metallothionein and gallium-nitrate

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.

Topics: Antineoplastic Agents; Cell Line, Tumor; Gallium; Gene Expression Regulation, Neoplastic; Heme Oxygenase (Decyclizing); Humans; Lymphoma; Metallothionein; Oxidative Stress

Several clinical trials have shown gallium nitrate to be an active agent in the treatment of lymphoma. Whereas gallium is known to target cellular iron homeostasis, the basis for lymphoma cell resistance to gallium is not known. Understanding mechanisms of resistance may suggest strategies to enhance the clinical efficacy of gallium. In the present study, we used a focused DNA microarray to compare the expression of genes related to metal metabolism in gallium-resistant and gallium-sensitive lymphoma cell lines developed by us. Gallium-resistant cells were found to display a marked increase in gene expression for metallothionein-2A and the zinc transporter ZnT-1. Cells exposed to gallium nitrate displayed an increase in the binding of metal-responsive transcription factor-1 to metal response element sequences involved in the transcriptional regulation of metallothionein and ZnT-1 genes. Gallium nitrate induced metallothionein-2A and ZnT-1 expression in cells. A role for metallothionein in modulating the antineoplastic activity of gallium was confirmed by showing that the induction of metallothionein expression by zinc provided partial protection against the cytotoxicity of gallium and by showing that the level of endogenous metallothionein in lymphoma cell lines correlated with their sensitivity to gallium nitrate. Immunohistochemical staining of lymphomatous tissues revealed metallothionein protein to be variably expressed in different lymphomas. Our studies show for the first time that gallium acts on pathways related to zinc metabolism and that metal-responsive transcription factor-1 activity and metallothionein expression contribute to the development of gallium drug resistance. Furthermore, the endogenous level of metallothionein in lymphoma may be an important determinant of clinical response to gallium nitrate.

Topics: Antineoplastic Agents; Blotting, Northern; Cation Transport Proteins; Cell Proliferation; DNA-Binding Proteins; DNA, Complementary; Drug Resistance, Neoplasm; Electrophoretic Mobility Shift Assay; Gallium; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Immunosuppressive Agents; Leukemia, Lymphoid; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Metallothionein; Oligonucleotide Array Sequence Analysis; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic