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Vol. 2, 235-243, March 2003     Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research

The Thioredoxin Redox Inhibitors 1-Methylpropyl 2-Imidazolyl Disulfide and Pleurotin Inhibit Hypoxia-induced Factor 1{alpha} and Vascular Endothelial Growth Factor Formation 1

Sarah J. Welsh2, Ryan R. Williams, Anne Birmingham, David J. Newman, D. Lynn Kirkpatrick and Garth Powis

Arizona Cancer Center, Tucson, Arizona 85724 [S. J. W., R. R. W., A. B., G. P.]; Natural Products Branch, National Cancer Institute, Frederick, Maryland 21702 [D. J. N.]; and ProlX Pharmaceuticals, Tucson, Arizona 85750 [D. L. K.]

2 To whom requests for reprints should be addressed, at Arizona Cancer Center, 1515 North Campbell Avenue, Tucson, AZ 85724. Phone: (520) 626-2446; Fax: (520) 626-4848; E-mail: swelsh{at}azcc.arizona.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in tumor growth by increasing resistance to apoptosis and the production of angiogenic factors such as vascular endothelial growth factor (VEGF). HIF-1 is a heterodimer comprised of oxygen-regulated HIF-1{alpha} and constitutively expressed HIF-1ß subunits. The redox protein thioredoxin-1 (Trx-1), which is found at high levels in many human cancers, increases both aerobic and hypoxia-induced HIF-1{alpha} protein in cells leading to increased expression of HIF-regulated genes. We have investigated whether two cancer drugs that inhibit Trx-1 signaling, PX-12 (1-methylpropyl 2-imidazolyl disulfide) and pleurotin, decrease HIF-1{alpha} protein levels and the expression of downstream target genes. Treatment of MCF-7 human breast cancer and HT-29 human colon carcinoma cells with PX-12 and pleurotin prevented the hypoxia (1% oxygen)-induced increase in HIF-1{alpha} protein. HIF-1-trans-activating activity, VEGF formation, and inducible nitric oxide synthase were also decreased by treatment with PX-12 and pleurotin under hypoxic conditions. PX-12 and pleurotin also decreased HIF-1{alpha} protein levels and HIF-1 trans-activation in RCC4 renal cell carcinoma cells that constitutively overexpress HIF-1{alpha} protein because of loss of the pVHL gene, indicating that HIF-1{alpha} is inhibited independently of the pVHL pathway. HIF-1{alpha} and VEGF protein levels in MCF-7 tumor xenografts in vivo were decreased by PX-12 treatment of mice. The results suggest that inhibition of HIF-1{alpha} by Trx-1 inhibitors may contribute to the growth inhibitory and antitumor activity of these agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of cancer cells to adapt to hypoxia is critically important for tumor growth (reviewed in Ref. 1). Hypoxia is known to exist in nearly all solid tumors and has been associated with poor prognosis in several tumor types (reviewed in Ref. 2). HIF-1 3 is a transcription factor that is activated by hypoxia and plays a critical role in the development of the cancer phenotype (reviewed in Ref. 3). HIF-1 is a heterodimer of the hypoxia-induced HIF-1{alpha} and the constitutively expressed HIF-1ß subunits (4). Both proteins are members of the basic helix-loop-helix superfamily of transcription factors in which the basic helix-loop-helix domains allow dimerization, and the basic domains bind to DNA (4). The HIF-1 complex interacts with coactivators such as CBP/p300 (5–7) mediated by hydroxylation of asparagine 851 of HIF-1{alpha} (8) and by SRC-1 (7, 9). Upon activation, the HIF-1 complex binds to target genes at sites containing the core recognition sequence 5'-RCGTG-3', also known as the HRE (10). HIF-1 activates the transcription of genes, the protein products of which play important roles in angiogenesis, vascular remodeling, glucose and energy metabolism, cell proliferation and survival, and erythropoiesis and iron homeostasis (reviewed in Ref. 1).

