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Research ArticleArticle

Novel Thiosemicarbazones Regulate the Signal Transducer and Activator of Transcription 3 (STAT3) Pathway: Inhibition of Constitutive and Interleukin 6–Induced Activation by Iron Depletion

Goldie Y. L. Lui, Zaklina Kovacevic, Sharleen V. Menezes, Danuta S. Kalinowski, Angelica M. Merlot, Sumit Sahni and Des R. Richardson
Molecular Pharmacology March 2015, 87 (3) 543-560; DOI: https://doi.org/10.1124/mol.114.096529

Abstract

Pharmacologic manipulation of metal pools in tumor cells is a promising strategy for cancer treatment. Here, we reveal how the iron-binding ligands desferrioxamine (DFO), di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibit constitutive and interleukin 6–induced activation of signal transducer and activator of transcription 3 (STAT3) signaling, which promotes proliferation, survival, and metastasis of cancer cells. We demonstrate that DFO, Dp44mT, and DpC significantly decrease constitutive phosphorylation of the STAT3 transcription factor at Tyr705 in the pancreatic cancer cell lines PANC-1 and MIAPaCa-2 as well as the prostate cancer cell line DU145. These compounds also significantly decrease the dimerized STAT3 levels, the binding of nuclear STAT3 to its target DNA, and the expression of downstream targets of STAT3, including cyclin D1, c-myc, and Bcl-2. Examination of upstream mediators of STAT3 in response to these ligands has revealed that Dp44mT and DpC could significantly decrease activation of the nonreceptor tyrosine kinase Src and activation of cAbl in DU145 and MIAPaCa-2 cells. In contrast to the effects of Dp44mT, DpC, or DFO on inhibiting STAT3 activation, the negative control compound di-2-pyridylketone 2-methyl-3-thiosemicarbazone, or the DFO:Fe complex, which cannot bind cellular iron, had no effect. This demonstrates the role of iron-binding in the activity observed. Immunohistochemical staining of PANC-1 tumor xenografts showed a marked decrease in STAT3 in the tumors of mice treated with Dp44mT or DpC compared with the vehicle. Collectively, these studies demonstrate suppression of STAT3 activity by iron depletion in vitro and in vivo, and reveal insights into regulation of the critical oncogenic STAT3 pathway.

Introduction

Identification of novel chemotherapeutics that target deregulated signaling pathways responsible for tumorigenesis and metastasis is urgently required, especially considering that cancer remains a major cause of mortality (Goldstein et al., 1989; Lee et al., 2008; Vincent et al., 2011). In this regard, novel di-2-pyridyl thiosemicarbazones (DpT) are well characterized as promising chemotherapeutic agents (Yuan et al., 2004; Whitnall et al., 2006; Kovacevic et al., 2011; Chen et al., 2012; Liu et al., 2012; Merlot et al., 2013a; Sun et al., 2013). Numerous studies have demonstrated their potent and selective antitumor activity and antimetastatic effects across a broad range of cancer cell-types in vitro and in vivo (Yuan et al., 2004; Whitnall et al., 2006; Kovacevic et al., 2011; Chen et al., 2012; Liu et al., 2012; Merlot et al., 2013a; Sun et al., 2013). Notably, the second-generation DpT analog di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC; Fig. 1A) displays marked antitumor activity and high tolerability in vivo that surpasses the former first-generation, lead compound di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1A) (Kovacevic et al., 2011; Lovejoy et al., 2012).

Fig. 1.
Fig. 1.

The iron chelators DFO, Dp44mT, and DpC decrease expression of phosphorylated STAT3 at Tyr705 in pancreatic and prostate cancer cells. (A) Line drawings of the chemical structures of the iron chelators DFO, Dp44mT, and DpC, and the negative control compound Dp2mT. (B–E) The levels of p-STAT3 and STAT3 protein in (B) PANC-1, (C) MIAPaCa-2, (D) DU145, and (E) mortal PaEC cells after a 24-hour/37°C incubation with control medium, DFO (250 µM), Dp44mT (5 µM), DpC (5 µM), DFO:Fe (1:1; 250 µM), Dp44mT:Fe (2:1; 5 µM), DpC:Fe (2:1; 5 µM), FeCl3 (250 µM), or Dp2mT (5 µM). The Western blots are typical of three independent experiments, with densitometric analysis representing mean ± S.D. Relative to control: *P < 0.05; **P < 0.01; ***P < 0.001.

Unlike desferrioxamine (DFO; Fig. 1A), the classic iron chelator used to treat iron overload disease, DpT thiosemicarbazones form redox active metal complexes that generate reactive oxygen species (ROS), which plays a key role in their cytotoxic activity (Yuan et al., 2004; Richardson et al., 2006; Lovejoy et al., 2011). The mechanism of action of these ligands also involves cellular iron depletion, which is known to affect cellular targets critical for cell proliferation, survival, and metastasis, including ribonucleotide reductase (Yu et al., 2011), cyclin D1 (Kulp et al., 1996; Gao and Richardson, 2001; Nurtjahja-Tjendraputra et al., 2007), p21 (Darnell and Richardson, 1999; Fu and Richardson, 2007), and the metastasis suppressor N-myc downstream regulated gene-1 (NDRG1) (Le and Richardson, 2004; Kovacevic et al., 2011; Kovacevic et al., 2013).

This group of thiosemicarbazones has also been demonstrated to induce apoptosis (Yuan et al., 2004; Noulsri et al., 2009) and autophagy (Sahni et al., 2014), as well as to inhibit the epithelial-to-mesenchymal transition (Chen et al., 2012) in cancer cells. Dp44mT could target these critical molecules and cellular processes through its demonstrated modulation of oncogenic signaling pathways, including the transforming growth factor-β (Kovacevic et al., 2013), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) (Yu and Richardson, 2011), Ras/ERK (Kovacevic et al., 2013), protein kinase B (AKT)/phosphatidylinositol-3-kinase (Dixon et al., 2013; Kovacevic et al., 2013), and ROCK1/pMLC2 pathways (Sun et al., 2013). Despite considerable advances in our understanding of the molecular mechanisms of action of thiosemicarbazones, they remain incompletely understood. Detailed characterization is lacking in particular for the second-generation thiosemicarbazone DpC, which is important to elucidate for clinical development.

The signal transducer and activator of transcription (STAT) family consists of seven proteins that transduce extracellular signals to regulate genes involved in cell growth, survival, and differentiation (Kamran et al., 2013). Of these, STAT3 is the most well characterized and has been linked to tumor progression (Kamran et al., 2013). STAT3 signaling can be activated by upstream receptor interactions (e.g., IL-6/Janus kinase [JAK]) and nonreceptor kinases (e.g., Src, c-Abl) that phosphorylate STAT3 at Tyr705 (p-STAT3) (Turkson and Jove, 2000). This leads to dimer formation, nuclear translocation, binding to STAT3-specific DNA-binding elements, and transcription of target genes (e.g., Bcl-2, cyclin D1, and c-myc) that promote cell proliferation, survival, migration, apoptosis inhibition, and immune evasion (Darnell et al., 1994; Ihle, 1995; Sasse et al., 1997; Zushi et al., 1998; Bromberg et al., 1999; Wang et al., 2004; Xie et al., 2004). Constitutive activation of STAT3 is a hallmark of many human malignancies, including those of the prostate, pancreas, and breast, as well as leukemias and melanomas (Bowman et al., 2000; Scholz et al., 2003). Indeed, several studies have demonstrated that STAT3 inhibition can induce apoptosis in a range of cancer types, including prostate and pancreatic cancers (Haura et al., 2005; Lewis et al., 2008). Understanding the role of STAT3 signaling in tumor progression and how this affects responsiveness to therapies is an active area of clinical investigation and is critical for the successful development of personalized cancer treatments.

Herein, for the first time, we demonstrate that inducing cellular iron depletion using the iron chelators DFO, Dp44mT, and DpC can inhibit STAT3 signaling in vitro in prostate and pancreatic cancer cells, as well as in vivo using pancreatic tumor xenografts grown in nude mice. These findings reveal key insights into the role of iron in tumor progression through STAT3 pathway inhibition and highlight its potential application in cancer therapy through the use of potent and novel thiosemicarbazones.

Materials and Methods

Cell Culture.

