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[Cancer Research 65, 11676-11688, December 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Motexafin Gadolinium and Zinc Induce Oxidative Stress Responses and Apoptosis in B-Cell Lymphoma Lines

Philip S. Lecane1, Mazen W. Karaman2, Mint Sirisawad1, Louie Naumovski1, Richard A. Miller1, Joseph G. Hacia2 and Darren Magda1

1 Pharmacyclics, Inc., Sunnyvale, California and 2 Institute for Genetic Medicine, University of Southern California, Los Angeles, California

Requests for reprints: Darren Magda, Pharmacyclics, Inc., 995 E. Arques Avenue, Sunnyvale, CA 94085. Phone: 408-774-3318; Fax: 408-328-3689; E-mail: dmagda{at}pcyc.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is an emerging appreciation of the importance of zinc in regulating cancer cell growth and proliferation. Recently, we showed that the anticancer agent motexafin gadolinium (MGd) disrupted zinc metabolism in A549 lung cancer cells, leading, in the presence of exogenous zinc, to cell death. Here, we report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines. We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptosis. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element–binding transcription factor-1 (MTF-1)–regulated and hypoxia-inducible transcription factor-1 (HIF-1)–regulated genes. Cultures cotreated with MGd and zinc acetate displayed further increases in the levels of MTF-1– and HIF-1–regulated transcripts as well as additional transcripts regulated by NF-E2–related transcription factor 2. These data provide insights into the molecular changes that accompany the disruption of intracellular zinc homeostasis and support a role for MGd in treatment of B-cell hematologic malignancies. (Cancer Res 2005; 65(24): 11676-88)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The intracellular abundance of loosely bound or free zinc can have a profound effect on cellular metabolism, survival, and growth. For example, elevated levels of intracellular free zinc have been proposed to inhibit glycolysis via glyceraldehyde phosphate dehydrogenase, the citric acid cycle via the {alpha}-ketoglutarate dehydrogenase complex and mitochondrial respiration (1–3). Free zinc can also inhibit the pentose cycle–dependent enzyme, thioredoxin reductase (4–7). Furthermore, zinc levels have been reported to modulate protein kinase C (8, 9), nuclear factor {kappa}B (10, 11), and p53 (12) activities and signaling pathways relevant to carcinogenesis (13, 14). In light of these effects, it is perhaps not surprising that zinc possesses both antiapoptotic or proapoptotic properties that are dependent on intracellular levels (6, 15).

Motexafin gadolinium (MGd, Xcytrin), an expanded porphyrin containing the lanthanide cation gadolinium, is currently in clinical trials for the treatment of several forms of cancer (16). MGd is an electron-affinic compound that mediates electron transfer from a variety of intracellular reducing species, such as ascorbate, NADPH, and thiols, to oxygen to form superoxide and hydrogen peroxide (17–20). Recently, we generated gene expression profiles of plateau phase A549 lung cancer cell cultures treated with MGd and observed marked induction of transcript levels of genes that play major roles in controlling intracellular free zinc levels (6). We also reported that MGd increased intracellular free zinc levels, modulated the cellular toxicity of zinc, and inhibited cellular bioreductive activity in several human cancer cell lines. Here, we describe effects of treatment with MGd and/or zinc acetate on intracellular levels of free zinc, oxidative stress, proliferation, cell cycle status, and cell death in B-cell lymphoma cell lines. Based on gene expression profiling and other functional analyses, we find that increased levels of oxidative stress and intracellular free zinc lead to the expression of genes under the control of metal response element–binding transcription factor-1 (MTF-1), hypoxia-inducible transcription factor-1 (HIF-1), and NF-E2-related factor 2 (NRF-2), and correlate with cell cycle arrest and apoptotic response. Overall, these studies lead us to suggest that cotreatment of Ramos cells with MGd and zinc acetate increases intracellular free zinc levels with a concomitant increase in oxidative stress levels that activate adaptive survival responses but eventually lead to cell death by disrupting redox balance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Cell Culture Reagents
Ramos, Raji, and DB B-cell lymphoma lines were purchased from American Type Culture Collection (Rockville, MD). DHL-4 and HF-1 lines were obtained from Ronald Levy (Stanford University, Stanford, CA). The HF-1 cell line was derived from a patient with follicular lymphoma (21). DHL-4 was derived from a patient with diffuse large cell lymphoma (22). Unless otherwise indicated, all cell culture reagents were purchased from Invitrogen (Carlsbad, CA). Cells were cultured in a 5% CO2 incubator at 37°C at a density between 0.2 x 106 and 1 x 106 cells/mL as previously described (6). MGd was prepared as a 2 mmol/L (2.3 mg/mL) formulation in 5% aqueous mannitol. Zinc acetate (ZnOAc2) and cobalt acetate (Aldrich Chemical, Milwaukee, WI) were used as 2 mmol/L formulations in 5% aqueous mannitol.