The activity of the HIF-1 complex is primarily controlled at the protein level. Under nonhypoxic conditions, pVHL binds to the oxygen degradation domain of HIF-1{alpha}, which recruits a ubiquitin-protein ligase complex containing elongin B, elongin C, and cullin, resulting in ubiquitination and degradation by the 26S proteosome pathway (11, 12). The interaction between human HIF-1{alpha} and pVHL is dependent on modification of proline-564 and proline-402 of the oxygen degradation domain of HIF-1{alpha} by prolyl hydroxylases (13). Under hypoxic conditions, HIF-1{alpha} is not hydroxylated, binding to pVHL is prevented, and HIF-1{alpha} protein levels increase and dimerize with HIF-1ß subunits, the expression of which is not changed by hypoxia (14). The MDM2 ubiquitin protein ligase is also recruited to HIF-1{alpha} by the binding of the tumor suppressor p53, which may also result in a decrease in HIF-1{alpha} levels (15, 16). Activation of the PI3K/Akt (protein kinase B) pathway has also been shown to induce expression of HIF-1{alpha} protein (17, 18) and recent studies have demonstrated a HSP90-dependent pathway for degradation of HIF-1{alpha} independent of the VHL pathway (19).

The molecular redox mechanisms of sensing and signaling changes in oxygen concentration are poorly understood. Prolyl hydroxylases responsible for HIF-1{alpha} hydroxylation require molecular oxygen and Fe2+, and availability of molecular oxygen could limit the activity of these enzymes, thus, preventing binding of pVHL resulting in increased HIF-1{alpha} protein levels (13). Hypoxia, paradoxically, leads to an increase in the production by mitochondria of reactive oxygen species (20, 21), which can regulate HIF-1 directly (6, 7, 22) or trigger a kinase cascade that leads to increased levels of HIF-1{alpha} protein under both normoxic and hypoxic conditions (23). The interaction between HIF-1{alpha} and CBP/p300 has also been shown to be redox regulated (6, 7, 23, 24).

HIF-1{alpha} protein is found in a wide variety of human primary tumors but not in normal tissue (25–31). The importance of HIF-1{alpha} in cancer is demonstrated by the high incidence of tumors such as renal cell carcinoma, pheochromocytoma, and hemangioblastoma of the central nervous system in individuals with loss of function of both alleles of the VHL gene leading to elevated HIF-1{alpha} levels (32). In addition, most cases of sporadic renal cell carcinoma are associated with an early loss of function of the VHL gene and increased HIF-1{alpha} levels (32–34). Reintroduction of the intact VHL gene into cells derived from renal carcinomas restores HIF-1{alpha} to normoxic levels (35) and decreases tumorigenicity (36, 37). HIF-1{alpha} levels are also increased in cancer cells with mutation or deletion of the tumor suppressor protein PTEN, which normally attenuates the activity of the PI3K/Akt pathway (17). Because of the importance of HIF-1{alpha} for tumor development and survival, it is an important target for cancer drug development (2, 38) but, to date, with few active agents reported.

Trx-1 is a ubiquitously expressed small redox protein with a conserved catalytic site (reviewed in Refs. 39, 40). Trx-1 undergoes reversible NADPH-dependent reduction by selenocysteine containing flavoprotein Trx-reductases (reviewed in Ref. 41). Trx-1 binds in a redox-dependent manner and regulates the activity of enzymes such as apoptosis signal-regulating kinase-1 (42) and protein kinases C {alpha}, {delta}, {epsilon}, and {zeta} (43). Trx-1 also increases the DNA binding of redox-sensitive transcription factors, including nuclear factor {kappa}B (44), the glucocorticoid receptor (45), and p53 (46). Mouse WEHI7.2 lymphoma cells transfected with human Trx-1 form tumors in immunodeficient scid mice that grow more rapidly and show less spontaneous and drug induced apoptosis than vector-alone transfected cells (47). A redox-inactive mutant Trx-1 acts as a dominant negative to inhibit human breast cancer MCF-7 and WEHI7.2 cell growth (48, 49). Trx-1 expression is increased in several human primary cancers, including lung, colon, cervix, liver, pancreatic, colorectal, and squamous cell cancer (47, 50–54). Increased Trx-1 levels have been correlated with increased proliferation and decreased apoptosis of human gastric tumors (53) and with decreased patient survival in non-small cell lung cancer (55). We have recently reported that increased expression of Trx-1 in cancer cells increases HIF-1{alpha} protein levels and trans-activating activity under both normoxic and hypoxic conditions (56).

