Clinical studies have shown gallium nitrate to have significant antitumor activity against non-Hodgkin's lymphoma and bladder cancer, thus indicating that gallium-based drugs have potential for further development as antineoplastic agents. In this study, we compared the cytotoxicity of gallium maltolate, a novel gallium compound, with gallium nitrate in lymphoma cell lines, including p53 variant and unique gallium nitrate-resistant cells. We found that gallium maltolate inhibited cell proliferation and induced apoptosis through the mitochondrial pathway at lower concentrations and more rapidly than gallium nitrate. Gallium maltolate produced an increase in intracellular reactive oxygen species (ROS) within 2 h of incubation with cells; this effect could be blocked by mitoquinone, a mitochondria-targeted antioxidant. The role of the transferrin receptor (TfR) in gallium maltolate's action was examined using monoclonal antibody (MoAb) 42/6 to block TfR function. However, although MoAb 42/6 reduced gallium maltolate-induced caspase-3 activity, it had only a minor effect on cell growth inhibition. Importantly, gallium maltolate induced apoptosis in cells resistant to gallium nitrate, and, unlike gallium nitrate, its cytotoxicity was not affected by cellular p53 status. Cellular gallium uptake was greater with gallium maltolate than with gallium nitrate. We conclude that gallium maltolate inhibits cell proliferation and induces apoptosis more efficiently than gallium nitrate. Gallium maltolate is incorporated into lymphoma cells to a greater extent than gallium nitrate via both TfR-independent and -dependent pathways; it has significant activity against gallium nitrate-resistant cells and acts independently of p53. Further studies to evaluate its antineoplastic activity in vivo are warranted.
Gallium nitrate is a metallodrug with clinical efficacy in non-Hodgkin's lymphoma. Its mechanisms of antineoplastic action are not fully understood. In the present study, we investigated the roles of transferrin receptor (TfR) targeting and apoptotic pathways in gallium-induced cell death. Although DoHH2 lymphoma cells displayed a 3-fold lower number of TfRs than CCRF-CEM lymphoma cells, they were 3-to 4-fold more sensitive to gallium nitrate. Despite a lower TfR expression, DoHH2 cells had greater TfR cycling and iron and gallium uptake than CCRF-CEM cells. In other lymphoma cell lines, TfR levels per se did not correlate with gallium sensitivity. Cells incubated with gallium nitrate showed morphologic changes of apoptosis, which were decreased by the caspase inhibitor Z-VAD-FMK and by a Bax-inhibitory peptide. Cells exposed to gallium nitrate released cytochrome c from mitochondria and displayed a dose-dependent increase in caspase-3 activity. An increase in active Bax levels without accompanying changes in Bcl-2 or Bcl-X L was seen in cells incubated with gallium nitrate. The endogenous expression of antiapoptotic Bcl-2 was greater in DoHH2 cells than in CCRF-CEM cells, suggesting that endogenous Bcl-2 levels do not correlate with cell sensitivity to gallium nitrate. Gallium-induced apoptosis was enhanced by the proteasome inhibitor bortezomib. Our results suggest that TfR function rather than TfR number is important in gallium targeting to cells and that apoptosis is triggered by gallium through the mitochondrial pathway by activating proapoptotic Bax. Our studies also suggest that the antineoplastic activity of combination gallium nitrate and bortezomib warrants further investigation.