Apoptosis Assays
Annexin V–propidium iodide. Cells from exponential phase cultures were treated with MGd, zinc, or control (5% mannitol) solution for 24 or 48 hours. After incubation, cells were harvested and washed twice with a solution of 0.5% bovine serum albumin (BSA) in HBSS. An aliquot of cells (1 x 106) was added to 500 µL diluted binding buffer from the Annexin V–propidium iodide kit (BD Biosciences, San Jose, CA). Cells were pelleted, resuspended in 100 µL of diluted binding buffer, and treated with the Annexin V–propidium iodide reagent as per the protocol of the manufacturer. Flow cytometry was done on a FACSCalibur instrument and data were analyzed using the CellQuest Pro software package (BD Biosciences).

Mitochondrial membrane potential. Loss of the mitochondrial membrane potential ({Delta}{Psi}m) of cells was measured by the use of JC-1 (Molecular Probes, Inc., Eugene, OR). Cells undergoing early apoptosis lose fluorescence in the 585 nm channel and gain it in the 530 nm channel. Briefly, cells cultured as described above were washed twice with complete medium, resuspended in 0.5 mL JC-1 solution (10 µg/mL in complete medium), and incubated at 37°C for 15 minutes. Cells were isolated by centrifugation, washed once, and then resuspended in 0.5 mL solution of 0.5% BSA in PBS and assayed immediately on the flow cytometer.

Cellular Proliferation
The proliferation of exponential phase cultures was assessed by colorimetric assay. In brief, 2 x 105 suspension cells per well were seeded on 96-well V-bottomed microtiter plates. Stock solutions of control vehicle, MGd, or ZnOAc2 in medium were added and plates were incubated at 37°C under a 5% CO2/95% air atmosphere. After 24 hours, medium was replaced with fresh medium. After 2 additional days, medium was exchanged with fresh medium (150 µL/well) supplemented with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/mL, Sigma Biochemical, St. Louis, MO). Plates were incubated at 37°C and viable cells were measured as described (23).

Intracellular Free Zinc
The concentration of intracellular free zinc was assessed using the ion-specific fluorescent probe, FluoZin-3-AM (FluoZin-3, Molecular Probes; ref. 24). Exponential-phase cultures were treated with control 5% mannitol vehicle or ZnOAc2 in the presence or absence of MGd, as described above, for 4 hours. Following treatment, cells were isolated by centrifugation. Cell pellets were washed and resuspended in a solution of 0.5% BSA in PBS. An aliquot of 106 cells (200 µL) was removed, centrifuged, and treated with FluoZin-3 reaction buffer as described (24). An aliquot of the cell suspension was supplemented with 2 µg/mL propidium iodide (Sigma Biochemical), incubated for 5 minutes, and subjected to two-variable flow cytometric analysis as described previously (6).

Measurement of Reactive Oxygen Species
Reactive oxygen species were measured in live cells as intracellular peroxides by monitoring the oxidation of 2',7'-dichlorofluorescin-diacetate to 2',7'-dichlorofluorescein (Molecular Probes). Cells (1 x 106/mL) were incubated in a solution of 1 µg/mL of 2',7'-dichlorofluorescin-diacetate in 0.5% BSA in HBSS for 15 minutes at 37°C. Two milliliters of additional 0.5% BSA in HBSS were added, cells were isolated by centrifugation, and the pellet was resuspended in a solution of 50 µg/mL of 7-aminoactinomycin D (7-AAD) in 0.5% BSA in HBSS. Cell suspensions were incubated at ambient temperature for 2 to 3 minutes and stored on ice until analysis. The fluorescent intensity in live (i.e., 7-AAD impermeable) cells was analyzed by flow cytometry.

