Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide

Abstract

Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia and has potential anticancer activity against a wide range of solid tumors. ATO exerts its effect mainly through elevated oxidative stress, but the exact molecular mechanism remains elusive. The thioredoxin (Trx) system comprising NADPH, thioredoxin reductase (TrxR), and Trx and the glutathione (GSH) system composed of NADPH, glutathione reductase, and GSH supported by glutaredoxin are the two electron donor systems that control cellular proliferation, viability, and apoptosis. Recently, the selenocysteine-dependent TrxR enzyme has emerged as an important molecular target for anticancer drug development. Here, we have discovered that ATO irreversibly inhibits mammalian TrxR with an IC(50) of 0.25 microM. Both the N-terminal redox-active dithiol and the C-terminal selenothiol-active site of reduced TrxR may participate in the reaction with ATO. The inhibition of MCF-7 cell growth by ATO was correlated with irreversible inactivation of TrxR, which subsequently led to Trx oxidation. Furthermore, the inhibition of TrxR by ATO was attenuated by GSH, and GSH depletion by buthionine sulfoximine enhanced ATO-induced cell death. These results strongly suggest that the ATO anticancer activity is by means of a Trx system-mediated apoptosis. Blocking cancer cell DNA replication and repair and induction of oxidative stress by the inhibition of both Trx and GSH systems are suggested as cancer chemotherapeutic strategies.

Fig. 1.

Inhibition of TrxR by ATO in vitro. (A) A 50 nM concentration of NADPH-reduced recombinant rat TrxR was incubated with different concentrations of ATO for different times, and then TrxR activity was assayed by DTNB reduction assay. Error bars represent the standard deviation of duplicate experiments. (B) A 50 nM concentration of recombinant rat TrxR in the presence (■) or absence (▵) of 200 μM NADPH was incubated with different concentrations of ATO for 30 min, and then TrxR activity was assayed by DTNB reduction assay. NADPH (200 μM), E. coli TrxR (25 nM), and E. coli Trx (2 μM) were incubated with different concentrations of ATO for 30 min, and then the activity was measured by the insulin reduction method (▴). (C) Peptide mass of TrxR–arsenic by MALDI mass spectrometry. Here 1 μM NADPH-reduced recombinant rat TrxR was incubated with 10 μM ATO, denatured in 8 M urea, and digested by trypsin and then subjected to mass spectra analysis.

Fig. 2.

Inhibition of TrxR in the MCF-7 cells by ATO. (A) MCF-7 cells were treated with different concentrations of ATO for 24 and 48 h. Cell proliferation and viability were determined by using the XTT method. (B and C) MCF-7 cells were treated with different concentrations of ATO for 12, 24, and 48 h. After the treatment, cells were lysed, and Trx (B) and TrxR (C) activities were determined with an endpoint insulin assay. Error bars represent the standard deviation of three independent experiments.

Fig. 3.

Trx redox state change in MCF-7 cells by the treatment of MCF-7. MCF-7 cells were treated with different concentrations of ATO for 48 h. The ATO-treated MCF-7 cells were lysed in guanidine lysis buffer containing 30 mM iodoacetamide so that protein SH groups were alkylated. Then samples in the presence or absence of DTT were separated on a 12% Bistris gel with Mes running buffer, and Trx was detected with goat anti-human Trx antibodies.

Fig. 4.

Effects of glutathione on the inhibition of TrxR and MCF-7 growth. (A) 10 nM concentration of NADPH-reduced recombinant rat TrxR in the presence of 2 μM human Trx or with 1 mM GSH was incubated with different concentrations of ATO for 30 min. Then the Trx system activity was assayed by the insulin reduction method. (B) MCF-7 cells were treated with 25 μM BSO for 24 h and then with different concentrations of ATO for another 48 h. The control cells were not treated with BSO.

Fig. 5.

Trx and GSH pathways interacting with ATO. Trx system including NADPH, TrxR, and Trx coupled with peroxiredoxins and the GSH system composed of NADPH, GR, GSH coupled with Grxs, GPxs, GSTs are the two main electron donor systems that mediate the cellular activity including DNA synthesis, protection against oxidative stress, and thus control cellular proliferation, viability, and apoptosis. The combination inhibition of ATO and BSO will lead to the disruption on both systems and block electron supply for DNA synthesis and give a rise of oxidative stress and thus induce cells into apoptosis.