630 Molecular pharmacology and antitumor activity of palmarumycin-based inhibitors of thioredoxin reductase Garth Powis,1 Peter Wipf,2 Stephen M. Lynch,2 Introduction 3 4 Anne Birmingham, and D. Lynn Kirkpatrick The cytosolic thioredoxin redox system is composed of thioredoxin-1 and thioredoxin reductase-1 reductase, 1Department of Experimental Therapeutics, M.D. Anderson Cancer Center, Houston, Texas; 2Department of Chemistry, which catalyzes the NADPH-dependent reduction of University of Pittsburgh, Pittsburgh, Pennsylvania; 3Arizona thioredoxin-1. Thioredoxin reductase-1 is an important Cancer Center; and 4ProlX Pharmaceuticals, Tucson, Arizona regulator of cancer cell growth and survival (1, 2). Thioredoxin-1 acting with peroxiredoxin-1 is an antioxi- dant that scavenges H2O2 (3). Thioredoxins are also able to Abstract reduce buried oxidized thiol residues in proteins (4) and The cytosolic thioredoxin redox system composed of regulate the activity of redox-sensitive transcription factors, thioredoxin-1 and the NADPH-dependent thioredoxin including p53 (5), nuclear factor-nB (6), the glucocorticoid reductase-1 reductase is an important regulator of cell receptor (7), activator protein-1 (8), hypoxia-inducible growth and survival. Thioredoxin-1 is overexpressed in factor-1 (HIF-1; ref. 9), Sp1 (10), and Nrf2 (11). Thiore- many human tumors where it is associated with doxin-1 also binds and inhibits the activity of the apoptosis- increased cell proliferation, decreased apoptosis, and inducing proteins, apoptosis signal-regulating kinase-1 (12) decreased patient survival. We hypothesized that thio- and, the tumor suppressor phosphatase and tensin homo- redoxin reductase-1 provides a target to inhibit the logue deleted on chromosome 10 (13), thus inhibiting activity of overexpressed thioredoxin-1 for the develop- apoptosis. Thioredoxin-1 is overexpressed in many human ment of novel anticancer agents. We found that the tumors (14–17) where it is associated with increased cell naphthoquinone spiroketal fungal metabolite palmarumy- proliferation, decreased apoptosis (18), and poor patient cin CP1 is a potent inhibitor of thioredoxin reductase-1, survival (19, 20). Thioredoxin reductase thus provides a but attempts to exploit the activity of palmarumycin CP1 target to regulate the activity of overexpressed thioredoxin- analogues as antitumor agents in vivo were hampered 1 (21, 22). by their insolubility. We have therefore developed PX- Thioredoxin reductase-1 is a selenocysteine-containing 916, a water-soluble prodrug of a palmarumycin CP1 flavoprotein with broad substrate specificity because of the analogue. PX-916 rapidly releases the parent compound ready accessibility of its COOH-terminal redox active site, at physiologic pH and in plasma but is stable at acid pH, which contains an essential selenocysteine residue (21). allowing its i.v. administration. PX-916 is a potent There are three thioredoxin reductase isoforms: the inhibitor of purified human thioredoxin reductase-1 and canonical cytoplasmic thioredoxin reductase-1, a mitochon- of thioredoxin reductase-1 activity in cells and tumor drial thioredoxin reductase-2, and a testes-specific thiore- xenografts when given to mice and inhibits the down- doxin reductase/glutathione reductase. The cellular level stream targets of thioredoxin-1 signaling, hypoxia- of thioredoxin reductase-1 is subject to complex regulation inducible factor-1A, and vascular endothelial growth (reviewed in ref. 23). The core promoter of the thioredoxin factor in tumors. PX-916 showed excellent antitumor reductase-1 gene contains several transcription factor activity against several animal tumor models with some activation sites, including those for the redox-sensitive cures. Thus, the study shows that water-soluble inhib- factors Oct-1 and Sp1 as well as others. Differential splicing itors of thioredoxin reductase-1, such as PX-916, can and alternative transcription start sites result in multiple block thioredoxin-1 signaling in tumors producing marked formsoftheenzyme.Post-transcriptionalregulation inhibition of tumor growth. [Mol Cancer Ther 2006; involving a selenocysteine insertion sequence element in 5(3):630–6] the 3V-untranslated region directs selenocysteine incorpo- ration, which is necessary for enzyme activity; thus, selenium supplementation can lead to increased thiore- doxin reductase-1 activity in cell culture and in selenium- deficient animals (24). Thioredoxin reductase-1 is necessary Received 11/28/05; revised 12/27/05; accepted 1/18/06. for cell proliferation. A thioredoxin reductase-1 knockout is Grant support: NIH grants CA52995 and CA90821. embryonic lethal in mice, and thioredoxin reductase- Requests for reprints: Garth Powis, Department of Experimental 1–deficient fibroblasts derived from the thioredoxin re- Therapeutics, M.D. Anderson Cancer Center, FC-6.3044, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: 713-745-3366; ductase-1 (À/À) embryos do