Cell Cycle Analysis
Exponential phase cultures were treated with control 5% mannitol vehicle or ZnOAc2 in the presence or absence of MGd as described above. Thirty minutes before harvest, cultures were treated with 5-bromo-2'-deoxyuridine (BrdUrd) at a final concentration of 10 µmol/L. Cells were isolated by centrifugation, washed once with 0.5% BSA in PBS, and the resulting cell pellets were fixed using 0.5 mL Cytofix/Cytoperm reagent (BD Biosciences). After incubation at ambient temperature for 30 minutes, cells were isolated by centrifugation, washed with 3% fetal bovine serum in PBS, resuspended in 10% DMSO in medium, and stored at –20°C until analysis. Cells were stained using a fluorescein-conjugated anti-BrdUrd antibody (clone PRB1, E-Bioscience, San Diego, CA) and 7-AAD. Cell cycle occupancy was analyzed by flow cytometry using fluorescein signal as a measure of DNA synthesis and 7-AAD signal as a measure of DNA content as described (25). For comparison, cultures were treated with 5-fluoro-2'-deoxyuridine, hydroxyurea, or irradiated using a 137Cs irradiator (Model 40 Gammacell, J.L. Shepherd & Associates, San Fernando, CA).

Hypoxia-inducible Transcription Factor-1 ELISA
Total HIF-1{alpha} protein was detected by sandwich ELISA using the DuoSet IC HIF-1{alpha} ELISA kit obtained from R&D Systems (Minneapolis, MN). All incubations were done at ambient temperature. Briefly, 96-well plates were coated with HIF-1{alpha} capture antibody overnight before blocking with 5% BSA in wash buffer. Protein lysates (50 µg protein per well prepared according to the instructions of the manufacturer) were added for 2 hours, whereupon plates were washed and a biotinylated detection antibody specific for HIF-1{alpha} was added. A streptavidin-horseradish peroxidase format was used for detection. The absorbance at 450 minus 570 nm was measured using a microplate reader (SpectraMax Plus, Molecular Devices, Palo Alto, CA). HIF-1{alpha} concentrations were calculated by linear regression using a standard curve prepared from HIF-1{alpha} standard supplied with the ELISA kit.

Western Blotting
Western blotting was done as described (26, 27). Antibodies against heme oxygenase 1 and metallothioneins 1 and 2 (clone E9) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and DAKO (Glostrup, Denmark), respectively. All membranes were blotted with an anti-Hsc70 (Santa Cruz Biotechnology) antibody to control for loading and transfer. Bands were imaged and quantified in the linear range and normalized to Hsc70 by using the Odyssey Infrared Imaging System (LICOR, Inc., Lincoln, NE).

Gene Expression Profiling
MGd (10 µmol/L), ZnOAc2 (25 or 50 µmol/L), the combinations, or control (5% mannitol) solution was added to Ramos cultures. Each treatment was done in triplicate. After 4 hours of incubation, all cultures were washed twice with 0.5% BSA in HBSS and total RNA was isolated and subjected to analysis on Human Genome U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA) as described (28). Microarray Suite version 5.0 software (Affymetrix) was used to generate raw gene expression scores and normalize the relative hybridization signal from each experiment as described (28). All gene expression scores were set to a minimum value of 50 to minimize noise associated with less robust measurements of rare transcripts. Both the parametric Student's t test and the permutation-based significance analysis of microarrays were used to determine genes differentially expressed in treatment versus control groups (29). We report data from genes that are at least 1.5-fold differentially expressed relative to controls using the Student's t test (P ≤ 0.005) because this empirically proved to be a more stringent criterion than significance analysis of microarray analysis using the same 1.5-fold cutoff and a <1% false discovery rate (data not shown). All scaled fluorescent intensity values and .cel files are available at http://hacialab.usc.edu/supplement/lecane_etal_2005/.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intracellular free zinc is elevated in Ramos cells treated with motexafin gadolinium and zinc. We have previously shown that treatment of Ramos cells with 10 µmol/L MGd and 50 µmol/L zinc led to synergistic increases in the level of free intracellular zinc within 6 hours of treatment as measured using the ion-specific dye FluoZin-3 (6). To further characterize this response and relate it to other cellular phenotypes, we analyzed the kinetics of intracellular free zinc accumulation. Cotreatment of Ramos cells with 10 µmol/L MGd and 50 µmol/L zinc led to a 4-fold increase in median FluoZin-3 fluorescence within 2 hours compared with control cells (Fig. 1A). This signal increased to ~8-fold within 12 hours and remained constant thereafter. Treatment with 10 µmol/L MGd or 50 µmol/L zinc alone led to small increases in FluoZin-3 fluorescence within 2 hours, which returned to baseline levels by 12 hours. As a negative control, we found no increase in cellular fluorescence at 530 nm in the absence of FluoZin-3 (data not shown).



