of p21 was determined by immunoblotting. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the vehicle‐treated control group; #P < 0.05, compared with the group treated with WMJ‐8‐B alone. (D) Cells were treated with vehicle or WMJ‐8‐B (10 μM) for the indicated periods. A ChIP assay was then performed. Typical traces representative of five independent experiments with similar results are shown. Mithramycin: mithramycin A. (E) Cells were treated with WMJ‐8‐B (10 μM) for the indicated periods. The cells were then lysed and immunoprecipitated with Sp1 antibodies followed by immunoblotting with acetyl‐lysine antibodies. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the vehicle‐treated control group. IB: immunoblotting. (F) Cells were treated with AA (10 μM) for 30 min followed by the treatment with WMJ‐8‐B (10 μM) for another 4 h. A ChIP assay was then performed. Typical traces representative of five independent experiments with similar results are shown. (G) Cells were treated with AA (10 μM) for 30 min followed by the treatment with WMJ‐8‐B (10 μM) for another 24 h. The protein level of p21 was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the vehicle‐treated control group; #P < 0.05, compared with the group treated with WMJ‐8‐B alone.

Effects of WMJ‐8‐B on SHP‐1 activation, STAT3 Tyr⁷⁰⁵ phosphorylation, survivin reduction, p21^cip/Waf1 induction and cell viability in two TNBC cell lines, HS578T and MDA‐MB‐468

In addition to MDA‐MB‐231 cells, we also determined the effects of WMJ‐8‐B in another two TNBC cell lines, HS578T and MDA‐MB‐468. As shown in Figure 8A, WMJ‐8‐B exhibited similar inhibitory effects on cell viability in MDA‐MB‐231, HS578T and MDA‐MB‐468 cells, after 48 h exposure. It also reduced survivin levels and increased the expression of p21 in HS578T and MDA‐MB‐468 cells (Figure 8B). Moreover, STAT3 Tyr⁷⁰⁵ phosphorylation was reduced in HS578T and MDA‐MB‐468 cells treated with WMJ‐8‐B exposure (Figure 8C). Similar to its effects in MDA‐MB‐231 cells, WMJ‐8‐B also increased SHP‐1 phosphatase activity in both TNBC cell lines (Figure 8D).

Figure 8.

The effect of WMJ‐8‐B on SHP‐1 activity, STAT3 Tyr⁷⁰⁵ phosphorylation, survivin and p21 levels and cell viability in HS578T and MDA‐MB‐468 TNBC cells. (A) MDA‐MB‐231, HS578T and MDA‐MB‐468 cells were treated with vehicle or WMJ‐8‐B at 10 μM for 48 h. Cell viability was then determined by an MTT assay. Each column represents the mean ± SEM of seven independent experiments performed in duplicate. Statistically significant differences were determined using the Kruskal–Wallis test. *P < 0.05, compared with the control group. Technical replicates were used to ensure the reliability of single values for each experiment. (B) Cells were treated with WMJ‐8‐B (10 μM) for 24 h. Protein levels of survivin and p21 were determined by immunoblotting. Each column represents the mean ± SEM of seven independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the control group. (C) Cells were treated with WMJ‐8‐B (10 μM) for 2 h. The extent of STAT3 Tyr⁷⁰⁵ phosphorylation was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the control group. (D) Cells were treated with WMJ‐8‐B (10 μM) for 10 min and then harvested for SHP‐1 phosphatase activity assay as described in the Methods section. Each column represents the mean ± SEM of five independent experiments. Statistically significant differences were determined using the Mann–Whitney test. *P < 0.05, compared with the control group. 8B: WMJ‐8B.