The human pancreatic cancer cell lines, PANC-1 and MIAPaCa-2, and the human prostate cancer cell line DU145 were purchased from the American Type Culture Collection (Manassas, VA). PANC-1 and MIAPaCa-2 cells were grown in Dulbecco’s modified Eagle medium (Life Technologies, Melbourne, Australia), and DU145 cells were grown in RPMI 1640 medium (Life Technologies). All media were supplemented with 10% fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), glutamine (2 mM), nonessential amino acids (100 mM), and sodium pyruvate (100 mM; all supplements from Life Technologies).

Primary human pancreatic epithelial cells were purchased from the Applied Cell Biology Research Institute (Kirkland, WA) and grown and maintained in Cell Systems Corporation (Kirkland, WA) Complete Medium containing 10% serum, as well as 2% CultureBoost-R (Applied Cell Biology Research Institute) to support cell growth. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.

Cell Treatments.

The thiosemicarbazones Dp44mT, DpC, and Dp2mT were synthesized and characterized using standard methods (Richardson et al., 2006; Lovejoy et al., 2012). DFO was purchased from Novartis (Basel, Switzerland). Dp44mT, DpC, and di-2-pyridylketone 2-methyl-3-thiosemicarbazone (Dp2mT) were dissolved in dimethylsulfoxide (DMSO) at 10 mM and then diluted in media containing 10% (v/v) fetal bovine serum (Sigma-Aldrich, Castle Hill, Australia) so that the final [DMSO] was ≤0.1% (v/v). Control studies have previously shown that these low DMSO levels do not affect cellular proliferation or iron metabolism (Richardson et al., 1995). IL-6 was purchased from R&D Systems (Minneapolis, MN), reconstituted in sterile phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), and used at a final concentration of 10 ng/ml. This IL-6 concentration was used as it has been previously shown to effectively activate STAT3 (Hahm and Singh, 2010).

Western Blotting.

Western blot analysis was performed using established procedures (Chen et al., 2012). The following primary antibodies used in this study were from Cell Signaling Technology (Beverly, MA): phospho-STAT3 (Tyr705), STAT3, phospho-Janus tyrosine kinase [p-JAK2] (Tyr1007/8), JAK2, phospho-Src (Tyr416), phospho-Src (Tyr527), Src, phospho-cAbl (Tyr245), cAbl, and Bcl-2. Antibodies against c-myc and cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA), and antibody against β-actin was purchased from Sigma-Aldrich. All primary antibodies were used at a 1:1000 dilution except for β-actin (1:10,000). All secondary antibodies were from Sigma-Aldrich and used at a 1:10,000 dilution.

Membranes were probed for β-actin as a loading control, and all sample data values were normalized to the corresponding β-actin values. Densitometric analysis was performed using Image Laboratory 4.0.1 software (Bio-Rad Laboratories, Hercules, CA).

Native PAGE.

Native PAGE was performed according to the method of Shin et al. (2009). Briefly, native cell extracts were prepared using ice-cold isotonic buffer containing 20 mM Tris (pH 7.0), 150 mM NaCl, 6 mM MgCl2, 0.8 mM phenylmethylsulfonyl fluoride, and 20% glycerol, and homogenized with a 27-gauge syringe. Lysates were cleared by centrifugation at 13,000 rpm for 30 minutes at 4°C. Samples (10 μg) were separated on 6% SDS-free PAGE gels, transferred to polyvinylidene difluoride membranes, and immunoblotted as described for Western blotting.

Chemiluminescent Electrophoretic Mobility Shift Assay.

To examine DNA-STAT3 interactions, nuclear protein extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Scoresby, Australia) according to the manufacturer’s instructions, and protein concentrations were determined using the BCA assay (Thermo Scientific). The double-stranded biotinylated oligonucleotides used to detect functional STAT3 DNA-binding were based on the STAT3 consensus sequence originally identified to bind the acute-phase response element in a variety of target gene promoters (Yu et al., 1995). These oligonucleotides were purchased from Life Technologies, with the following sequences: STAT3 specific probe, 5′-GATCCTTCTGGGAATTCCTAGATC-3′; STAT3 mutant probe, 5′-GATCCTTCTGGGCCGTCCTAGATC-3′; nonspecific probe, 5′-AGTCTAGAGTCACAGTGAGTCGGCAAAATTTGAGCC-3′. Equal amounts of nuclear extract (10 μg) were incubated with 20 fmol biotin-end-labeled DNA in a final volume of 20 μl with binding buffer [10 mM Tris pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 10% glycerol, 0.1 μg/μl poly(dI-dC), and 0.5 mg/ml BSA] and allowed to bind for 30 minutes at 4°C. The protein-DNA complexes were separated on a 6% native PAGE gel in 0.5× Tris borate-EDTA, then transferred onto a positively charged nylon membrane (GE Healthcare, Rydalmere, Australia) for 30 minutes in 0.5× Tris borate-EDTA. The DNA was crosslinked to the membrane using a UV Stratalinker 1800 (Stratagene, Santa Clara, CA) and detected using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific).

Immunofluorescence.

Cells were visualized by immunofluorescence as described previously elsewhere (Chen et al., 2012). Briefly, cells were fixed with 4% (w/v) paraformaldehyde (Sigma-Aldrich) for 10 minutes at 20°C then permeabilized using 0.1% (v/v) Triton X-100 for 5 minutes at 20°C. Blocking was performed using 5% BSA in PBS for 1 hour at 20°C (room temperature), and then the cells were incubated with primary antibodies overnight at 4°C. The cells were subsequently incubated with fluorescent secondary antibody for 1 hour at 20°C, washed with PBS, and set onto slides using mounting solution containing 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Imaging was performed using an Olympus Zeiss AxioObserver Z1 fluorescence microscope (Carl Zeiss GmbH, Jena, Germany) with a 63× oil objective. Mean fluorescence intensities were measured using ImageJ (http://imagej.nih.gov/ij/; Schneider et al., 2012), with average values taken from three random images per sample.

Preparation of Tumor Xenografts in Nude Mice.

Our in vivo study was approved by the Sydney University Animal Ethics Committee. Tumor xenografts were established in 8-week-old female nude mice (BALBc nu/nu), which were routinely fed basal rodent chow, watered ad libitum, and housed under a 12-hour light/dark cycle. Briefly, 2 × 106 PANC-1 cells grown in culture were harvested and suspended in Matrigel (BD Biosciences, San Jose, CA) and injected subcutaneously into the right flank of each mouse. Tumor size and volume were monitored daily, and treatments began when tumors reached an average size of 90 mm3. The mice were separated into three groups (n = 6), with each group receiving either Dp44mT (0.4 mg/kg), DpC (5 mg/kg), or the vehicle control intravenously via tail vein injection 5 days/week. This treatment regimen was established based on previous studies performed in our laboratory using these agents that had demonstrated that this administration schedule was well tolerated and showed high antitumor efficacy (Whitnall et al., 2006; Kovacevic et al., 2011; Lovejoy et al., 2012). The Dp44mT and DpC treatments were prepared by dissolving each of the compounds in 30% propylene glycol/0.9% saline, and the vehicle control treatment consisted of 30% propylene glycol/0.9% saline only (Whitnall et al., 2006; Kovacevic et al., 2011; Lovejoy et al., 2012). After 22 days of treatment, the mice were euthanized, and the tumors were harvested, fixed in 10% neutral buffered formalin for 24 hours, then processed in paraffin wax.

Immunohistochemistry.

Tumor tissue sections were sectioned at 5 μm onto Superfrost Plus slides (Menzel-Gläser Braunschweig, Germany), deparaffinized through xylene rinses, and rehydrated through graded ethanols. Antigen retrieval was performed by boiling slides in a water bath for 30 minutes using Dako Target Retrieval Solution (Dako, Carpinteria, CA) at pH 9.0 for p-STAT3 staining, and pH 6.0 for STAT3 staining. Endogenous peroxidase activity was blocked by 5 minutes of incubation with 3% hydrogen peroxide, followed by a wash with 1× Tris buffered saline (TBS)/Tween (0.05%). The samples were incubated with primary antibody for 30 minutes at 20°C, followed by a TBS/Tween wash. Bound antibody was detected using Dako Envision+ anti-rabbit horseradish peroxidase–labeled polymer secondary antibody (Dako) for 30 minutes at 20°C. Slides were rinsed in a series of TBS/Tween washes, then incubated with DAB+ chromogen (Dako) for 10 minutes. Finally, the slides were counterstained with Harris hematoxylin, dehydrated in graded ethanols, cleared through xylene rinses, and mounted. IHC staining was performed using the following primary antibodies from Cell Signaling Technology: p-STAT3 and STAT3.