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  		Figure 1. MGd treatment alters levels of intracellular free zinc, oxidative stress, and apoptotic response of Ramos cells to zinc. Exponential phase Ramos cultures were treated with control vehicle (Mannitol), zinc acetate (Zinc, 50 µmol/L), MGd (10 µmol/L), or the combination for up to 24 hours in duplicate experiments. Columns: A, fold increase of FluoZin-3 fluorescence in live-gated cells. B, fold increase of dichlorofluorescein (DCF) fluorescence in live-gated cells. C, percentage of Annexin V–stained cells. D, percentage of live-gated cells exhibiting green (nonaggregated) JC-1 fluorescence characteristic of lost mitochondrial membrane potential. Bars, SD.

 
Motexafin gadolinium and zinc increase oxidative stress in Ramos cells. We measured oxidative stress in Ramos cells treated with MGd and/or zinc over time by monitoring the conversion of dichlorofluorescin acetate to dichlorofluorescein (Fig. 1B). Cultures of Ramos cell treated with both 10 µmol/L MGd and 50 µmol/L zinc displayed 1.8-fold increase in median dichlorofluorescein signal within 2 hours, which gradually diminished over the 24-hour time course of the experiment to background levels. Treatment with MGd or zinc alone also increased dichlorofluorescein signal, albeit not to the same degree as the combination. Treatment of Ramos cells with hydrogen peroxide also increased dichlorofluorescein and FluoZin-3 fluorescence (see Supplementary Data).

Motexafin gadolinium and zinc treatment leads to apoptosis in Ramos cells. To determine the rate at which cotreated Ramos cells undergo cell death, Ramos cultures treated as above were analyzed using FITC-labeled Annexin V reagent to detect early and late apoptotic events (Fig. 1C). In addition, the dye JC-1 was used to assess mitochondrial function (Fig. 1D). In cultures treated with MGd and zinc, 21% of Ramos cells exhibited a positive Annexin V signal within 8 hours of treatment. This fraction increased to 30% within 12 hours and 68% by 24 hours. Analogous results were obtained using JC-1, with 38% of cells exhibiting nonaggregated (green) JC-1 fluorescence characteristic of mitochondrial dysfunction within 8 hours of combined treatment with MGd and zinc. This fraction increased to 52% by 12 hours and 74% by 24 hours. No significant change in Annexin V signal or JC-1 fluorescence was observed within 4 hours or as a result of treatment with MGd or zinc alone.

Motexafin gadolinium and zinc treatment leads to cell cycle arrest in Ramos cells. Next, we sought to examine the kinetics of growth rate responses to cotreatment with MGd and zinc acetate. To do this, Ramos cultures cotreated as above were labeled with BrdUrd and 7-AAD to determine cell cycle occupancy (25). As shown in Fig. 2, cotreatment with MGd and zinc halted BrdUrd incorporation in S-phase cells actively synthesizing DNA within 8 hours. It also led to inhibition of cell entry and progression through G1 and G2-M phases (as determined by DNA content analysis; data not shown). The effects of treatment with 5-fluoro-2'-deoxyuridine, hydroxyurea, and ionizing radiation are shown for comparison.



