Extracellular thiol-assisted selenium uptake dependent on the x(c)- cystine transporter explains the cancer-specific cytotoxicity of selenite

Abstract

The selenium salt selenite (SeO(3)(2-)) is cytotoxic in low to moderate concentrations, with a remarkable specificity for cancer cells resistant to conventional chemotherapy. Our data show that selenium uptake and accumulation, rather than intracellular events, are crucial to the specific selenite cytotoxicity observed in resistant cancer cells. We show that selenium uptake depends on extracellular reduction, and that the extracellular environment is a key factor specific to selenite cytotoxicity. The extracellular reduction is mediated by cysteine, and the efficacy is determined by the uptake of cystine by the x(c)(-) antiporter and secretion of cysteine by multidrug resistance proteins, both of which are frequently overexpressed by resistant cancer cells. This mechanism provides molecular evidence for the existence of an inverse relationship between resistance to conventional chemotherapy and sensitivity to selenite cytotoxicity, and highlights the great therapeutic potential in treating multidrug-resistant cancer.

Fig. 1.

Selenite sensitivity is related to selenium uptake and extracellular thiols. Error bars display ± 0.95 confidence intervals. (A) Extracellular thiols, produced by cells at 0–20 h, measured with DTNB. (B) Total selenium uptake in cells treated with 5 μM selenite at 5 h measured with inductively coupled plasma mass spectroscopy, correlated to extracellular thiol production rate at 5 h measured with DTNB.

Fig. 2.

Modulation of the extracellular redox state alters selenite sensitivity and selenium uptake. Error bars display ± 0.95 confidence intervals. ***P < .001. (A) Viability measured by XTT in lung cancer cells treated with 5 μM selenite for 20 h. GSH (75 μM) and TCEP (75 μM) were used as extracellular reductants. DTNB (500 μM) was used as an extracellular thiol scavenger. *In relation to selenite treatment. (B) Total selenium uptake, measured at 5 h, in extracellular redox state–modulated cells treated with 5 μM selenite and TCEP (75 μM) in H611 cells or DTNB (500 μM) in H157 cells.

Fig. 3.

Lung cancer cells expressing the x_c⁻ cystine/glutamate antiporter have a reduced extracellular microenvironment, mainly through cysteine secretion. Error bars display ± 0.95 confidence intervals. (A) Total growth medium cystine/cysteine levels and reduced cysteine levels in lung cancer cells at 0–20 h, measured with HPLC. (B) Total growth medium reduced/oxidized glutathione levels and reduced glutathione levels in lung cancer cells at 0–20 h, measured with HPLC. (C) Total extracellular thiol levels in cells treated with monosodium glutamate (60 mM) at 0–20 h. (D) Western blot analysis of the x_c⁻ cystine/glutamate antiporter–specific xct subunit. Actin was used as a loading control.

Fig. 4.

Inhibition of the x_c⁻ cystine/glutamate antiporter inhibits selenium uptake and selenite toxicity. Error bars display ± 0.95 confidence intervals. ***(^xxx)P < .001 (A) Viability measured by XTT in lung cancer cells treated with 5 μM selenite for 20 h. MSG (60 mM) was used as an inhibitor of the x_c⁻ cystine/glutamate antiporter, and TCEP (75 μM) with MSG was used as an extracellular thiol impact control. *In relation to selenite treatment; ^xIn relation to selenite + MSG. (B) Total selenium uptake, measured at 5 h, in H157 cells treated with 5 μM selenite with MSG or with MSG + TCEP.

Fig. 5.

MRP, but not Pgp, is involved in cysteine secretion from lung cancer cells. Error bars display ± 0.95 confidence intervals. ***P < .001 in relation to no inhibitor. (A) Extracellular redox state measured with DTNB at 5 h in H157 cells treated with/without the MRP-specific inhibitor MK571. (B) Extracellular redox state measured with DTNB at 5 h in H157 cells treated with/without the Pgp-specific inhibitor verapamil.

Fig. 6.

Model for selenite cytotoxicity. The x_c⁻ cystine/glutamate antiporter facilitates the uptake of cystine, which is reduced intracellularly to cysteine by NADPH-dependent redox systems. A significant fraction of intracellular cysteine is resecreted into the extracellular compartment by MRPs, causing a reductive extracellular microenvironment. Extracellular reduction of selenite leads to a high-affinity uptake of a reduced form of selenite, possibly selenide, causing selenium accumulation and toxicity.