Evaluation of Immunohistochemical Staining.

As the IHC p-STAT3 staining demonstrated nuclear localization in this study, we quantified the nuclear p-STAT3 staining in ImageJ (Schneider et al., 2012) using the ImmunoRatio plug-in (Tuominen et al., 2010), as previously described elsewhere (Jahangiri et al., 2013). The average values were taken from three random images per sample, and background correction was performed for each slide using a blank field image.

As STAT3 staining was not exclusively localized in the nucleus, the above method of quantification was not appropriate. Instead, STAT3 staining was scored for each sample by considering both the percentage and intensity of cells displaying positive staining. Scores for the percentage of positive cells were assigned based on standard procedures (Krajewska et al., 1996; Gong et al., 2005) as follows: 0–10% = 0; 11–25% = 1; 26–50% = 2; 51–75% = 3; and 75–100% = 4. Scores for staining intensity were assigned as follows: light brown, weak staining = 1; brown, moderate staining = 2; and dark brown, intense staining = 3. The individual scores for percentage and intensity were multiplied to obtain an IHC score, as previously described elsewhere (Krajewska et al., 1996; Gong et al., 2005). Two researchers independently scored three random fields for each sample in a blinded manner, with the average IHC score being presented.

Statistical Analysis.

Data are expressed as mean ± S.D. Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Data were compared against the respective control in each experiment by one-way analysis of variance followed by Dunnett’s post-test. P < 0.05 was considered statistically significant.

Results

Previous studies have revealed that the p38 mitogen-activated protein kinase (MAPK), AKT, and transforming growth factor β (TGF-β) pathways are molecular targets that play a role in the antiproliferative activity of DFO and Dp44mT (Yu and Richardson, 2011; Dixon et al., 2013; Kovacevic et al., 2013). To further elucidate the molecular mechanisms of thiosemicarbazones, we considered evidence that suggested crosstalk between these and other major signaling pathways, including the STAT3 pathway (Jain et al., 1998; Zhao et al., 2008; Assinder et al., 2009), and we examined the potential role of the latter pathway in mediating the antiproliferative activities of the potent DpT thiosemicarbazones DpC and Dp44mT. In the current investigation, we used the pancreatic cancer cell lines PANC-1 and MIAPaCa-2 as well as the prostate cancer cell line DU145 because the potent antitumor efficacy of Dp44mT and DpC have previously been demonstrated in these cell types (Whitnall et al., 2006; Kovacevic et al., 2011; Dixon et al., 2013; Sun et al., 2013). Furthermore, these cell lines display constitutive phosphorylation of STAT3 that leads to its activation (Mora et al., 2002; Lian et al., 2004).

The cells were incubated with DFO (250 μM), Dp44mT (5 μM), or DpC (5 μM) for 24 hours at 37°C (Fig. 1A). These conditions have been previously demonstrated to efficiently induce iron depletion and increase the expression of iron-regulated molecules (Richardson et al., 1994; Yuan et al., 2004; Kovacevic et al., 2011). Furthermore, we have shown that Dp44mT uptake by cells saturates at 5–10 μM (Merlot et al., 2013b). At this concentration, the level of agent is pharmacologically relevant in humans, as the structurally related thiosemicarbazone Triapine has been observed in the serum at similar levels (Wadler et al., 2004; Chao et al., 2012). The much higher DFO concentration was used because of its limited membrane permeability compared with Dp44mT and DpC, which display high permeability and marked iron chelation efficacy (Yuan et al., 2004; Kovacevic et al., 2011; Lovejoy et al., 2012; Sun et al., 2013). As a negative control, under the same conditions, cells were also incubated with Dp2mT (5 μM) (Fig. 1A), a structural analog of Dp44mT and DpC that cannot bind metal ions and displays negligible antitumor activity (Yuan et al., 2004; Richardson et al., 2006; Chen et al., 2012; Sun et al., 2013).

Dp44mT and DpC Decrease Constitutive STAT3 Phosphorylation at Tyr705.

Initial studies revealed that incubation of PANC-1, MIAPaCa-2, and DU145 cells with DFO, Dp44mT, or DpC significantly (P < 0.001–0.01) decreased levels of phosphorylated (Tyr705) STAT3 (i.e., p-STAT3; Fig. 1, B–D). Under the same conditions, total STAT3 expression was also significantly (P < 0.05) decreased after incubation with Dp44mT or DpC, but not with DFO. In contrast, the negative control compound for Dp44mT—namely, Dp2mT, which cannot bind metal ions (Yuan et al., 2004; Chen et al., 2012)—did not significantly (P > 0.05) alter p-STAT3 or STAT3 levels in any of the cell types examined (Fig. 1, B–D). Herein, levels of STAT3 activation were determined by densitometric analysis of the Western blots and calculation of the p-STAT3/STAT3 ratio.

To understand the role of iron depletion in the decrease of the p-STAT3/STAT3 ratio induced by these compounds, the cells were also incubated with the iron complexes of these ligands that were prepared by precomplexing the chelators with FeCl3 using standard methods (Yu and Richardson, 2011). Because DFO is a hexadentate ligand (Merlot et al., 2013a), the iron complex of DFO (DFO:Fe) was prepared in a 1:1 ligand/iron molar ratio to saturate the coordination sphere at a concentration of 250 μM. In contrast, the iron complexes of the tridentate chelators Dp44mT and DpC (Richardson et al., 2006; Lovejoy et al., 2012; Merlot et al., 2013a) (i.e., Dp44mT:Fe and DpC:Fe, respectively) were prepared in 2:1 ligand/iron molar ratios to saturate the coordination sphere at a final concentration of 5 μM. As another relevant control, cells were also incubated with FeCl3 alone (250 μM), which resulted in no significant (P > 0.05) effects on p-STAT3 or STAT3 levels versus the control (Fig. 1, B–D).

For all three cell-types, incubation with the nonredox-active DFO:Fe complex (Kalinowski and Richardson, 2005) resulted in p-STAT3/STAT3 levels that were significantly (P < 0.001–0.01) greater than those found for DFO alone, and comparable to its respective control (Fig. 1, B–D). This finding indicates that the ability of DFO to inhibit constitutively activated STAT3 depends on its ability to bind intracellular iron. In contrast, incubation of PANC-1, MIAPaCa-2, or DU145 cells with the Dp44mT:Fe or DpC:Fe complex significantly (P < 0.001–0.05) decreased the p-STAT3/STAT3 ratio compared with the control. The extent of the decrease in the p-STAT3/STAT3 ratio by the complexes was similar (PANC-1) or slightly less (MIAPaCa-2, DU145) than that observed for Dp44mT or DpC alone. These results suggest that, in contrast to the nonredox active DFO:Fe complex, the well characterized ability of Dp44mT:Fe and DpC:Fe complex to generate ROS (Yuan et al., 2004; Richardson et al., 2006; Lovejoy et al., 2011) may also play a role in the decreased p-STAT3/STAT3 ratio.

To compare the effects of these compounds on mortal, nontransformed cells, we also examined the effects of DFO, Dp44mT, or DpC on primary cultures of normal pancreatic epithelial cells (PaEC; Fig. 1E). Incubation of PaEC with DFO, Dp44mT, and DpC for 24 hours at 37°C did not result in any significant (P > 0.05) alteration in p-STAT3 or STAT3 levels relative to the control. In fact, p-STAT3 expression was barely detectable in these normal cells (Fig. 1E) compared with their malignant counterparts (Fig. 1, B–D). This observation is consistent with 1) previous reports that STAT3 is overactivated in pancreatic carcinoma specimens, but not in normal pancreatic tissues (Lian et al., 2004); and 2) that these compounds can selectively target cancer cells over normal cells in vitro and in vivo (Yuan et al., 2004; Kovacevic et al., 2011; Lovejoy et al., 2012; Dixon et al., 2013).

Collectively, these studies demonstrate that iron chelation decreases the p-STAT3/STAT3 ratio and that the redox activity of the Dp44mT:Fe and DpC:Fe complexes may be important in inducing this effect.

The Antioxidant and Glutathione Precursor N-acetylcysteine Prevents the Ability of the Dp44mT- or DpC-Fe Complexes to Decrease the p-STAT3/STAT3 Ratio in PANC-1 and MIAPaCa-2 Cells.