WMJ‐8‐B inhibited MDA‐MB‐231 tumour xenograft growth in vivo

We next performed the transwell invasion assay to determine whether the invasive potential of MDA‐MB‐231 cells is altered in the presence of WMJ‐8‐B. As shown in Figure 9A, using serum (10% FBS) as the chemoattractant, WMJ‐8‐B significantly reduced the number of cells that invaded through the gelatin‐coated transwell membrane barrier. We used a mouse xenograft tumour model to further determine the in vivo effects of WMJ‐8‐B. MDA‐MB‐231 cells were injected into the mammary fat pad of nude mice. After the tumours had grown to an average size of about 200 mm³, either vehicle or WMJ‐8‐B (20 mg·kg⁻¹·day⁻¹) was injected i.p. for 34 days. At the end of the 34 day treatment, mice were killed and xenografts were collected. Compared with the vehicle‐treated control group, WMJ‐8‐B markedly suppressed MDA‐MB‐231 tumour xenograft growth (Figure 9B) and reduced tumour weight (Figure 9C). WMJ‐8‐B at 20 mg·kg⁻¹·day⁻¹ had no significant effects on mouse body weight as compared with the vehicle‐treated control group within 34 days (Supporting Information Fig. S3). We also determined the protein levels of survivin and cleaved caspase 3 in the excised tumours. As shown in Figure 9D, the survivin level was decreased while the cleaved caspase 3 was increased in MDA‐MB‐231 xenografts from WMJ‐8‐B‐treated mice. We further examined the STAT3 Tyr⁷⁰⁵ phosphorylation status in the excised MDA‐MB‐231 xenografts. As shown in Figure 9E, STAT3 Tyr⁷⁰⁵ phosphorylation was reduced in MDA‐MB‐231 xenografts from WMJ‐8‐B‐treated mice. These results suggest that WMJ‐8‐B treatment is capable of suppressing tumour growth in vivo through, at least in part, inactivating STAT3 signalling and reducing the level of survivin. To explore whether WMJ‐8‐B exhibits acute toxicity, the effects of 7 days of treatment with WMJ‐8‐B, at a 10‐fold higher concentration (200 mg·kg⁻¹·day⁻¹, i.p.), on the major organs in mice were examined. No obvious pathohistological changes in heart, liver, spleen, lung and kidney were detected on microscopic examination after administration of this higher concentration of WMJ‐8‐B (Supporting Information Fig. S4). Together, these findings suggest that WMJ‐8‐B at 20 mg·kg⁻¹·day⁻¹ is capable of suppressing tumour growth in vivo without causing obvious pathological abnormalities in organs.

Discussion

Although the mechanisms underlying the anti‐tumour effects of hydroxamate derivatives remain largely unknown, there is increasing evidence suggesting they have beneficial effects in cancer treatment and potential in the development of novel anti‐tumour drugs. In the present study, we demonstrated that WMJ‐8‐B, a novel aliphatic hydroxamate derivative with HDAC inhibitory properties, increases tubulin acetylation and disrupts microtubule assembly. We also noted that WMJ‐8‐B activates SHP‐1‐STAT3‐survivin and Sp1‐p21^cip/Waf cascades to induce G2/M cell cycle arrest and apoptosis in MDA‐MB‐231 TNBC cells. Moreover, WMJ‐8‐B suppressed the growth of MDA‐MB‐231 human breast cancer xenografts in vivo.

In keeping with previous studies that aliphatic hydroxamate derivatives induce p21^cip/Waf activation and suppress cell proliferation in cancer cells (Jiang et al., 2012; Huang, Huang, Hsu, Ou, Huang & Hsu, 2015b), we noted that WMJ‐8‐B increased p21^cip/Waf level while decreasing survivin in MDA‐MB‐231, HS578T and MDA‐MB‐468 TNBC cells. WMJ‐8‐B is capable of inducing not only growth arrest but also cell apoptosis through the inactivation of the STAT3‐survivin signalling. STAT3 phosphorylation mechanisms may vary among different breast cancer cell lines. STAT3 is constitutively Tyr⁷⁰⁵‐phosphorylated and activated in approximately half of breast carcinomas (Diaz et al., 2006; Gritsko et al., 2006; Leslie et al., 2006). Abnormal activation of Src and JAKs has been implicated in STAT3 activation in breast cancer cells (Dolled‐Filhart et al., 2003; Yu and Jove, 2004; Kortylewski et al., 2005). STAT3 activation may also occur via phosphorylation of serine residues by MAPKs (Tkach et al., 2013). We noted, in this study, that WMJ‐8‐B, although not significantly, slightly increased STAT3 Ser⁷²⁷ phosphorylation. In addition, WMJ‐8‐B induced ERK activation, which may contribute to Sp1 activation and the induction of p21^cip/Waf. The link between ERK signalling and STAT3 activation remains to be established in MDA‐MB‐231 cells exposed to WMJ‐8‐B. It is also worthy of further investigation to clarify whether STAT3 phosphorylation on other residues is also altered in the presence of WMJ‐8‐B. It has been reported that MDA‐MB‐231 TNBC cell line has high levels of STAT3 Tyr⁷⁰⁵ phosphorylation, whereas the MCF‐7 breast cancer cell line expresses lower levels of STAT3 Tyr⁷⁰⁵ phosphorylation (Berishaj et al., 2007). These observations explain, at least in part, why the inhibitory effects of WMJ‐8‐B on cell viability and survivin level were less pronounced in MCF‐7 cells.