Several inhibitors of Trx-1 have been developed (57–59). PX-12 (Fig. 1A) is a potent inhibitor of the Trx-1 by irreversibly thioalkylation of Cys73 of Trx-1 (60), causing inhibition of Trx-dependent cell growth (58). PX-12 has antitumor activity against human tumor xenografts in scid mice (60) and is currently undergoing Phase I clinical trials. Pleurotin (NSC-131233; Fig. 1B) was identified by the COMPARE program as having a pattern of cell killing activity similar to PX-12 in the National Cancer Institute human cell line panel (61). Pleurotin is an irreversible inhibitor of thioredoxin reductase with a Ki of 0.28 µM.



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  		Fig 1. Structure of PX-12 (A) and pleurotin (B).

 
We have investigated the effects of the Trx-1 redox signaling inhibitors PX-12 and pleurotin on HIF-1{alpha} and its downstream targets. Both inhibitors decreased HIF-1{alpha} protein, HIF-1 trans-activating activity and expression of the downstream targets of HIF-1, VEGF, and iNOS in vitro. The inhibitors also decreased expression of HIF-1{alpha} and VEGF proteins and microvessel density in tumor xenografts in vivo. Thus, the growth inhibitory and antitumor activity of inhibitors of Trx-1 may be mediated, in part, by inhibition of HIF-1{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Hypoxia Treatment.
MCF-7 human breast cancer and HT-29 colon cancer cells were obtained from the American Tissue Type Collection (Manassas, VA). Human renal cell carcinoma RCC4 cells and RCC4/VHL into which the wild-type pVHL gene has been transfected (32) were obtained from Dr. Peter Ratcliffe (Welcome Trust Centre for Human Genetics, Oxford, United Kingdom). Cells were grown under humidified 95% air, 5% CO2 at 37°C in DMEM supplemented with 10% fetal bovine serum, and 1 mg/ml G418 for the RCC4 and RCC4/VHL cells. Cells in culture flasks were exposed to hypoxia for various times in a humidified chamber at 37°C with a gas mixture containing 1% oxygen in a 5% CO2/74% N2/21% argon maintained using an oxygen sensor (Pro:Ox 110; Reming Bioinstruments Co., Redfield, NY).

Cell Growth Inhibition.
Cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (62). Cells were exposed to a range of concentrations of PX-12 or pleurotin for 16 h in air or hypoxia (1% oxygen). The cells were then washed with warm drug-free medium and grown in air for the remainder of the 72-h incubation.

VEGF ELISA.
MCF-7 or HT-29 cells (107) were lysed at 4°C for 1 h in 200 µl of lysis buffer [150 mM NaCl, 50 mM Tris buffer (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.1 mM sodium orthovanadate, 1% NP40, and 0.2% SDS]. The lysate was centrifuged for 15 min at 4°C at 10,000 x g, and the supernatant was stored at -80°C until required. A 20-µl aliquot was removed for analysis of the protein using the Bio-Rad Protein Detection System (Bio-Rad, Hercules, CA). Human VEGF in cell lysates and VEGF secreted into the growth medium was measured using an ELISA kit that detects VEGF165 and VEGF121 isoforms (Human VEGF-ELISA; R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. VEGF in cell lysates was expressed as pg VEGF protein/mg of total cell protein and VEGF in the medium corrected to pg VEGF protein/mg of total cell protein measured in cells from the same flask.