Considering that incubation of PANC-1, MIAPaCa-2, and DU145 cells with Dp44mT:Fe or DpC:Fe also significantly decreased the p-STAT3/STAT3 ratio (Fig. 1, B–D), we further examined whether the redox activity of these complexes (Yuan et al., 2004; Richardson et al., 2006; Lovejoy et al., 2011) was at least partly responsible for this effect (Fig. 2). This hypothesis was investigated by using the antioxidant N-acetylcysteine (NAC), which has been well characterized to increase intracellular glutathione (GSH) and quench ROS (Schafer and Buettner, 2001; Watts and Richardson, 2001). Previous studies have demonstrated that NAC markedly reduces the antiproliferative activity of Dp44mT and prevents its ability to induce lysosomal membrane permeabilization due to its ability to act as a GSH donor (Lovejoy et al., 2011).

Fig. 2.
Fig. 2.

The antioxidant NAC prevents the ability of Dp44mT- or DpC-Fe complexes to suppress STAT3 phosphorylation in pancreatic cancer cells. The levels of p-STAT3 and STAT3 protein are shown as determined by Western blotting in (A) PANC-1, (B) MIAPaCa-2, and (C) DU145 cells after a preincubation with control medium alone or NAC (10 mM) for 1 hour at 37°C, followed by a second incubation with control medium alone, Dp44mT:Fe (2:1; 1 or 5 µM), or DpC:Fe (2:1; 1 or 5 µM) in control medium or in the continued presence of NAC (10 mM) for 24 hours at 37°C. The Western blots are typical of three independent experiments, with densitometric analysis representing mean ± S.D. Relative to control: *P < 0.05; **P < 0.01; ***P < 0.001.

In this investigation, cells were initially preincubated for 1 hour at 37°C with control medium alone or NAC (10 mM) dissolved in medium. Then, the cells were incubated with Dp44mT:Fe (1 or 5 µM) or DpC:Fe (1 or 5 µM) in control medium alone, or in medium containing NAC (10 mM) for a further 24 hours at 37°C.

Consistent with the results in Fig. 1B, incubation of PANC-1 cells with Dp44mT:Fe or DpC:Fe decreased the p-STAT3/STAT3 ratio, particularly at 5 µM relative to 1 µM (Fig. 2A). However, incubation of cells with Dp44mT:Fe (1 µM) or DpC:Fe (1 µM) in the presence of NAC prevented the decrease in the p-STAT3/STAT3 ratio in PANC-1 cells (Fig. 2A). This finding suggests the ability of Dp44mT:Fe and DpC:Fe to inhibit STAT3 activation could depend on their ability to generate ROS (Yuan et al., 2004). When the concentration of the Dp44mT:Fe and DpC:Fe complexes was increased to 5 µM, NAC no longer significantly prevented their ability to decrease the p-STAT3/STAT3 ratio (Fig. 2A). This observation suggested ROS generation by the complexes at this higher concentration was markedly greater than that induced at 1 µM and could not be sufficiently rescued by NAC.

A similar result was observed with the MIAPaCa-2 cells (Fig. 2B), with both Dp44mT:Fe and DpC:Fe significantly (P < 0.01–0.05) decreasing the p-STAT3/STAT3 ratio relative to the control, as shown in Fig. 1C. The presence of NAC inhibited the decrease in the p-STAT3/STAT3 ratio for both Dp44mT:Fe (1 and 5 µM) and DpC:Fe (1 and 5 µM) (Fig. 2B). In examining the DU145 cells, we found that both Dp44mT:Fe and DpC:Fe complexes also markedly (P < 0.01) reduced the p-STAT3/STAT3 ratio at the 5 µM concentration (Fig. 2C), which was consistent with the results in Fig. 1D. However, in contrast to the PANC-1 and MIAPaCa-2 cells, incubation of the DU145 cells with the Dp44mT:Fe or DpC:Fe complexes in the presence of NAC did not significantly (P > 0.05) rescue the effect of these agents on reducing the p-STAT3/STAT3 ratio (Fig. 2C).

Considering these results, it could be suggested that DU145 cells have an altered redox state, which may modulate their response to NAC and the complexes. Indeed, a previous study showed that DU145 cells have higher basal GSH levels and a reducing redox environment when compared with other prostate cancer cells, enhancing their tolerability to ROS (Jayakumar et al., 2014). Alternatively, the Dp44mT:Fe and DpC:Fe complexes may have additional molecular effects in this cell line that are not accounted for by ROS production alone.

DFO, Dp44mT, and DpC Inhibit STAT3 Dimerization.

Activation of STAT3 by phosphorylation at Tyr705 induces dimerization, which allows subsequent nuclear translocation (Ihle, 1995; Bromberg et al., 1999). Thus, native PAGE was used to assess whether the decrease in p-STAT3/STAT3 levels induced by the ligands (Fig. 1, B–D) resulted in a reduction in STAT3 dimers, as this is vital for STAT3 activity (Haura et al., 2005).

Incubation of PANC-1, MIAPaCa-2 and DU145 cells with DFO, Dp44mT, or DpC for 24 hours significantly (P < 0.001–0.01) decreased dimerized STAT3 levels compared with the untreated controls (Fig. 3). Notably, Dp44mT and particularly DpC were significantly (P < 0.05) more effective than DFO at decreasing STAT3 dimer formation. Further, incubation of these cells with Dp44mT:Fe or DpC:Fe complexes also resulted in significantly (P < 0.001–0.01) decreased levels of dimerized STAT3, whereas incubation with either DFO:Fe, FeCl3, or Dp2mT did not significantly (P > 0.05) alter dimerized STAT3 levels compared with the control (Fig. 3).

Fig. 3.
Fig. 3.

DFO, Dp44mT, and DpC inhibit STAT3 dimerization in pancreatic and prostate cancer cells. We used native PAGE to determine STAT3 dimer levels in (A) PANC-1, (B) MIAPaCa-2, and (C) DU145 cells after a 24-hour/37°C incubation with control medium, DFO (250 µM), Dp44mT (5 µM), DpC (5 µM), DFO:Fe (1:1; 250 µM), Dp44mT:Fe (2:1; 5 µM), DpC:Fe (2:1; 5 µM), FeCl3 (250 µM), or Dp2mT (5 µM). The Western blots are typical of three independent experiments, with densitometric analysis representing mean ± S.D. Relative to control: **P < 0.01; ***P < 0.001.

These findings agree with the decrease in the p-STAT3/STAT3 ratio observed in Fig. 1, indicating that the ability of DFO to inhibit STAT3 dimer formation depends solely on its iron-binding activity. In contrast, the ability of Dp44mT and DpC to inhibit STAT3 dimer formation could also be dependent on ROS generation in addition to their known ability to chelate intracellular iron (Yuan et al., 2004; Whitnall et al., 2006; Kovacevic et al., 2011; Lovejoy et al., 2012). It is notable that the effect observed after treating cells with the ligands could not be reproduced by adding the agents directly to control cell lysates, indicating a biologic response that required cellular and metabolic integrity.

DFO, Dp44mT, and DpC Inhibit STAT3 DNA-Binding Activity.

To examine whether the chelator-induced decrease in p-STAT3/STAT3 and STAT3 dimer levels resulted in decreased STAT3 DNA-binding activity, we performed an electrophoretic mobility shift assay using a biotin-labeled, double-stranded DNA probe containing the STAT3 consensus-binding motif (Fig. 4). In these studies, incubation of nuclear extracts with this probe resulted in a pronounced band (Fig. 4A, lane 2) that was not present with the probe alone (Fig. 4A, lane 1).

Fig. 4.
Fig. 4.

DFO, Dp44mT, and DpC inhibit STAT3 DNA-binding activity in pancreatic and prostate cancer cells. (A) Electrophoretic mobility shift assay (EMSA) examining STAT3 DNA-binding activity of PANC-1 cells in the presence of relevant controls to confirm specific DNA binding. Asterisk indicates antibody-induced supershift of STAT3-DNA band. Control Ab, isotype control antibody; Non-spec. comp., nonspecific competitor oligonucleotide; Spec. Ab, antibody against STAT3; Spec. comp., specific competitor oligonucleotide. (B–D) EMSA examining STAT3 DNA-binding activity after incubation of (B) PANC-1, (C) MIAPaCa-2, and (D) DU145 cells for 24 hours at 37°C with control medium, DFO (250 µM), Dp44mT (5 µM), or DpC (5 µM). Nuclear lysates were then prepared and EMSA performed. The results are typical of three independent experiments, with densitometric analysis representing mean ± S.D. Relative to control: **P < 0.01; ***P < 0.001.