Similar to previous reports (Liu et al., 2013; Fan et al., 2015), we demonstrated that SHP‐1‐mediated STAT3 inactivation contributes to survivin reduction and cell death in MDA‐MB‐231 cells after WMJ‐8‐B exposure. These observations indicate that SHP‐1 plays a pivotal role in STAT3 Tyr⁷⁰⁵ dephosphorylation and subsequent signalling events in the presence of WMJ‐8‐B. We also noted that WMJ‐8‐B significantly reduced STAT5 Tyr⁶⁹⁴ phosphorylation and the levels of the cell cycle regulator cyclinD1 (unpublished data). Whether WMJ‐8‐B‐induced activation of SHP‐1 also leads to STAT5 Tyr⁶⁹⁴ dephosphorylation and cyclinD1 reduction, as suggested in other studies (Paling and Welham, 2002; Leslie et al., 2006), remains to be confirmed. PTP‐1B, another PTP, also dephosphorylate and thereby inactive STAT3 signalling (Gu et al., 2003), but the exact role of PTP‐1B in breast cancer remains to be clarified since it may exhibit both tumour‐suppressing and tumour‐promoting effects. A recent study showed that PTP‐1B expression in breast cancer is associated with a significantly improved clinical outcome (Soysal et al., 2013). Whether PTP‐1B or other PTPs contribute to WMJ‐8‐B′s actions in MDA‐MB‐231 cells needs to be further investigated.

The precise mechanisms underlying WMJ‐8‐B′s activation of SHP‐1 or Sp1 signalling in MDA‐MB‐231 cells remains to be elucidated. Su et al. (2016) recently showed that SHP‐1 expression is inversely correlated with STAT3 phosphorylation in human TNBC cell lines and clinical breast cancer specimens. We noted, in the siRNA experiments, that WMJ‐8‐B slightly increased SHP‐1 levels in MDA‐MB‐231 cells in the absence of SHP‐1 siRNA. This suggests that WMJ‐8‐B may activate SHP‐1 via augmentation of SHP1 in MDA‐MB‐231 cells. Moreover, the WMJ‐8‐B‐increased SHP‐1 phosphatase activity was reduced in the presence of the HAT inhibitor AA, indicating that HDACs inhibition contributes to WMJ‐8‐B‐activated SHP‐1 and subsequent cellular events. It is likely that SHP‐1 activity may be modulated by its acetylation as is the case with MKP‐1, which is another protein phosphatase whose activity is enhanced via acetylation (Cao et al., 2008; Jeong et al., 2014). Moreover, acetylation of Sp1 may affect the transactivation of its target genes including p21^cip/Waf1 (Swingler et al., 2010; Waby et al., 2010). WMJ‐8‐B, like other hydroxamate‐based HDAC inhibitors, may inhibit HDACs to increase the acetylation of cellular signalling molecules other than H3, α‐tubulin and Sp1 as shown in this study. The ability of WMJ‐8‐B to enhance Sp1 binding to the p21^cip/Waf1 promoter region and increase p21^cip/Waf1 were reduced in the presence of AA. It appears that WMJ‐8‐B regulates Sp1 or SHP‐1 signalling through, at least in part, inhibiting the activity of HDACs. The link between SHP‐1‐STAT3‐survivin and Sp1‐p21^cip/Waf1 pathways and the different mechanisms through which WMJ‐8‐B affects these two cascades remain to be investigated. It is likely that these two signalling cascades converge in growth arrest and cell death. Additional work is needed to establish whether WMJ‐8‐B acetylation of SHP‐1 or other signalling molecules contributes to its growth‐inhibitory and apoptotic effects. Similarly, the relationship between SHP‐1 and Sp1 signalling in WMJ‐8‐B‐induced MDA‐MB‐231 cell death needs to be further characterized.

Aberrant activity of HDACs, most notably HDAC1, 2, 3 and 6, contributes to cell survival and tumour progression in several types of cancers including breast cancer (West and Johnstone, 2014). We demonstrated that WMJ‐8‐B‐induced α‐tubulin acetylation was significantly reduced in cells transfected with HDAC4‐flag or HDAC6‐flag. We also noted that AA restored the survivin levels in WMJ‐8‐B‐stimulated MDA‐MB‐231 cells (unpublished data). These observations suggest that WMJ‐8‐B‐induced MDA‐MB‐231 breast cancer cell death may involve alterations in the cellular acetylation status. In addition to HDAC4 or HDAC6, further investigations are needed to explore whether other HDAC isoforms contribute to the anti‐tumour mechanisms of WMJ‐8‐B.

