Role of oxidative stress in the induction of metallothionein-2A and heme oxygenase-1 gene expression by the antineoplastic agent gallium nitrate in human lymphoma cells

Abstract

The mechanisms of action of gallium nitrate, an antineoplastic drug, are only partly understood. Using a DNA microarray to examine genes induced by gallium nitrate in CCRF-CEM cells, we found that gallium increased metallothionein-2A (MT2A) and heme oxygenase-1 (HO-1) gene expression and altered the levels of other stress-related genes. MT2A and HO-1 were increased after 6 and 16 h of incubation with gallium nitrate. An increase in oxidative stress, evidenced by a decrease in cellular GSH and GSH/GSSG ratio, and an increase in dichlorodihydrofluorescein (DCF) fluorescence, was seen after 1-4 h of incubation of cells with gallium nitrate. DCF fluorescence was blocked by the mitochondria-targeted antioxidant mitoquinone. N-Acetyl-L-cysteine blocked gallium-induced MT2A and HO-1 expression and increased gallium's cytotoxicity. Studies with a zinc-specific fluoroprobe suggested that gallium produced an expansion of an intracellular labile zinc pool, suggesting an action of gallium on zinc homeostasis. Gallium nitrate increased the phosphorylation of p38 mitogen-activated protein kinase and activated Nrf-2, a regulator of HO-1 gene transcription. Gallium-induced Nrf-2 activation and HO-1 expression were diminished by a p38 MAP kinase inhibitor. We conclude that gallium nitrate induces cellular oxidative stress as an early event which then triggers the expression of HO-1 and MT2A through different pathways.

Figure 1. Gallium nitrate induces the expression of metallothionein and heme oxygenase-1 in CCRF-CEM cells

A and B. DNA arrays showing induction of gene expression with gallium nitrate (GaN). Biotinylated cDNA probes prepared from CCRF-CEM cells incubated without (Control, A) or with 250 µmol/L gallium nitrate (B) for 24 h were hybridized to human metal transport and homeostasis gene array membranes and hybridization signals were detected as detailed under Methods. The positions of the HO-1 and metallothionein genes are shown. Asterisks mark the location of the beta-actin gene. C and D. Verification that gallium nitrate increases metallothionein-2A (MT2A) expression. C. CCRF-CEM cells were incubated with increasing concentrations of gallium nitrate for 24 h and analyzed for the MT2A mRNA levels by Northern blotting. D. Time-dependent induction of MT2A expression by gallium nitrate. CCRF-CEM cells were incubated with 100 µmol/L gallium nitrate for the times shown and analyzed for MT2A protein levels by Western blotting. E and F. Verification that gallium nitrate increases heme oxygenase-1 (HO-1) expression. E. Concentration-dependent upregulation of HO-1 expression by gallium nitrate. CCRF cells were incubated with increasing concentrations of gallium nitrate for 6 and 24 h and analyzed for HO-1 protein content by Western blotting; F. Time-dependent upregulation of HO-1 expression by gallium nitrate. CCRF-CEM cells were incubated with 100 or 300 µmol/L gallium nitrate for the times shown and analyzed for HO-1 protein content by Western blotting. G and H. EGR1 protein expression is not increased by gallium nitrate. G. Western blot. Cells were incubated with gallium nitrate for 24 h at the concentrations shown and analyzed by Western blotting for EGR1. H. Western blot showing that HO-1 but not EGR1 is increased by gallium nitrate. Cells were incubated with gallium nitrate for 24 h. The membrane was immunoblotted sequentially with antibodies against EGR1 and HO-1.

Figure 2. Gallium nitrate induces oxidative stress in cells

A and B. Effect of gallium nitrate on cellular GSH levels and GSH/GSSG ratio. CCRF-CEM cells were treated with 100 or 300 µmol/L gallium nitrate for the times shown and then analyzed for GSH and GSSG levels as described under Methods. Data represents means ±S.E. (n = 3). C. Gallium nitrate increases intracellular ROS. ROS production was detected in intact CCRF-CEM cells by DCF fluorescence. Cells were loaded with 10 µmol/L 6-carboxy-DCF-AM for 1 h, incubated with gallium nitrate for the specified times, and analyzed for DCF fluorescence by spectrofluorimeter as described under Methods. Values shown are means +/− S.E. of a representative experiment performed in triplicate.