not proliferate in vitro (25). Fax: 713-745-1710. E-mail: [email protected] Furthermore, cancer cell growth is inhibited by thioredoxin Copyright C 2006 American Association for Cancer Research. reductase-1 antisense (26), thioredoxin reductase-1 small doi:10.1158/1535-7163.MCT-05-0487 interfering RNA (27) and by a mutant redox inactive Mol Cancer Ther 2006;5(3). March 2006 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Molecular Cancer Therapeutics 631 duction Laboratories (Lexington, KY), and rabbit polyclon- al anti-human vascular endothelial growth factor (VEGF) was from Santa Cruz Biotechnology (Santa Cruz, CA). Immunohistochemistry for tumor HIF-1a and VEGF was done as described previously (35). Figure 1. Compound structures. thioredoxin reductase-1 (28). There are reports that levels of thioredoxin reductase-1 are increased by epidermal growth factor (29) and hypoxia (30) in cancer cells, although tumors show only moderately increased levels of thioredoxin reductase-1 (23, 30). We have identified the naphthoquinone spiroketal fungal metabolite palmarumycin CP1 as a potent inhibitor of thioredoxin reductase-1, but attempts to exploit the activity of palmarumycin CP1 analogues as antitumor agents in vivo were hampered by their insolubility (31). We have recently reported the synthesis of a series of water-soluble palmarumycin CP1 analogue prodrugs (32). We now report the molecular pharmacology and antitumor activity of one of these prodrugs in cancer cells and in human tumor xenografts. Materials and Methods Materials The palmarumycin CP1 analogues PX-911, PX-916, and PX-960 (Fig. 1) were synthesized as described previously (31, 32). PX-916 was dissolved at 3 mg/mL in 5% ethanol, 10 mmol/L sodium phosphate (pH 4.0) for i.p. and oral administration and in 5% ethanol, 0.9% NaCl, 10 mmol/L sodium phosphate (pH 4.0) for i.v. administration and used within 30 minutes. Human placental thioredoxin reductase- 1, specific activity 33 Amol NADPH oxidized/min/mg protein at room temperature, was prepared as described previously (33). Human recombinant thioredoxin-1 was prepared as described previously (34). Mouse monoclonal anti-human HIF-1a antibody was purchased from Trans- Table 1. Inhibition of thioredoxin reductase-1 and cell growth by palmarumycin CP1 analogues Figure 2. Inhibition of cellular and tumor thioredoxin reductase by PX- 916. MCF-7 human breast cancer cells grown in medium containing Compound Inhibition of Inhibition of 1 Amol/L selenium for 7 d were treated with PX-916 and cellular human MCF-7 breast thioredoxin reductase activity was measured. A, time course of the thioredoxin cancer cell inhibition of thioredoxin reductase on exposure to 1 Amol/L PX-916. B, concentration dependence of the inhibition of thioredoxin reductase on reductase-1 IC50 growth IC50 exposure to various concentrations of PX-916 for 17 h. Points, mean of (Amol/L) (Amol/L) three determinations; bars, SE. *, P V 0.05; **, P V 0.01, compared with control. C, MCF-7 human breast cancer xenografts were grown in female Palmarumycin CP1 0.35 1.0 scid mice implanted with 90-d 17h-estradiol slow release pellets until they f 3 PX-911 3.2 9.2 were 300 mm . The mice were given a single dose of PX-916 (25 mg/kg i.v.) and tumors were harvested at various times. Thioredoxin PX-916 0.28 3.1 reductase activity was measured in tumor homogenates. Points, mean of PX-960 0.27 4.1 three mice at each time point; bars, SE. **, P < 0.01, compared with pretreatment value. Mol Cancer Ther 2006;5(3). March 2006 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. 632 Palmarumycin-Based Inhibitors of Thioredoxin Reductase MCF-7 human breast cancer cells were obtained from the American Tissue Type Collection (Manassas, VA). All cells were tested to be Mycoplasma free using a PCR ELISA kit (Roche Diagnostics, Inc., Indianapolis, IN) and grown in j 95% humidified air with 5% CO2 at 37 C in McCoy’s 5A medium supplemented with 10% fetal bovine serum. For studies of effects of the palmarumycin CP1 analogues on cellular thioredoxin reductase activity, MCF-7 human breast cancer cells were grown in medium containing 1 Amol/L selenium for 7 days, which increased cellular thioredoxin reductase activity by f5-fold as reported previously (36). Figure 3. Antitumor activity of PX-916. A, female Beige nude mice were inoculated s.c. with 107 A673 rhabdomyosarcoma cells. When tumors were 100 mm3 on day 8 (arrow), dosing was begun with vehicle alone (o) or 30 mg/kg/d i.p. PX-916 for five doses (E). Points, mean of six mice per group; bars, SE. B, female scid mice were inoculated s.c. with 107 SHP-77 human small cell lung cancer cells. When tumors were 150 mm3 on day 17 (arrow), dosing was begun with vehicle alone (o), 10 mg/kg/d i.v. PX-916 for eight doses (n), or 25 mg/kg/d i.v. PX-916 for five doses (E). Points, mean of eight mice per group; bars, SE. C, female scid mice implanted 1 d previously with a 90-d 17h-estradiol slow release pellet were inoculated s.c.