In conclusion, we demonstrated in this study that WMJ‐8‐B exhibits anti‐tumour effects via SHP‐1‐STAT3‐survivin and Sp1‐p21 signalling cascades in MDA‐MB‐231 breast cancer cells. The inhibition of HDACs by WMJ‐8‐B may also contribute to its ability to induce cell death. The precise mechanisms underlying these activities remain to be fully investigated, but together, these findings support the role of WMJ‐8‐B as a potential lead compound in the development of anti‐tumour agents.

Author contributions

Y.F.C., S.W.H., W.J.H. and M.J.H. designed the experiments. Y.F.C., M.C.Y. and Y.F.H. performed the experiments. Y.F.C., S.W.H., Y.F.H. and M.J.H. analysed the data. W.J.H. contributed reagents/synthesized WMJ‐8 compounds. Y.F.C., G.O., W.J.H. and M.J.H. wrote the paper.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1 Effects of WMJ‐8‐B on STAT3 Ser727 phosphorylation in MDA‐MB‐231 cells. Cells were treated with vehicle or WMJ‐8‐B at 10 μM for indicated periods. The phosphorylation status of STAT3 Ser727 was determined by immunoblotting. Each column represents the mean ± S.E.M. of six independent experiments.

Figure S2 ERK signalling contributes to WMJ‐8‐B‐induced Sp1 activation and p21^cip/Waf1 induction in MDA‐MB‐231 cells. (A) Cells were treated with U0126 (3 μM) for 30 min followed by the treatment with WMJ‐8‐B (10 μM) for another 24 h. Protein levels of survivin and p21 were determined by immunoblotting. Each column represents the mean ± S.E.M. of five independent experiments. *P < 0.05, compared with the vehicle‐treated control group; #P < 0.05, compared with the group treated with WMJ‐8‐B alone. Mann–Whitney test (B) Cells were transiently transfected with Sp1‐luc and renilla‐luc for 24 h. After transfection, cells were treated with U0126 (3 μM) for 30 min followed by the treatment with WMJ‐8‐B (10 μM) for another 24 h. Reporter assay was performed. Each column represents the mean ± S.E.M. of six independent experiments performed in duplicate *P < 0.05, compared with the vehicle‐treated control group; #P < 0.05, compared with the group treated with WMJ‐8‐B alone. Mann–Whitney test (C) Cells were treated with U0126 (3 μM) for 30 min followed by the treatment with WMJ‐8‐B (10 μM) for another 4 h. A ChIP assay was performed. Typical traces representative of five independent experiments with similar results are shown. (D) Cells were treated with vehicle or WMJ‐8‐B at 10 μM for indicated periods. The phosphorylation status of ERK1/2 was determined by immunoblotting. Each column represents the mean ± S.E.M. of six independent experiments. *P < 0.05, compared with the vehicle‐treated control group. Kruskal–Wallis test.

Figure S3 Effects of WMJ‐8‐B on mouse body weight in the mouse xenograft model. Mouse xenograft model was performed as described in ‘Methods’ section. The body weights of the nude mice were examined daily within 34 days treatment of vehicle or WMJ‐8‐B. Values represent the mean ± S.E.M. n = 6 for vehicle‐treated control group and n = 6 for WMJ‐8‐B‐treated group.

Figure S4 WMJ‐8‐B did not cause obvious pathological abnormalities in organs. Mice were administered intraperitoneally once daily with vehicle (6 mice) or WMJ‐8‐B (200 mg/kg/day) (6 mice) for 7 days. At the end of treatment, 2 mice in the control group and 6 mice in the WMJ‐8‐B‐treated group were killed by carbon dioxide euthanasia. The organs including heart, liver, spleen, lung and kidney were removed for haematoxylin and eosin (H&E) staining. Results shown are representative of 2 vehicle‐treated mice and 6 WMJ‐8‐B‐treated mice.

Table S1 The half‐life and intrinsic clearancea of WMJ‐8‐B for human liver microsomes.

Chuang, Y.‐F. , Huang, S.‐W. , Hsu, Y.‐F. , Yu, M.‐C. , Ou, G. , Huang, W.‐J. , and Hsu, M.‐J. (2017) WMJ‐8‐B, a novel hydroxamate derivative, induces MDA‐MB‐231 breast cancer cell death via the SHP‐1‐STAT3‐survivin cascade. British Journal of Pharmacology, 174: 2941–2961. doi: 10.1111/bph.13929.