Figure 3. Effect of NAC on gallium-induced upregulation of MT2A and HO-1

A – D. NAC blocks the increase in MT2A and HO-1 expression by gallium nitrate. CCRF-CEM cells were preincubated for 2 h with or without NAC and then incubated with or without gallium nitrate for 16 h at the concentrations shown. In coincubations, cells were exposed to both gallium nitrate and NAC simultaneously for 16 h. A and B. Western blots showing HO-1 (panel A) and MT2A (panel B) protein levels in cells. C. Northern blot showing steady-state MT2A mRNA levels in cells. D. Quantification of the MT2A band densities from the Northern blot in Figure 3C normalized to the corresponding β-actin level.

Figure 4. Effect of NAC on the cytotoxicity of gallium nitrate

A. NAC increases the antiproliferative effects of gallium nitrate cells. CCRF-CEM cells were pretreated with 10 or 20 mol/L of NAC for 2 h and then incubated with increasing concentrations of gallium nitrate plus NAC for 48 h. Cell growth was determined by counting the number of cells using a hemocytometer. B. NAC increases gallium-induced activation of caspase-3. CCRF-CEM cells were preincubated with 20 mmol/L NAC for 2 h and then incubated with increasing concentrations of gallium nitrate plus NAC for 24 h. Caspase-3 activity in intact cells was assayed as described under Methods. Values shown represent the mean ± S.E. (n = 3).

Figure 5. Gallium-induced increase in MT2A expression involves changes in cellular zinc homeostasis

A – F. Zinquin fluorescence. CCRF-CEM cells were incubated for 16 h without additives (A), or with 100 µM ZnSO4 (B), 100 µmol/L gallium nitrate (C), 200 µmol/L gallium nitrate (D), or 300 µmol/L gallium nitrate (E and F). Cells were then allowed to take up Zinquin for 40 min, as described under Methods. In panel F, prior to the addition of Zinquin, 50 µM TPEN was added for 90 minutes to cells that had incubated with 300 µmol/L of gallium nitrate. Cells were examined by fluorescence microscope. G. Zinquin fluorescence is produced by zinc and not by gallium. Gallium nitrate or zinc sulfate solutions at the concentrations shown were incubated with 3 µmol/L Zinquin for 40 min and then analyzed for fluorescence by spectrofluorimetry. H. Gallium-induced MT2A protein expression is reduced by TPEN. CCRF cells were incubated for 16 h with 300 µmol/L gallium nitrate, TPEN, or both agents in the combinations shown. MT2A and β-actin levels were measured by Western blotting.

Figure 6. Involvement of p38 MAP kinase and Nrf2 in the induction of HO-1 gene expression by gallium nitrate

A – C. Effects of MAP kinase inhibitors on HO-1 expression. CCRF-CEM cells were incubated for 16 h with 100 µmol/L gallium nitrate in the absence or presence of: A. p38 kinase inhibitor SB203580; B. ERK inhibitor PD98059; and C. JNK inhibitor SP600125 at the concentrations shown. Cellular HO-1 level was determined by Western blotting. D. Gallium nitrate increases the phosphorylation of p38 MAP kinase. Cells were incubated for 6 h with increasing concentrations of gallium nitrate. Cell lysates were analyzed by Western blotting for unphosphorylated (p38) and phosphorylated forms (p-p38) of p38 MAP kinase. E. Gallium nitrate increases Nrf-2 levels in the nucleus. Cells were incubated with or without SB203580 for 2 h. Following this, gallium nitrate was added to cells as shown and the incubation continued for an additional 6 h. Nuclear proteins were extracted as described under Methods and analyzed for Nrf-2 levels by Western blotting.