Western Blotting.
Nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) according to the manufacturer’s instructions. Total cell lysates were prepared as described above. Western blotting was performed as previously described (56) using mouse antihuman HIF-1{alpha} monoclonal antibody (1 µg/ml; Transduction Labs, Lexington, KY), mouse antihuman HIF-1ß monoclonal antibody (1 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), mouse antihuman iNOS monoclonal antibody (5 µg/ml; Transduction Labs), goat antihuman actin polyclonal antibody (0.5 µg/ml; Santa Cruz Biotechnology), goat antihuman lamin A polyclonal antibody (0.5 µg/ml; Santa Cruz Biotechnology), and mouse antihuman VHL monoclonal antibody (1 µg/ml; Neomarkers, Fremont, CA). Antimouse or antigoat horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia, Uppsala, Sweden) were used at a dilution of 1:5000 for detection by chemiluminescence, and blots were quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

HRE Reporter Assay.
The pGL3 firefly luciferase reporter plasmid containing the HRE from phosphoglycerate kinase (62) was supplied by Dr. Ian Stratford (University of Oxford, Oxford, United Kingdom). Plasmid DNA was prepared using a commercial kit (Qiagen, Valencia, CA). The empty pGL3 control plasmid and the pRL-CMV Renilla luciferase containing plasmid used to control for transfection efficiency were obtained from Promega (Madison, WI). Cells were transfected with 5 µg of HIF-1 reporter plasmid or pGL3 control plasmid, and 0.025 µg of pRL-CMV Renilla luciferase plasmid using LipoTAXI mammalian transfection reagent (Stratagene, TX). Twenty-four h later, cells were exposed to hypoxia for 16 h as described previously. We have previously found that 16 h of hypoxia is optimal for increases in HIF-1{alpha} protein levels (56). Firefly and Renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

Immunohistochemistry.
Human breast carcinoma MCF-7 cells (107) cells in 0.2 ml of matrigel were injected s.c. in the flanks of scid mice, and tumors were allowed to grow to 0.5 g. The mice then received an i.p. injection of either vehicle alone or 12 mg/kg PX-12 in 0.01 N HCl, polyethylene glycol-400 (2.0 mg/ml). Four h (or 24 h for measurement of microvessel density) later, the tumors were excised, fixed in formalin, and embedded in paraffin. Sections were stained with mouse antihuman HIF-1{alpha} monoclonal antibody (10 µg/ml; Transduction Labs), a rabbit antihuman VEGF polyclonal antibody (3 µg/ml; Santa Cruz Biotechnology), and a mouse antihuman PECAM monoclonal antibody (7 µg/ml; Novocastra, Newcastle Upon Tyne, United Kingdom), using an automated immunostainer system (ES; Ventana Medical Systems, Tucson, AZ). The intensity of staining was quantified using the Simple PCI analysis software (Compix, Cranberry Township, PA). Light levels and exposure times were kept constant throughout image acquisition. HIF-1{alpha} and VEGF staining was determined by measuring total gray levels per image after setting the threshold such that only staining above background was detected. Microvessel density was determined as described previously (56). Student’s t test was then applied to determine statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of HIF-1{alpha} Protein.
PX-12 and pleurotin inhibited the growth of MCF-7 and HT-29 cells with IC50s in MCF-7 cells of 1.9 ± 0.8 and 0.9 ± 1.0 µM, respectively, and of HT-29 cells of 2.9 ± 2.0 and 0.9 ± 1.2 µM, respectively. HIF-1{alpha} protein was not measurable in MCF-7 breast cancer cells grown in air but was measurable after exposure of the cells to hypoxia (1% oxygen) for 16 h, thus confirming previous observations (56). Exposure to PX-12 and pleurotin during hypoxia both caused dose-dependent decreases in hypoxia-induced HIF-1{alpha} protein levels in MCF-7 breast cancer cells (Fig. 2, A and B). The IC50s (±SE, n = 3) were 7.2 ± 0.9 µM for PX-12 and 7.6 ± 1.1 µM for pleurotin. HIF-1ß levels were not affected by exposure to hypoxia or pleurotin for 16 h but were slightly decreased by PX-12 under hypoxia with an IC50 of >10 µM. Similar effects of PX-12 and pleurotin on HIF-1{alpha} and HIF-1ß protein levels were seen in HT-29 colon cancer cells (data not shown). Neither drug caused a change in pVHL levels or in HIF-1{alpha} mRNA levels (data not shown).