The mechanism of drug resistance to gallium nitrate is not known. Since gallium can be incorporated into ferritin, an iron storage protein that protects cells from iron toxicity, we investigated whether ferritin expression was altered in gallium-resistant (R) CCRF-CEM cells. We found that the ferritin content of R cells was decreased, while heavy chain ferritin mRNA levels and iron regulatory protein-1 (IRP-1) RNA binding activity were increased. IRP-1 protein levels were similar in gallium-sensitive (S) and R cells, indicating that R cells contain a greater proportion of IRP-1 in a high affinity mRNA binding state. 59 Fe uptake and transferrin receptor expression were decreased in R cells. In both S and R cells, gallium inhibited cellular 59 Fe uptake, increased ferritin mRNA and protein, and decreased IRP-1 binding activity. Gallium uptake by R cells was markedly diminished; however, the sensitivity of R cells to gallium could be restored by increasing their uptake of gallium with excess transferrin. Our results suggest that R cells have developed resistance to gallium by down-regulating their uptake of gallium. In parallel, iron uptake by R cells is also decreased, leading to changes in iron homeostasis. Furthermore, since gallium has divergent effects on iron uptake and ferritin synthesis, its action may also include a direct effect on ferritin mRNA induction and IRP-1 activity.Gallium nitrate, a group IIIa metal salt with antineoplastic activity (1), is currently undergoing evaluation as a chemotherapeutic agent. A number of clinical studies have shown gallium to be effective in the treatment of lymphoma and bladder cancer (2-4). Although gallium is in clinical use, information regarding its action at the cellular and molecular levels is largely incomplete. It has been known for some time that gallium resembles iron in certain respects. Gallium binds avidly to the iron transport protein Tf 1 (5) and is incorporated into cells by Tf receptor-dependent and -independent transport systems (6 -8). Furthermore, we have shown that gallium inhibits the growth of several leukemic cell lines by inhibiting cellular iron uptake and by blocking the activity of ribonucleotide reductase (9 -11).Iron taken up by cells is stored in ferritin, a high molecular weight protein composed of 24 subunits of H and L chains (12). Ferritin sequesters excess intracellular iron and protects cells from the toxicity of iron overload. An increase in the delivery of iron to cells therefore serves as a powerful stimulus for ferritin synthesis. It is now established that iron-dependent ferritin and transferrin receptor gene expression is regulated by the binding of two iron regulatory proteins (IRPs), IRP-1 and IRP-2, to sequences termed iron-responsive elements (IREs) present at the 5Ј-untranslated region of ferritin mRNA and the 3Ј-untranslated region of Tf receptor mRNA (13-17). In irondepleted cells, IRPs bind with high affinity to the IREs, resulting in suppression of ferritin mRNA translation and stabilization of Tf receptor mRNA (incr...
Gallium, a metal with antineoplastic activity, binds transferrin (Tf) and enters tumor cells via Tf receptor1 (TfR1); it disrupts iron homeostasis leading to cell death. We hypothesized that TfR1 on brain microvascular endothelial cells (BMEC) would facilitate Tf-Ga transport into the brain enabling it to target TfR-bearing glioblastoma. We show that U-87 MG and D54 glioblastoma cell lines and multiple glioblastoma stem cell (GSC) lines express TfRs, and that their growth is inhibited by gallium maltolate (GaM) After 24 hours of incubation with GaM, cells displayed a loss of mitochondrial reserve capacity followed by a dose-dependent decrease in oxygen consumption and a decrease in the activity of the iron-dependent M2 subunit of ribonucleotide reductase (RRM2). IHC staining of rat and human tumor-bearing brains showed that glioblastoma, but not normal glial cells, expressed TfR1 and RRM2, and that glioblastoma expressed greater levels of H- and L-ferritin than normal brain. In an orthotopic U-87 MG glioblastoma xenograft rat model, GaM retarded the growth of brain tumors relative to untreated control ( = 0.0159) and reduced tumor mitotic figures ( = 0.045). Tumors in GaM-treated animals displayed an upregulation of TfR1 expression relative to control animals, thus indicating that gallium produced tumor iron deprivation. GaM also inhibited iron uptake and upregulated TfR1 expression in U-87 MG and D54 cells We conclude that GaM enters the brain via TfR1 on BMECs and targets iron metabolism in glioblastoma thus inhibiting tumor growth. Further development of novel gallium compounds for brain tumor treatment is warranted. .
Recent studies showed that gallium and iron uptake are decreased in gallium-resistant (R) CCRF-CEM cells; however, the mechanisms involved were not fully elucidated. In the present study, we compared the cellular uptake of 59Fe-transferrin (Tf) and59Fe-pyridoxal isonicotinoyl hydrazone (PIH) to determine whether the decrease in iron uptake by R cells is caused by changes in Tf receptor (TfR)-dependent or TfR-independent iron uptake. We found that both 59Fe-Tf and 59Fe-PIH uptake were decreased in R cells. The uptake of 59Fe-Tf but not59Fe-PIH could be blocked by an anti-TfR monoclonal antibody. After 59Fe-Tf uptake, R cells released greater amounts of 59Fe than gallium-sensitive (S) cells. However, after 59Fe-PIH uptake 59Fe release from S and R cells was similar. 125I-Tf exocytosis was greater in R cells. At confluency, S and R cells expressed equivalent amounts of TfR; however, at 24 and 48 hours in culture, TfR expression was lower in R cells. Our study suggests that the decrease in Tf-Fe uptake by R cells is caused by a combination of enhanced iron efflux from cells and decreased TfR-mediated iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells, both uptake systems may be controlled at some level by similar regulatory signal(s).
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