To assess whether the STAT3 DNA-binding activity observed in Fig. 4A (lane 2) was a specific interaction, a series of controls was used. First, addition of a 50-fold molar excess of unlabeled specific competitor oligonucleotide (i.e., Spec. comp.; Fig. 4A) markedly and significantly (P < 0.001) reduced the intensity of this band (Fig. 4A, lane 3), whereas addition of a 50-fold molar excess of a nonspecific competitor oligonucleotide (i.e., Nonspec. comp.; Fig. 4A) did not significantly (P > 0.05) alter the intensity of the STAT3 band compared with the control in lane 2 (Fig. 4A, lane 4). Second, incubation with a biotin-labeled probe containing a mutated STAT3-binding motif did not result in the appearance of any clear bands (Fig. 4A, lane 5). Third, addition of an antibody against STAT3 (i.e., Spec. Ab; Fig. 4A) to the binding reaction resulted in a significant (P < 0.001) reduction of the STAT3 band, but also the apparent presence of a smear that ran higher in the gel (see asterisk), suggesting an antibody-induced supershift of STAT3 (Fig. 4A, lane 6). Fourth, as a further control, incubation of an isotype control antibody (i.e., control Ab; Fig. 4A) to the binding reaction under the same conditions (Fig. 4A, lane 7) did not significantly (P > 0.05) alter the appearance of the band compared with the control in lane 2 (Fig. 4A). Collectively, these controls demonstrate the identified STAT3 band was representative of STAT3 DNA-binding activity.

To assess the effect of the ligands on STAT3 DNA-binding activity, PANC-1 cells were incubated with DFO (250 μM), Dp44mT (5 μM), or DpC (5 μM) for 24 hours at 37°C, nuclear lysates were prepared, and electrophoretic mobility shift assay was performed (Fig. 4B). Incubation with these ligands significantly (P < 0.001) reduced the intensity of the STAT3 band compared with control cells (Fig. 4B; compare lanes 3–5 with lane 2). Moreover, this effect was also observed in MIAPaCa-2 (Fig. 4C) and DU145 (Fig. 4D) cells, indicating that DFO, Dp44mT, and DpC could significantly (P < 0.001–0.01) reduce binding of STAT3 to its target DNA in each cell line examined. Thus, these data agree with the effects of the ligands on STAT3 activation (p-STAT3/STAT3 ratio; Fig. 1) and STAT3 dimerization (Fig. 3), and demonstrate that they inhibit STAT3 DNA-binding activity, which may compromise its downstream oncogenic effects.

DFO, Dp44mT, and DpC Downregulate the Expression of the Downstream STAT3 Targets, c-myc, Cyclin D1, and Bcl-2.

Considering the results indicating these ligands can decrease STAT3 activation, dimerization, and DNA binding (Figs. 1–4), we examined whether this subsequently affected the expression of multiple downstream STAT3 targets that regulate cell cycle progression and apoptosis, namely, c-myc, cyclin D1, and Bcl-2 (Darnell, 1997; Sasse et al., 1997; Zushi et al., 1998; Bromberg et al., 1999; Zhong et al., 1999). In these studies, incubation of cells with DFO (250 μM), Dp44mT (5 μM), or DpC (5 μM) for 24 hours at 37°C significantly (P < 0.001–0.05) reduced the protein levels of c-myc, and cyclin D1 in PANC-1 (Fig. 5A), MIAPaCa-2 (Fig. 5B), and DU145 (Fig. 5C) cells. Notably, Bcl-2 was also significantly (P < 0.01) reduced by Dp44mT and DpC in each of the three cell types, whereas DFO only significantly (P < 0.01) reduced Bcl-2 levels in DU145 cells (Fig. 5C). These findings are in agreement with previous reports that cellular iron depletion using chelators can regulate cyclin D1 and Bcl-2 levels, which could mediate the potent antiproliferative activity of these compounds (Gao and Richardson, 2001; Yuan et al., 2004; Kovacevic et al., 2011; Dixon et al., 2013).

Fig. 5.
Fig. 5.

DFO, Dp44mT, and DpC downregulate the expression of downstream STAT3 targets. The expression of c-myc, cyclin D1, and Bcl-2 proteins in (A) PANC-1, (B) MIAPaCa-2, (C) DU145, or (D) PaEC cells after a 24-hour/37°C incubation with control medium, DFO (250 µM), Dp44mT (5 µM), DpC (5 µM), DFO:Fe (1:1; 250 µM), Dp44mT:Fe (2:1; 5 µM), DpC:Fe (2:1; 5 µM), FeCl3 (250 µM), or Dp2mT (5 µM). The Western blots are typical of three independent experiments, with densitometric analysis representing mean ± S.D. Relative to control: *P < 0.05; **P < 0.01; ***P < 0.001.

The iron complex DFO:Fe did not significantly alter the expression of these downstream targets compared with the control, indicating that the ability of DFO to sequester cellular iron is important for its ability to inhibit STAT3 signaling and its downstream effectors. In contrast, incubation of cells with the Dp44mT:Fe and DpC:Fe complexes significantly (P < 0.001–0.05) reduced expression of the downstream targets c-myc, cyclin D1, and Bcl-2 in all three cell-types (Fig. 5, A–C). Again, this suggests that the redox activity of Dp44mT:Fe and DpC:Fe complexes could play a role in Dp44mT- and DpC-mediated suppression of downstream STAT3 signaling.

Use of FeCl3 as a control for the Dp44mT:Fe and DpC:Fe complexes indicated that it did not significantly (P > 0.05) affect c-myc expression in PANC-1 or MIAPaCa-2 cells (Fig. 5, A and B), nor did it significantly (P < 0.05) affect cyclin D1 or Bcl-2 expression in any cell types examined. Interestingly, for DU145 cells only, FeCl3 was found to significantly (P < 0.001) decrease c-myc expression (Fig. 5C). This observation may reflect cell-type heterogeneity that results in enhanced sensitivity of c-myc to high intracellular iron levels in DU145 cells. For all cell types, incubation with the negative control compound Dp2mT, which cannot bind metals (Yuan et al., 2004; Chen et al., 2012), resulted in no significant (P > 0.05) alteration in the levels of c-myc, cyclin D1, or Bcl-2, demonstrating the key role of iron chelation by Dp44mT and DpC in decreasing the expression of these proteins (Fig. 5).

To compare the activity of these agents in mortal, nontransformed cells, we also examined the effects of DFO, Dp44mT, and DpC on these downstream target molecules in primary PaEC cultures (Fig. 5D). In agreement with the results indicating that iron chelation did not significantly alter STAT3 activation in these cells (Fig. 1E), c-myc and Bcl-2 expression levels were not significantly (P > 0.05) altered compared with the control in response to any of the ligands examined (Fig. 5D). Interestingly, cyclin D1 expression was significantly (P < 0.001) increased in response to Dp44mT, DpC, Dp44mT:Fe, and DpC:Fe in PaEC (Fig. 5D), which may possibly reflect a prosurvival response of these normal cells to the stress conditions induced by these compounds.

Pretreatment of PANC-1 and DU145 cells with Dp44mT or DpC Inhibits IL-6–Induced Activation of STAT3.

IL-6 is implicated in the hyperactivation of STAT3 signaling in tumor progression by stimulating this pathway through a receptor-mediated mechanism (Turkson and Jove, 2000). To investigate whether cellular iron depletion could also inhibit IL-6–induced activation of STAT3, we preincubated PANC-1 and DU145 cells with Dp44mT, DpC, or Dp2mT in serum-free medium or with the serum-free medium alone for 24 hours at 37°C to remove cytokines/growth factors (Hahm and Singh, 2010), then stimulated the cells with IL-6 (10 ng/ml) in the serum-free medium or the serum-free medium alone for 30 minutes at 37°C (Fig. 6).

Fig. 6.
Fig. 6.

Pretreatment of PANC-1 and DU145 cells with Dp44mT or DpC inhibits IL-6–induced STAT3 phosphorylation. The expression of p-STAT3 and STAT3 protein levels as determined by Western blotting of (A) PANC-1, (B) DU145, and (C) MIAPaCa-2 cells after a 24-hour/37°C incubation with serum-free medium alone, Dp44mT (5 µM), or DpC (5 µM) in serum-free medium, followed by a 30-minute/37°C incubation with serum-free medium alone or IL-6 (10 ng/ml) in serum-free medium. The Western blots are typical of three independent experiments, with densitometric analysis representing mean ± S.D. Relative to control: **P < 0.01; ***P < 0.001.