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  		Figure 2. Treatment of Ramos cells with MGd and zinc inhibits DNA synthesis. Exponential phase Ramos cultures were treated with zinc acetate (50 µmol/L) and MGd (10 µmol/L) for up to 8 hours. BrdUrd (10 µmol/L) was added 30 minutes before harvest. After the indicated time intervals, cells were washed, fixed, and stained with fluorescein-conjugated anti-BrdUrd antibody (Fluorescein) and 7-AAD. Cell cycle occupancy was analyzed by flow cytometry using fluorescein signal as a measure of DNA synthesis and 7-AAD signal as a measure of DNA content. The effect of treatment with control vehicle, hydroxyurea (HU, 250 µmol/L), ionizing radiation (XRT, 10 Gy), and 5-fluoro-2'-deoxyuridine (FUDR, 10 µmol/L) after 8 hours is shown for comparison. Representative of two experiments.

 
Motexafin gadolinium modulation of zinc activity in other lymphoma cell lines. The effect of MGd and zinc on proliferation was examined in Ramos and several other hematologic cell lines (see Supplementary Data). The B-cell lines (Ramos, Raji, DB, DHL-4, and HF-1) seemed to be more sensitive than the Jurkat T-cell line and the myeloid cell lines K562 and HL-60. In all cell lines, sensitivity to zinc was increased by MGd, whereas low concentrations of zinc or MGd alone had no significant effect.

The five B-cell lines were further tested for changes in intracellular free zinc levels and oxidative stress after 4 hours and apoptosis after 24 and 48 hours of treatment with 10 µmol/L MGd and 50 µmol/L zinc acetate (Fig. 3). Increases in FluoZin-3, dichlorofluorescein, and Annexin V–FITC fluorescence relative to control were in the following order: Ramos > DHL-4 > DB > Raji > HF-1. This roughly matches the sensitivity of these lines to treatment with zinc in the proliferation assay. However, there was no significant change in median dichlorofluorescein fluorescence in the DB or HF-1 lines at 4 hours. No changes in FluoZin-3, dichlorofluorescein, or Annexin V–FITC fluorescence were observed in Jurkat, K562, or HL-60 lines under these conditions (data not shown).



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  		Figure 3. MGd treatment alters levels of oxidative stress, intracellular free zinc, and apoptotic response of B-cell lines to zinc. Exponential phase cultures were treated with control vehicle, zinc acetate (50 µmol/L), MGd (10 µmol/L), or the combination in duplicate experiments. Columns: A, fold increase of FluoZin-3 fluorescence in live-gated cells after 4 hours of treatment. B, fold increase of dichlorofluorescein fluorescence in live-gated cells after 4 hours of treatment. C, percentage of Annexin V–stained cells after 24 and 48 hours of treatment.

 
Gene expression profiling of motexafin gadolinium–treated Ramos cells. To assess the effects of MGd or zinc treatment on gene expression profiles, total cellular RNA was isolated from exponential phase Ramos cultures treated in triplicate with control vehicle (5% mannitol), 10 µmol/L MGd, 25 or 50 µmol/L zinc acetate, or the zinc and MGd combinations for 4 hours and analyzed on oligonucleotide microarrays (28). Twenty-nine transcripts (25 of which had annotated functions) showed differential expression in response to MGd treatment that reached our criteria for statistical significance (≥1.5-fold, P ≤0.005; Table 1). The most prominent consequence of MGd treatment was the up-regulation of MTF-1-regulated genes (30–33), including metallothionein and zinc transporter family transcripts, as was observed previously in A549 lung cancer plateau phase cultures (6). The levels of six transcripts were down-regulated, including SLC39A10, which encodes a transporter involved in the uptake of zinc. Interestingly, a splice variant of this transporter was significantly increased, presumably reflecting additional mechanisms operating to regulate levels of intracellular free zinc. In addition, we observed HIF-1-related transcripts displaying significant changes, including DDIT4, EGLN1, and PFKFB3 (Table 1). Similar expression patterns were observed in response to 50 µmol/L zinc.