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  		Fig 2. Effect of the Trx-1 inhibitors PX-12 and pleurotin on HIF-1{alpha} and HIF-1ß levels. MCF-7 cells were exposed to normoxia (20% oxygen) or hypoxia (1% oxygen) for 16 h in the presence of varying concentrations of PX-12 (A) or pleurotin (B). HIF-1{alpha} and HIF-1ß protein levels in cell nuclear extracts were detected using Western blotting. Blots are representative of two separate experiments. Lamin A was used as a loading control.

 
Inhibition of HIF-1 Trans-activation.
HIF-1 trans-activating activity was measured in cells by transiently transfecting a reporter construct expressing luciferase under the control of multiple copies of the HRE. Hypoxia had no effect on background luciferase activity measured using a control vector lacking the HRE. Both PX-12 (Fig. 3A) and pleurotin (Fig. 3B) caused significant inhibition of hypoxia-induced HIF-1 trans-activating activity with IC50s (±SE, n = 3) of 11.5 ± 1.1 and 9.0 ± 0.6 µM, respectively. Neither agent had a significant effect upon HIF-1 trans-activating activity under aerobic conditions (Fig. 3, A and B).



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  		Fig 3. Effect of thioredoxin inhibitors on trans-activation of HIF-1. MCF-7 cells were transiently transfected with vector-expressing firefly luciferase under the control of the HRE from phosphoglycerate kinase (PGK) or an empty vector control (neo) and exposed to PX-12 (A) or pleurotin (B) in normoxia (20% oxygen) or hypoxia (1% oxygen). The cells were cotransfected with a vector expressing the Renilla luciferase. The firefly luciferase: Renilla luciferase activities were then determined. Data represent the mean ± SE of three separate experiments. * denotes significant differences from neo (normoxia) values (P <= 0.001).

 
Cells Constitutively Expressing HIF-1{alpha}.
Wild-type RCC4 renal cell carcinoma cells that have a mutated VHL show constitutive overexpression of HIF-1{alpha} protein under aerobic conditions, but they still have a small hypoxia-induced increase of HIF-1{alpha} protein (Fig. 4A). RCC4/VHL cells stably transfected with wild-type VHL (32) show no detectable HIF-1{alpha} under aerobic conditions and a large hypoxia-inducible increase in HIF-1{alpha} protein (Fig. 4A). Treatment of wild-type RCC4 cells with PX-12 (Fig. 4B) for 16 h gave a significant decrease in HIF-1{alpha} protein under both normoxic and hypoxic conditions with IC50s of 6.7 ± 0.5 µM and 7.1 ± 0.8 µM, respectively. HIF-1 trans-activating activity was significantly decreased (P <= 0.01) under both normoxic and hypoxic conditions by 10 µM PX-12 in RCC4 cells (Fig. 4B). Similar results were observed using pleurotin (data not shown for Western blotting).



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  		Fig 4. Effect of Trx-1 inhibition in cells overexpressing HIF-1{alpha} protein. Human renal cell carcinoma RCC4 cells, overexpressing HIF-1{alpha} protein, and RCC4/VHL cells into which wild-type VHL has been transfected, resulting in lower expression of HIF-1{alpha} protein, were exposed to normoxia (20% oxygen) or hypoxia (1% oxygen) in the presence of various concentrations of PX-12 or pleurotin. A, nuclear extracts were prepared, and Western blotting was performed to examine the effect on HIF-1{alpha} protein. B, trans-activation by the HIF-1 complex was also determined under normoxic and hypoxic conditions after exposure to 10 µM PX-12 or pleurotin using a luciferase reporter under the control of a HRE. Blots are representative of at least two separate experiments.

 
VEGF and iNOS.
Neither PX-12 or pleurotin had any significant effect on VEGF formation by MCF-7 or HT-29 cells under aerobic conditions. However, PX-12 and pleurotin caused dose-dependent decreases in the hypoxia-induced VEGF formation in both cell lines with IC50s for total VEGF formation (cells and medium) in MCF-7 and HT-29 cells of 10.4 ± 2.5 and 10.1 ± 2.2 µM for PX-12 and 5.2 ± 2.5 and 4.7 ± 2.3 µM for pleurotin, respectively (Fig. 5). PX-12 and pleurotin also caused a dose-dependent decrease in iNOS protein under both normoxic and hypoxic conditions in MCF-7 cells (Fig. 6). The IC50s for inhibition of iNOS were 18.1 ± 4.5 µM for PX-12 and 9.8 ± 4.1 µM for pleurotin.