Preincubation with the control medium alone followed by incubation with IL-6 for 30 minutes markedly and significantly (P < 0.001) increased the p-STAT3/STAT3 ratio in PANC-1 (Fig. 6A; lane 2) and DU145 (Fig. 6B; lane 2) cells compared with the same cells incubated with medium alone (Fig. 6, A and B; lane 1). Notably, the low p-STAT3 levels in Fig. 6 relative to Fig. 1, are consistent with serum starvation (Hahm and Singh, 2010). Preincubation of cells with Dp44mT or DpC significantly (P < 0.001) inhibited IL-6–induced activation of STAT3 in PANC-1 and DU145 cells (Fig. 6, A and B; lanes 4 and 6). In contrast to Dp44mT and DpC, Dp2mT, which does not bind metal ions (Yuan et al., 2004; Richardson et al., 2006; Chen et al., 2012; Sun et al., 2013), had no significant (P > 0.05) effect on STAT3 activation induced by IL-6 relative to the control (Fig. 6, A and B; lane 8).

In contrast to the PANC-1 and DU145 cell types, MIAPaCa-2 cells showed a different response to the ligands and incubation with IL-6 (Fig. 6C). In fact, IL-6 stimulation of cells preincubated with Dp44mT or DpC resulted in a significant (P < 0.001–0.01) increase in p-STAT3/STAT3 levels compared with cells preincubated with control media alone (Fig. 6C; compare lane 2 with lanes 4 and 6). This observation was unexpected given the previous results demonstrating that Dp44mT and DpC could inhibit constitutive STAT3 activation and its downstream targets in this particular cell line (Figs. 1C, 2B, 3B, 4C, 5B) and may reflect tumor cell heterogeneity. Alternatively, this result may reflect a compensatory mechanism in the MIAPaCa-2 cells in response to Dp44mT or DpC that leads to a more marked IL-6 response, which is then subsequently inhibited by these agents downstream, as demonstrated in Figs. 1C, 2B, 3B, 4C, and 5B.

The positive findings in PANC-1 and DU145 cells using IL-6 (Fig. 6, A and B) were further confirmed by immunofluorescence staining that demonstrated a marked induction of p-STAT3 expression (green fluorescence) upon incubation of control cells with IL-6, which was predominantly nuclear as demonstrated by the merged stain showing overlap with DAPI staining (blue fluorescence; Fig. 7, A and B). Compared with their respective control cells, the mean p-STAT3 staining intensities in cells treated with IL-6 were significantly (P < 0.01) increased by 3.9-fold in PANC-1 cells and 3.5-fold in DU145 cells. However, stimulation with IL-6 of PANC-1 or DU145 cells preincubated with Dp44mT or DpC did not result in marked p-STAT3 staining, with mean p-STAT3 staining intensities that were not significantly different compared with the control cells (Fig. 7, A and B).

Fig. 7.
Fig. 7.

Pretreatment of PANC-1 and DU145 cells with Dp44mT or DpC inhibits IL-6–induced nuclear translocation of p-STAT3. Immunofluorescence microscopy examining p-STAT3 staining was performed in (A) PANC-1 and (B) DU145 cells and (C) MIAPaCa-2 cells under the same conditions as described in Fig. 6. Individual and merged images were taken to show staining of p-STAT3 (green) with the cell nucleus (blue) as stained by DAPI. The scale bar in the bottom left corner of the first image represents 20 µm and is the same across all images. The results are typical of three independent experiments.

Using MIAPaCa-2 cells, the mean p-STAT3 staining was also significantly (P < 0.05) increased 2.1-fold upon treatment with IL-6 when compared with their respective control cells (Fig. 7C). However, as demonstrated in Fig. 6C, preincubation of these cells with either Dp44mT or DpC resulted in a significantly (P < 0.01–0.05) greater 2.4-fold and 2.5-fold increase in p-STAT3 levels in response to IL-6 treatment, respectively, when compared with the IL-6–treated control cells (Fig. 7C). Together, the results from immunoblotting and fluorescence microscopy demonstrate that pretreatment of PANC-1 and DU145 cells with Dp44mT or DpC inhibits IL-6–induced STAT3 activation, and this process is stimulated by these ligands in MIAPaCa-2 cells.

DFO, Dp44mT, and DpC Decrease Activation of Kinases Upstream of STAT3, Namely, Src and cAbl.

It is possible that the observed decrease in STAT3 activation induced by DFO, Dp44mT, and DpC may be mediated by their effect on upstream kinases known to promote STAT3 signaling (Yu and Richardson, 2011). To investigate this hypothesis, we examined the effect of these compounds on key protein tyrosine kinases that phosphorylate and activate STAT3, namely, the nonreceptor tyrosine kinases Src and cAbl, and the receptor-associated Janus tyrosine kinase (JAK2) (Turkson et al., 1998; Haura et al., 2005; Srinivasan et al., 2008; Hedvat et al., 2009). Specifically, activation of Src and cAbl were examined through assessing levels of an activating phosphorylation of Src (p-SrcY416), an inactivating phosphorylation of Src (p-SrcY527) (Hunter, 1987), and an activating phosphorylation of cAbl (p-cAblY245) (Brasher and Van Etten, 2000). Activation of JAK2 was also assessed by investigating the levels of phospho-JAK2Y1007/8 (p-JAK2), an activating phosphorylation that occurs in response to receptor-mediated cytokine or growth factor stimulation (Feng et al., 1997; Hedvat et al., 2009). It is important to note that JAK2, rather than JAK1, was investigated because numerous studies have identified that the JAK2/STAT3 cascade has been specifically implicated in pancreatic (Thoennissen et al., 2009; Corcoran et al., 2011) and other cancers (Xiong et al., 2008; Zhang et al., 2009; Colomiere et al., 2009; Marotta et al., 2011) as promoting tumor progression.

Incubation with Dp44mT, DpC, Dp44mT:Fe, or DpC:Fe significantly (P < 0.001–0.01) decreased the p-SrcY416/Src ratio relative to the control, with no significant (P > 0.05) alteration being observed with total Src levels in PANC-1 (Fig. 8A), MIAPaCa-2 (Fig. 8B), and DU145 (Fig. 8C) cells. In contrast, incubation of these cells with DFO, DFO:Fe, FeCl3, or Dp2mT did not significantly (P > 0.05) alter levels of p-SrcY416 or total Src compared with the untreated control. Further, none of the treatments were found to significantly (P > 0.05) alter p-SrcY527 levels, indicating that these compounds did not affect Src activity by regulating the phosphorylation of this site. Examination of cAbl activity revealed that incubation with Dp44mT, DpC, Dp44mT:Fe, and DpC:Fe could significantly (P < 0.001–0.05) reduce the p-cAbl/cAbl ratio with no significant (P > 0.05) alterations to total cAbl levels in MIAPaCa-2 (Fig. 8B) and DU145 (Fig. 8C) cells, but not in PANC-1 cells (Fig. 8A). Interestingly, incubation of DU145 cells with FeCl3 alone resulted in significantly (P < 0.05) decreased p-cAbl and cAbl levels (Fig. 8C). This result appeared analogous to that seen with c-myc (Fig. 5C), and again, may reflect an enhanced sensitivity of these molecules to high cellular iron levels in DU145 cells (Li et al., 2011).

Fig. 8.
Fig. 8.

DFO, Dp44mT, and DpC decrease activation of kinases upstream of STAT3. The levels of p-SrcY416, p-SrcY527, Src, p-cAbl, cAbl, p-JAK2, and JAK2 were measured by Western blot analysis in (A) PANC-1, (B) MIAPaCa-2, and (C) DU145 cells after a 24-hour/37°C incubation with control medium, DFO (250 µM), Dp44mT (5 µM), DpC (5 µM), DFO:Fe (1:1; 250 µM), Dp44mT:Fe (2:1; 5 µM), DpC:Fe (2:1; 5 µM), FeCl3 (250 µM), or Dp2mT (5 µM). The Western blots are typical of three independent experiments, with densitometric analysis representing mean ± S.D. Relative to control: *P < 0.05; **P < 0.01; ***P < 0.001.

In all three cell types, incubation with any of the chelators, their corresponding iron complexes, FeCl3, or the negative control compound Dp2mT did not significantly (P > 0.05) alter p-JAK2 or JAK2 levels (Fig. 8). This indicates that the ability of iron chelators to inhibit STAT3 activation in these cells likely does not occur through inhibition of constitutive JAK2 activation.