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  		Table 1. Genes differentially expressed in response to MGd treatment

 
Depending on treatment condition, the numbers of significantly changed transcripts were as follows: 25 µmol/L zinc acetate (3) < MGd alone (29) < MGd + 25 µmol/L zinc acetate (278) ~ 50 µmol/L zinc acetate (347) << MGd + 50 µmol/L zinc acetate (1,226). Whereas only one annotated transcript (SLC39A10) was differentially regulated in response to treatment with 25 µmol/L zinc acetate, 347 transcripts were differentially expressed in response to treatment with the higher concentration of 50 µmol/L zinc acetate (284 up-regulated and 63 down-regulated). A total of 12 of 29 (41%) of the transcripts significantly changed by MGd treatment were also changed (≥1.5-fold in the same direction; P ≤ 0.005) by treatment with 50 µmol/L zinc acetate (Fig. 4A). However, 28 of 29 (97%) MGd-responsive genes were also differentially expressed in the same direction in Ramos cultures treated with 50 µmol/L zinc using less stringent criteria (≥1.2-fold, P ≤ 0.05).



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  		Figure 4. Treatment of Ramos cells with MGd and zinc alters RNA and protein expression. Exponential phase cultures were treated with control vehicle, zinc acetate (25 or 50 µmol/L), MGd (10 µmol/L), or the combination. A, Venn diagram showing relationship between transcripts significantly altered by treatment with 10 µmol/L MGd or 50 µmol/L zinc. B, Venn diagram showing relationship between transcripts significantly altered by treatment with 10 µmol/L MGd and 25 or 50 µmol/L zinc. C, levels of HIF-1{alpha} protein relative to control vehicle after 4 hours treatment as measured by ELISA. Effect of incubation in 2% oxygen atmosphere (Hypoxia) or with cobalt acetate (Cobalt) is shown for comparison. Bars, 1 SD. D, representative Western blots showing levels of heme oxygenase 1 (HO1) and metallothioneins 1 and 2 (MT) after 16 hours treatment. Hsc70 served as a loading control.

 
To simplify presentation and interpretation, a selected group of the 1,226 differentially expressed transcripts in the 10 µmol/L MGd + 50 µmol/L zinc acetate group are shown in Table 2. A total of 64% (178 of 278) of the differentially expressed transcripts in the 10 µmol/L MGd and 25 µmol/L zinc acetate group were shared with the 10 µmol/L MGd and 50 µmol/L zinc acetate group (Fig. 4B). Using less stringent criteria (1.2-fold, P ≤ 0.05), 253 of 278 (91%) of the transcripts differentially expressed in the former group were shared with the latter group. This is especially interesting given that changes in cell viability were observed in the 10 µmol/L MGd and 50 µmol/L zinc acetate group but not in the 10 µmol/L MGd and 25 µmol/L zinc acetate group. Overall, we observed a trend toward larger magnitudes of differential gene expression in response to cotreatment with MGd and zinc relative to individual treatments (Tables 1 and 2).


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  		Table 2. Transcriptional responses of Ramos cells cotreated with MGd and zinc

 
Levels of hypoxia-inducible transcription factor-1{alpha}, metallothioneins, and heme oxygenase-1. We showed that some of the transcriptional changes determined by DNA microarray analysis were also reflected in alterations of protein expression. Cellular levels of HIF-1{alpha} in Ramos cells were measured by ELISA following treatment with MGd and zinc for 4 hours (Fig. 4C). Total cellular HIF-1{alpha} levels were increased 1.5- to 3-fold by treatment with MGd, zinc, or the combination. As expected, HIF-1{alpha} levels were also increased by treatment with cobalt acetate or by the use of hypoxic culture conditions. Levels of metallothioneins and heme oxygenase-1 were shown by Western blot to be increased following cotreatment with MGd and zinc for 16 hours (Fig. 4D). Metallothionein and heme oxygenase-1 are proteins with expression induced by MTF-1 and NRF-2, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MGd is an electron-affinic anticancer agent that is being tested clinically in a variety of settings, including the treatment of B-cell lymphoma. Although the current study is limited by its reliance on cell lines that might not be representative of primary tumor cells, this approach has the advantage of avoiding heterogeneity that can confound analysis of drug activity in patient samples. Thus, we first sought to understand the effect of drug treatment in cultured B-cell lines as a prelude to further studies involving primary cancer cells. As we had previously shown that the cellular activity of 10 µmol/L MGd was enhanced in the presence of 25 and 50 µmol/L exogenous zinc (6), we chose to focus on these zinc concentrations in our studies. They are also relevant given that (a) standard tissue culture conditions are deficient in zinc, having an estimated 3- to 6-fold lower concentration (~4 µmol/L) compared with normal human plasma samples and (b) interstitial fluid zinc concentrations can vary greatly in vivo (1, 34). Within 2 hours of MGd and/or zinc treatment, Ramos cells showed significant increases in intracellular free zinc (Fig. 1A). These levels continued to increase for at least 12 hours and remained high, at least in the group cotreated with MGd and zinc. This could represent a catastrophic loss of zinc homeostasis in these cells due to an overwhelming of cellular stress responses. Four other B-cell lines treated with MGd and zinc displayed increased intracellular free zinc levels after 4 hours (Fig. 3A) albeit to variable degrees.

