Gallium nitrate as a novel agent for the treatment of mantle cell lymphoma: Targets and mechanisms of action
Authors: Thomas P. Joseph, Janine P. Wereley, Christopher R. Chitambar
Journal: Cancer Research (2005)

Proc Amer Assoc Cancer Res, Volume 46, 2005

5879 

Background: Mantle cell lymphoma (MCL) is a clinically aggressive malignancy characterized by an overexpression of cyclin D1. Since effective treatment options for MCL are limited, we examined the potential of the drug gallium nitrate (GaN) as a therapeutic agent for this disease. Methods and Results: Our studies revealed that over a 48-h incubation, GaN inhibited the proliferation of MCL cell lines HB12, JVM2, Z138C, and Granta with IC50s of 50, 85, 100, and 400 μM, respectively. Cellular transferrin receptor (TfR) expression, a known target for Ga uptake, was measured by transferrin (Tf) binding assay and found to be lower in Granta cells (least sensitive to GaN) and higher in the other cell lines more sensitive to GaN. TfR affinity for Tf, as determined by Scatchard analysis, was similar in all cell lines. Radiogallium (Ga-67) uptake by individual MCL cell lines did not correlate with their relative differences in sensitivity to GaN, suggesting that gallium’s cytotoxicity is ultimately determined by its action on intracellular targets rather than its uptake per se . In all cell lines, GaN, at concentrations of 100 and 200 μM, inhibited cellular iron uptake over 24 h by 26-36% and 35-47% respectively. However, this effect on iron uptake did not explain the differences in Ga-induced growth inhibition among the cell lines. Analyses with Western blotting, flow cytometry and functional assays revealed that GaN produced a dose-dependent decrease in cyclin D1 expression, an increase in proapoptotic Bax expression and an activation of caspase-3. No effect of GaN on Bcl-2 levels was noted, nor was a correlation found between endogenous steady-state Bcl-2 expression and cell sensitivity to GaN. Cells exposed to GaN displayed an increase in their ability to generate fluorescent dichlorofluorescein (DCF) from nonfluorescent carboxy-H2DCF diacetate, consistent with the production of reactive oxygen species (ROS) within these cells. Bortezomib, a proteosome inhibitor with clinical activity in MCL, significantly enhanced the cytotoxicity of GaN in Granta cells but not in the other cell lines more sensitive to GaN. After a 48-h incubation, Granta cell growth inhibition was 18% with GaN 200 μM, 36% with bortezomib 4 nM, and 76% with the combination of both drugs. The addition of bortezomib to cells incubated with GaN also increased cellular ROS production and caspase-3 activity. Conclusions: Our studies indicate that GaN induces apoptosis in MCL cells through mechanisms that involve an increase in Bax levels, the generation of ROS, and a downregulation of cyclin D1. Since cyclin D1 plays a key role in the pathogenesis of MCL, the ability of GaN to decrease its level suggests that this drug may have preferential clinical activity against MCL. Furthermore, since the cytotoxicity of GaN can be enhanced by bortezomib, clinical trials using these two drugs in combination to treat MCL should be considered.