Collectively, these findings indicate the ability of Dp44mT, DpC, Dp44mT:Fe, and DpC:Fe to inhibit STAT3 activation could potentially be mediated by the upstream inhibition of Src activation in PANC-1, MIAPaCa-2, and DU145 cells as well as inhibition of cAbl activation in MIAPaCa-2 and DU145 cells.

Dp44mT and DpC Inhibit STAT3 Signaling In Vivo in a PANC-1 Tumor Xenograft Model.

To further validate the data observed in vitro, we used a previously established PANC-1 tumor xenograft model in nude mice that assessed the antitumor activity of Dp44mT and DpC (Kovacevic et al., 2011). In this study, PANC-1 tumor xenografts were established in nude mice and allowed to grow to 90 mm3, when treatment was initiated. Each group of mice (n = 6) received either the vehicle alone, Dp44mT (0.4 mg/kg, 5 days/week), or DpC (5 mg/kg, 5 days/week) intravenously (via tail vein) over 22 days. At the end of the experiment, the mice were sacrificed, and tumors were harvested.

In these studies, Dp44mT and DpC reduced PANC-1 tumor growth to 29.9 ± 8.8% (n = 6) and 19.3 ± 1.4% (n = 6) of the control, respectively, after 22 days. Tumor tissues were examined by immunohistochemistry for p-STAT3 and STAT3 levels (Fig. 9). The staining for p-STAT3 was predominantly located in nuclei, and thus staining was quantified in each sample as the percentage of DAB-positive nuclei to the total nuclear area in each sample, as per established methods (Tuominen et al., 2010; Schneider et al., 2012; Jahangiri et al., 2013). Staining of p-STAT3 in control-treated tumor samples displayed 10.09 ±1.73% (n = 18, six tumor samples/treatment group, with an average taken from three random images/sample) DAB-positive nuclei/total nuclear area. The Dp44mT- and DpC-treated tumor samples displayed significantly (P < 0.01–0.05) lower values of 5.60 ± 2.07% (n = 18) and 5.20 ± 3.24% (n = 18) DAB-positive nuclei/total nuclear area, respectively (Fig. 9A).

Fig. 9.
Fig. 9.

Dp44mT and DpC inhibit STAT3 signaling in vivo in a PANC-1 tumor xenograft model. PANC-1 tumor xenografts were grown subcutaneously in nude mice that were separated into three groups, with each group receiving Dp44mT (0.4 mg/kg), DpC (5 mg/kg), or the vehicle control intravenously as described in detail in Materials and Methods. Immunohistochemistry was performed on formalin-fixed tumor tissue sections to examine (A) p-STAT3 or (B) STAT3 protein expression in tumors obtained from mice treated with the vehicle control, Dp44mT, or DpC. Scale bar: 1 mm. Quantitation of p-STAT3 staining and scoring of STAT3 staining was performed as described. The analysis of p-STAT3 or STAT3 staining represents the mean ± S.D. (n = 18; six tumor samples/treatment group, with an average taken from three random images/sample). Relative to control: *P < 0.05; **P < 0.01; ***P < 0.001.

IHC staining of total STAT3 in the control-treated tumor samples was noticeably marked and intense in both the cytoplasm and nuclei, with an average IHC score of 10.89 ± 1.77 (n = 18). However, STAT3 staining was found to be significantly (P < 0.001–0.01) lower in tumor samples from mice treated with Dp44mT or DpC, with IHC scores from these groups of 6.38 ± 2.70 (n = 18) and 5.50 ± 2.06 (n = 18), respectively (Fig. 9B). Collectively, these results demonstrate that Dp44mT and DpC can also inhibit STAT3 signaling in vivo, which corroborates well with our findings in vitro.

Discussion

In this study, we have further elucidated the antitumor mechanism of the novel di-2-pyridyl thiosemicarbazones Dp44mT and DpC, which are emerging as a promising class of anticancer agents that demonstrate marked and selective antitumor and antimetastatic efficacy in vitro and in vivo in a range of cancer cell types (Whitnall et al., 2006; Kovacevic et al., 2011; Chen et al., 2012; Liu et al., 2012; Lovejoy et al., 2012; Sun et al., 2013). Here, for the first time, we have identified that these agents can inhibit the STAT3 pathway, which could be a crucial mechanism by which they exhibit their potent antitumor activity (Fig. 10A).

Fig. 10.
Fig. 10.

(A) Schematic summary of the effect of the chelators on the STAT3 signaling pathway. The ligands DFO, Dp44mT, and DpC, decrease (1) phosphorylated and activated STAT3; (2) STAT3 dimerization; (3) STAT3-DNA binding; and (4) expression of the downstream STAT3 targets c-myc, cyclin D1, and Bcl-2. Both Dp44mT and DpC can also inhibit activation of the upstream nonreceptor tyrosine kinases Src and cAbl, suggesting that these molecules could potentially mediate, at least in part, the observed effects on STAT3 activation. (B) Schematic of the multiple effects of the DpT class of thiosemicarbazones (in addition to the effects on the STAT3 signaling pathway) that lead to the inhibition of tumor cell proliferation. The effects of these agents include 1) enhancement of iron mobilization from cells; 2) inhibition of iron uptake from transferrin; 3) upregulation of the growth and metastasis suppressor N-myc downstream regulated gene 1 (NDRG1); 4) lysosomal membrane permeabilization mediated by the generation of redox active copper complexes leading to impaired autophagy and the induction of apoptosis; 5) inhibition of multiple oncogenic cellular signaling pathways such as phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and transforming growth factor-β (TGF-β) pathways, and upregulation of tumor-suppressive phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and SMAD4; (6) inhibition of cyclin D1 expression; and (7) inhibition of the rate-limiting step of DNA synthesis catalyzed by the iron-containing enzyme ribonucleotide reductase (RR).

We have demonstrated that DFO, Dp44mT, and DpC, inhibit the STAT3 pathway by markedly decreasing constitutive phosphorylation and STAT3 activation in three cancer cell lines—PANC-1, MIAPaCa-2, and DU145 (Fig. 1, B–D)—but not in primary cultures of mortal PaEC (Fig. 1E). We also showed these compounds inhibit STAT3 dimerization (Fig. 3), the binding of nuclear STAT3 to its target DNA (Fig. 4, B–D), and subsequently attenuate the expression of the downstream STAT3 targets c-myc, cyclin D1, and Bcl-2 (Fig. 5, A–C). These effects were dependent on the ability of the ligands to bind iron, because incubation of cells with the inert DFO:Fe complex, which is not redox active (Kalinowski and Richardson, 2005), or the control compound Dp2mT, which does not bind metal ions (Yuan et al., 2004; Chen et al., 2012), did not lead to inhibition.

Further studies herein demonstrate that the redox-active Dp44mT:Fe and DpC:Fe complexes inhibit STAT3 phosphorylation, dimerization, and expression of downstream STAT3 targets. These observations suggest the ability of these complexes to form redox-active species within cells also plays a role in their STAT3-inhibitory properties. In fact, the decreased STAT3 activation induced by Dp44mT:Fe and DpC:Fe could be prevented by the antioxidant NAC in the PANC-1 and MIAPaCa-2 cells (Fig. 2), indicating that ROS generation is involved in mediating this effect. Interestingly, ROS can inhibit STAT3 signaling by oxidation of conserved cysteines on JAK and STAT proteins that decrease their activation, implicating STAT3 as a mediator of redox homeostasis (Mamoon et al., 2007; Li et al., 2010). More recently, STAT3 transcriptional activity has been shown to be directly regulated by the redox function of the APE1/Ref-1 endonuclease (Cardoso et al., 2012).

Our studies demonstrating the effect of the DpT agents on the STAT3 pathway through their ability to bind iron and redox cycle to generate ROS illustrate a unique mechanism of action that is very different compared with other drugs targeting this molecule. In fact, previous attempts to target STAT3 in cancer therapy have included direct chemical inhibitors of STAT3, JAK and Src, IL-6 receptor antagonists, antisense strategies, decoy phosphopeptides, decoy duplex oligonucleotides, dominant negative proteins, RNA interference, chemopreventive agents, and G-quartet oligodeoxynucleotides (Jing and Tweardy, 2005; Sansone and Bromberg, 2012; Wang et al., 2012; Siveen et al., 2014). Recently, resistance to standard anticancer therapies has been correlated to constitutive or unabated activation of STAT3, indicating that combination therapy with STAT3 inhibitors may be important for patient treatment (Tan et al., 2014). Hence, this finding underlines the importance of developing new STAT3 inhibitors such as Dp44mT and DpC that demonstrate potent and selective antitumor activity.