We also observed substantial increases in levels of reactive oxygen species in Ramos cells within 2 hours of treatment with 10 µmol/L MGd and/or 50 µmol/L zinc (Fig. 1B). However, in contrast to the intracellular free zinc levels described above, reactive oxygen species decreased over the course of the 24-hour treatment. However, these data could be strongly influenced by apoptosis-related events or alterations in cellular metabolism that occur at later times.

In keeping with their differences in intracellular free zinc, four other B-cell lines displayed variable degrees of oxidative stress after 4 hours of cotreatment with MGd and zinc (Fig. 3B). Interestingly, treatment of Ramos cultures with hydrogen peroxide also led to transient increases in oxidative stress and sustained increases in intracellular free zinc (see Supplementary Data). This is consistent with observations that zinc can induce oxidative stress in cultured mammalian cells, and, conversely, that thiol oxidation can mobilize zinc (35–37).

In addition, we sought to relate changes in intracellular free zinc and oxidative stress to cellular growth rate. We observed a large reduction in the number of Ramos cells actively synthesizing DNA in S-phase after treatment with MGd and 50 µmol/L zinc by 4 hours (Fig. 2). Other cell lines tested also displayed decreased DNA synthesis under these conditions (see Supplementary Data). Interestingly, treatment with 50 to 100 µmol/L zinc inhibited the proliferation of four additional B-cell lines, an acute myelogenous leukemia line (K562), and a T-cell lymphoma line (Jurkat), but not in an acute promyelocytic leukemia line (HL60; see Supplementary Data). In all lines except HL60, MGd cotreatment potentiated the inhibition by zinc. The effect of MGd and zinc differed strikingly from that of 5-fluoro-2'-deoxyuridine or ionizing radiation, both of which permitted BrdUrd incorporation into DNA and changed cell cycle distribution with accumulation of cells in G1-S and G2-M, respectively. It also differed from hydroxyurea, which inhibited BrdUrd incorporation but allowed passage through G2M. This suggests that increased intracellular free zinc inhibits proliferation at multiple checkpoints.

Importantly, we observed that the increased oxidative stress and intracellular free zinc levels induced by cotreatments with MGd and zinc preceded mitochondrial dysfunction and early events of apoptosis and thus were not a consequence of them. Furthermore, increased intracellular free zinc and oxidative stress roughly correlate with cell death, with Ramos the most sensitive line, followed by DHL-4 and the others (Fig. 3C). However, intracellular free zinc levels seemed to be better predictors of proliferative and apoptotic response. K562, HL60, and Jurkat lines did not exhibit changes in oxidative stress, intracellular free zinc, or apoptosis under these conditions (data not shown).

To better understand the molecular changes accompanying loss of zinc homeostasis before apoptosis, we examined the effect of 4-hour treatment with MGd and/or zinc on gene expression in Ramos cultures. There was a striking overlap in transcriptional responses to 10 µmol/L MGd or 50 µmol/L zinc (Table 1). Depending on the stringency of our criteria, up to 97% of MGd-responsive genes were also differentially expressed in the same direction in cells treated with 50 µmol/L zinc. This indicates that MGd acts as a "zinc mimetic" with regard to the transcriptional responses induced in Ramos cells.