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  		Fig 5. Effects of thioredoxin inhibitors on VEGF levels. MCF-7 and HT 29 cells were exposed to a range of concentrations of PX-12 (A) or pleurotin (B) under normoxic (20% oxygen; closed symbols) or hypoxic (1% oxygen; open symbols) conditions for 16 h. VEGF levels in cell lysates (circles) or secreted into the medium (triangles) were determined by ELISA. Data represent the mean ± SE from three separate experiments.

 


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  		Fig 6. Effect of Trx-1 inhibitors on iNOS. MCF-7 cells were exposed to a range of concentrations of PX-12 of pleurotin under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. The cells were lysed, and iNOS expression was determined using Western blotting. Actin was used as a loading control. Blots are representative of results from two separate experiments.

 
Inhibition of Trx-1 Decreases HIF-1{alpha} and VEGF Protein Levels and Microvessel Density in Vivo.
Staining for HIF-1{alpha} protein in control MCF-7 tumor xenografts showed patches of cells with darkly stained nuclei throughout the xenograft (Fig. 7A). However, after treatment of the mice with 12 mg/kg PX-12, the intensity of staining was significantly decreased (P <= 0.001) both overall and within these patches, although any staining present remained localized within the nuclei of the cells (Fig. 7, B and G). VEGF protein staining was more uniform throughout the tumor xenografts, although staining was increased in areas showing increased HIF-1{alpha} staining (Fig. 7C). Treatment of mice with 12 mg/kg PX-12 significantly decreased VEGF protein staining (P <= 0.001; Fig. 7, D and G). Microvessel density was also significantly decreased (P <= 0.001) in PX-12-treated xenografts 24 h after drug injection compared with untreated controls (Fig. 7, E–G). No significant decrease in microvessel density was observed after 4 h treatment with PX-12 (data not shown).



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  		Fig 7. In vivo effects of PX-12. MCF-7 cells were grown as xenografts in scid mice to ~0.5 g. Groups of four mice were then treated as controls (A, C, and E) or with 12 mg/kg PX-12 i.p. (B, D, and F). Four (for HIF-1{alpha} and VEGF) or 24 (for measurement of microvessel density) h later, tumors were removed, fixed in 4% formaldehyde, and embedded in paraffin and HIF-1{alpha} (A and B). VEGF (C and D) or PECAM (E and F) proteins were detected using immunohistochemistry. Representative staining is shown. The graph (G) shows the mean HIF-1{alpha}, VEGF and microvessel density (MVD) staining or (±SE, n = 4). (-) denotes no drug treatment, (+) denotes drug treatment. * shows a significant difference compared with untreated controls (P <= 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HIF-1{alpha} protein is increased in several human tumors compared with normal tissue (25–31). The HIF-1 transcription complex has been implicated in many important aspects of tumor growth, including increased angiogenesis, metastasis, and inhibited apoptosis (reviewed in Refs. 1, 3). The activity of the HIF-1 complex is primarily controlled by levels of HIF-1{alpha}, which is tightly regulated in normal cells but elevated in tumor cells through a number of mechanisms (reviewed in Ref. 2). Because increases in HIF-1{alpha} protein have been shown to result in elevated HIF-1 trans-activation leading to increased angiogenesis and poor prognosis in patients (3), the HIF-1 pathway has become an important target for cancer chemotherapy (2, 38).

We have recently shown that Trx-1 increases HIF-1{alpha} protein in both aerobic and hypoxic cancer cells leading to increased angiogenesis (56). A redox inactive mutant Trx-1 decreased HIF-1{alpha} protein and transactivation. In this study we have shown that PX-12 and pleurotin, two inhibitors of the Trx-1 redox system, inhibit HIF-1{alpha} protein and HIF-1 transactivation both in vitro and in vivo resulting in decreased expression of downstream target genes, including the angiogenesis inducers VEGF and iNOS. Microvessel density was also decreased by PX-12 treatment in vivo, suggesting decreased angiogenesis. PX-12 is an inhibitor of Trx-1 (59, 60) and pleurotin is an inhibitor of Trx-1 reductase (61).

Previous studies have shown that Trx-1 can affect HIF-1 trans-activation (6, 7, 22) acting through a dual function DNA repair endonuclease and redox regulatory protein Ref-1 (63). Trx-1 directly reduces Ref-1 (64) and promotes the binding of the transcription coactivator complex CBP/p300 to the COOH-terminal trans-activation domain of HIF-1{alpha} leading to increased HIF-1 trans-activation (5, 6, 22). Although inhibition of Ref-1 reduction by Trx-1 could account, at least in part, for the inhibition of HIF-1{alpha} trans-activation observed in this study, it does not explain the marked decrease in HIF-1{alpha} protein levels seen upon inhibition of Trx-1 by PX-12 and pleurotin.

The mechanism of the decrease in HIF-1{alpha} protein by the Trx-1 redox inhibitors is not known. It is unlikely to involve pVHL because PX-12 and pleurotin decreased HIF-1{alpha} protein levels in RCC4 cells, which lack pVHL. Previous studies have shown the existence of a heat shock protein 90-dependent pathway for degradation of HIF-1{alpha} independent of pVHL (19). In addition, recent studies have shown that hypoxia activates the PI3K/AKT pathway and that this pathway is involved in the stabilization and activation of HIF (Refs. 12, 13, 17, 18, 65, and references within). Trx-1 binds to and inhibits the tumor suppressor protein PTEN, leading to activation of the PI3K pathway through AKT (66). Trx-1 may therefore affect HIF-1{alpha} protein through either of these pathways, although this was not investigated in this study.

Importantly, PX-12 and pleurotin caused significant decreases in expression of HIF-1{alpha} and VEGF (in vitro and in vivo) and microvessel density in vivo. VEGF is thought to be of critical importance to the progression of cancer (67). VEGF is the major angiogenic factor, leading to the development of new blood vessels from preexisting capillary beds in solid tumors and their metastases and is an autocrine factor in hematological tumors (68, 69). Inhibition of VEGF activity by functional blocking antibodies, expression of antisense VEGF mRNA, or disruption of VEGF receptor signaling (70, 71) reduces neoangiogenesis and tumor growth. Activation of VEGF is mediated by a 47-bp hypoxia response element located 985–939 bp 5' to the VEGF transcription initiation site (72). A HIF-1 binding site has also been demonstrated in this region (73). Therefore, the changes in VEGF protein levels observed in this study can presumably be accounted for by the changes in the trans-activating ability of HIF-1. However, HIF-independent mechanisms may also play a role in controlling VEGF production (74) and could explain why PX-12 showed a smaller decrease in VEGF levels compared with HIF-1{alpha} levels in vivo in this study. It also seems likely that the decrease in VEGF could account for the decrease in microvessel density observed in PX-12 treated xenografts in this study because microvessel density is used as a measure of angiogenesis in tumors (27).

In summary, we have shown that inhibition of the Trx-1 redox system leads to decreased HIF-1{alpha} protein and trans-activation of downstream targets both in vitro and in vivo, leading to decreased angiogenesis. Inhibition of the Trx-1 redox system therefore represents a novel and effective way of treating cancer cells leading to decreased angiogenesis.


   Footnotes
 
1 Supported by NIH Grants CA98920, CA17094, and CA52995. Back

3 The abbreviations used are: HIF-1, hypoxia-inducible factor-1; CBP, Creb-binding protein; HRE, hypoxia response element; pVHL, von Hippel-Lindau protein; PI3K, phosphatidylinositol 3'-kinase; Trx-1, thioredoxin-1; PX-12, 1-methylpropyl 2-imidazolyl disulfide; VEGF, vascular endothelial growth factor; iNOS, inducible nitric oxide synthase; Ref-1, redox factor-1. Back

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/12/02; revised 12/ 3/02; accepted 1/ 7/03.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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