To investigate whether the decreased STAT3 activation observed with DFO, Dp44mT, and DpC was due to the reduced activity of well known upstream activators of this protein, our studies examined the effect of these agents on JAK2, Src, and cAbl activation. These agents did not significantly alter constitutive phosphorylation of JAK2 in any of the three cell types (Fig. 8). However, Dp44mT and DpC were found to decrease activation of the nonreceptor kinase Src in all cell lines as well as decrease activation of cAbl in MIAPaCa-2 and DU145 cells (Fig. 8). This observation suggests the ability of Dp44mT, DpC, Dp44mT:Fe, and DpC:Fe to inhibit STAT3 activation in these cells could potentially be mediated by the upstream inhibition of Src activation. However, considering that cAbl activation was not significantly altered in response to Dp44mT or DpC in PANC-1 cells, and that DFO did not significantly alter Src and cAbl activation, these latter proteins may not be the only contributors to inhibiting the STAT3 pathway.

In addition to inhibiting constitutive activation of STAT3 in tumor cells, Dp44mT and DpC could inhibit IL-6–induced activation of STAT3 in PANC-1 and DU145 cells (Figs. 6 and 7). This finding is critical given that several studies have indicated that IL-6–mediated activation of STAT3 is a principal pathway implicated in promoting tumorigenesis (Sansone and Bromberg, 2012). Interestingly, this effect was not observed with MIAPaCa-2 cells, where Dp44mT and DpC enhanced subsequent IL-6–induced STAT3 phosphorylation compared with the control (Figs. 6C and 7C). This latter observation was unexpected given that these compounds inhibit constitutive STAT3 activation in MIAPaCa-2 cells, and may reflect cell-type genetic heterogeneity resulting in a differential response to the combined stimulus of Dp44mT or DpC with IL-6. Importantly, this effect is not pro-oncogenic because these cells are sensitive to the antitumor activity of these compounds in vitro and in vivo (Kovacevic et al., 2011). In fact, Dp44mT and DpC markedly inhibit STAT3 activation (Figs. 1C, 2B, 3B, 4C) as well as expression of downstream tumorigenic effectors c-myc, cyclin D1, and Bcl-2 (Fig. 5B) in MIAPaCa-2 cells. Moreover, Dp44mT and DpC increase the expression of the cyclin-dependent kinase inhibitor p21, the apoptotic markers cleaved PARP and Bax, and the growth and metastasis suppressor NDRG1, which mediates the pronounced inhibition of proliferation by these compounds in MIAPaCa-2 cells (Kovacevic et al., 2011).

In light of a recent study demonstrating the potential of Dp44mT and DpC to be effective for pancreatic cancer treatment in vivo (Kovacevic et al., 2011), we used tumors from mice treated with Dp44mT, DpC, or the vehicle alone and performed IHC staining to examine p-STAT3 and STAT3 levels. Both p-STAT3 and STAT3 staining were significantly lower in tumors from mice treated with Dp44mT and DpC compared with the vehicle (Fig. 9). These studies suggest Dp44mT and DpC prevent tumor growth, in part, by inhibition of STAT3.

Apart from the effect of these agents on the STAT3 pathway, it has been demonstrated that these compounds have multiple molecular targets in tumor cells, which leads to their marked antiproliferative efficacy (Fig. 10B). These include: 1) enhancement of iron mobilization from cells (Yuan et al., 2004; Richardson et al., 2006; Lovejoy et al., 2011); 2) inhibition of iron uptake from transferrin [both (1) and (2)], leading to cellular iron depletion, which is essential for growth (Yuan et al., 2004; Richardson et al., 2006; Lovejoy et al., 2011); 3) upregulation of the potent growth and metastasis suppressor NDRG1 (Le and Richardson, 2004; Kovacevic et al., 2011; Kovacevic et al., 2013), leading to inhibition of the epithelial-mesenchymal transition (Chen et al., 2012); 4) lysosomal membrane permeabilization mediated by the generation of redox active copper complexes (Yuan et al., 2004; Richardson et al., 2006; Lovejoy et al., 2011), resulting in impaired autophagy (Gutierrez et al., 2014) and the induction of apoptosis (Gutierrez et al., 2014; Sahni et al., 2014); 5) inhibition of multiple oncogenic cellular signaling pathways such as phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and transforming growth factor-β (TGF-β) pathways and upregulation of tumor-suppressive phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and SMAD4 (Dixon et al., 2013; Kovacevic et al., 2013); 6) inhibition of cyclin D1 expression (Kulp et al., 1996; Gao and Richardson, 2001; Nurtjahja-Tjendraputra et al., 2007); and 7) inhibition of the rate-limiting step of DNA synthesis catalyzed by the iron-containing enzyme ribonucleotide reductase (Yu et al., 2011). The relative contribution of each of these targets to the inhibition of tumor cell growth is yet to be deciphered, but may depend on the type and stage of the neoplasm. In fact, the multiple molecular targets of these agents probably explains their marked effect on a broad range of aggressive tumor types (Whitnall et al., 2006; Kovacevic et al., 2011; Lovejoy et al., 2012) and their ability to overcome drug resistance (Whitnall et al., 2006; Kovacevic et al., 2011; Lovejoy et al., 2012).

Considering other molecular targets of thiosemicarbazones, it is notable that the loss of STAT1 expression, which is closely related to STAT3, corresponds to advanced-stage pancreatic cancer with lymph node metastasis and poor prognosis (Sun et al., 2014). Conversely, STAT1 signaling has also been described as a tumor promoter (Kovacic et al., 2006), and thus the importance of STAT1 in pancreatic and prostate tumor progression remains to be more extensively characterized. However, it may be of interest to examine the potential role of STAT1 in mediating the activities of thiosemicarbazones in future studies.

Our investigation is the first to examine in detail the regulation of the STAT3 pathway by cellular iron in pancreatic and prostate cancer (Fig. 10A). Importantly, our findings provide further rationale for the use of novel thiosemicarbazones in chemotherapy. Moreover, because STAT3 inhibition increases the response to conventional therapies and delays pancreatic cancer progression (Venkatasubbarao et al., 2013), Dp44mT and DpC could be an effective chemotherapeutic strategy in combination with existing anticancer agents.

Acknowledgments

The authors wish to thank Sanaz Maleki for her valuable assistance with immunohistochemical staining.

Authorship Contributions

Participated in research design: Lui, Kovacevic, Richardson.

Conducted experiments: Lui, Kovacevic, Menezes.

Contributed new reagents or analytic tools: Richardson.

Performed data analysis: Lui, Kovacevic, Richardson.

Wrote or contributed to the writing of the manuscript: Lui, Kovacevic, Menezes, Kalinowski, Merlot, Sahni, Richardson.

Footnotes

    • Received October 22, 2014.
    • Accepted January 5, 2015.

  • This work was supported by a National Health and Medical Research Council of Australia (NHMRC) Project Grant [APP1060482], NHMRC Senior Principal Research Fellowship [APP1062607], and a NHMRC Peter Doherty Biomedical Post-Doctoral Fellowship [APP1037323] and a Cancer Institute New South Wales Early Career Development Fellowship [12/ECF/2-17].

  • dx.doi.org/10.1124/mol.114.096529.

Abbreviations

BSA
bovine serum albumin
DAPI
4′,6-diamidino-2-phenylindole
DFO
desferrioxamine
DMSO
dimethylsulfoxide
Dp2mT
di-2-pyridylketone 2-methyl-3-thiosemicarbazone
Dp44mT
di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone
DpC
di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone
DpT
di-2-pyridyl thiosemicarbazone
GSH
glutathione
IHC
immunohistochemical
JAK
Janus kinase
JAK2
Janus tyrosine kinase
NAC
N-acetylcysteine
PaEC
normal pancreatic epithelial cells
PBS
phosphate-buffered saline
ROS
reactive oxygen species
TBS
Tris buffered saline

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Antitumor Thiosemicarbazones Inhibit STAT3 Signaling

Goldie Y. L. Lui, Zaklina Kovacevic, Sharleen V. Menezes, Danuta S. Kalinowski, Angelica M. Merlot, Sumit Sahni and Des R. Richardson
Molecular Pharmacology March 1, 2015, 87 (3) 543-560; DOI: https://doi.org/10.1124/mol.114.096529
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