Treatment with MGd or zinc both resulted in a strong and sustained induction of MTF-1-regulated metallothionein and zinc transporter 1 (ZnT1) genes, which play major roles in regulating intracellular free zinc levels, as well as HIF-1-regulated genes (e.g., PFKB3, DDIT4, and EGLN1; Table 1). PFKFB3 is a kinase/phosphatase that modulates the concentration of fructose-2,6-bisphosphate, a key modulator of the glycolytic rate in proliferating cells (38). DDIT4 is a proapoptotic protein recently reported to be a negative regulator of the mammalian target of rapamycin pathway (39, 40). EGLN1 (also known as PHD2) is a prolyl hydroxylase that plays a key role in regulating HIF-1 activity by targeting HIF-1{alpha} for ubiquitin-mediated degradation (41). Although it is not as effective in this regard as the better known "hypoxia-mimetic" cobalt, zinc can inhibit the activity of HIF-associated hydroxylases by displacing iron from the active site of these enzymes (42, 43). Indeed, we measured greater cellular HIF-1{alpha} levels by ELISA in Ramos cultures treated with either zinc or MGd (Fig. 4C). Thus, we propose that MGd induces hypoxia-mimetic transcriptional responses in this system as a result of HIF-1 stabilization due to increased intracellular free zinc and/or generation of reactive oxygen species.

It is notable that levels of transcripts under the control of MTF-1 (e.g., metallothionein family members) and under the control of HIF-1 (e.g., DDIT4) are increased synergistically in some instances by MGd and zinc treatment (Tables 1 and 2). These changes could contribute to the observed biological effects of the combined treatment. Indeed, the increased activation of HIF-1 would be expected to alter cellular metabolism to favor glycolysis over oxidative phosphorylation via the induction of transcripts, such as PFKFB3 and PGK1 (38). HIF-1 is often considered to be essential for tumor growth and, indeed, its inhibition is the subject of ongoing drug development activities (44). However, under the appropriate conditions, HIF-1 activation can have negative consequences for tumor growth by induction of targets linked to apoptosis, such as BNIP3, E2IG5, PMAIP1, and DDIT4, or through metabolic alteration of cells in the low nutrient context of the tumor microenvironment (45).

In addition to the MTF-1- and HIF-1-regulated transcripts discussed above, cotreatment with MGd and zinc resulted in the expression of NRF-2-regulated transcripts, such as GCLM, HMOX1, and NQO3A2, which all have antioxidant response elements in their promoters (46, 47). Additional transcripts, such as TXNRD1, CTH, GSR, and a variety of transporters (e.g., SLC7A11) presumably involved in cellular uptake of amino acids required for glutathione synthesis, are also induced. The induction of NRF-2 activity may be related to its nuclear translocation following disruption of the cytoplasmic Keap-1–NRF-2 complex. The capacity of Keap-1 to bind NRF-2 is regulated by critical cysteine residues shown to be modified under oxidative stress conditions (48, 49). It has been proposed that induction of thioredoxin reductase (TXNRD1) and increased glutathione levels serves to restore Keap-2 binding of NRF-2 as part of a feedback loop (48). Induction of NRF-2 response genes could therefore reflect the altered redox state of the cells under conditions where this enzyme is inhibited.

Overall, our data indicate that the primary effect of moderately increased free zinc in Ramos cells is the activation of MTF-1 and HIF-1. Induction of free zinc at higher levels increases oxidative stress, leading to the activation of NRF-2. Paradoxically, it is conceivable that the activation of transcriptional cascades under the control of MTF-1, HIF-1, and NRF-2 by MGd could be protective toward reactive oxygen species in some circumstances (i.e., through "adaptive resistance" mechanisms and the induction of "cell life" transcripts; refs. 50, 51). Cells already under oxidative stress, on the other hand, would be expected to display increased sensitivity to MGd as a consequence of preexisting elevated levels of intracellular free zinc, depleted stores of reducing equivalents, and dependence on key antioxidant enzymes, such as thioredoxin reductase (52). If so, this would imply that a considerable degree of selectivity might be achieved in targeting this drug to diseases involving oxidative stress.


   Acknowledgments
 
Grant support: V Foundation for Cancer Research, National Institute of Environmental Health Sciences grant P30-ES07048, and a grant from Pharmacyclics (J.G. Hacia).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 8/ 4/05. Revised 9/14/05. Accepted 9/30/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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