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NEOPLASIA
From the Department of Microbiology and Immunology,
Division of Hematology and Oncology, Sylvester Cancer Center,
University of Miami School of Medicine, Florida; and the H. Lee Moffitt
Cancer Center and Research Institute, University of South Florida
College of Medicine, Tampa, FL.
Multiple myeloma (MM) is a clonal B-cell malignancy characterized
by slow-growing plasma cells in the bone marrow (BM). Patients with MM
typically respond to initial chemotherapies; however, essentially all
progress to a chemoresistant state. Factors that contribute to the
chemorefractory phenotype include modulation of free radical
scavenging, increased expression of drug efflux pumps, and changes in
gene expression that allow escape from apoptotic signaling. Recent data
indicate that arsenic trioxide (As2O3) induces
remission of refractory acute promyelocytic leukemia and apoptosis of
cell lines overexpressing Bcl-2 family members; therefore, it was
hypothesized that chemorefractory MM cells would be sensitive to
As2O3. As2O3 induced
apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and
U266/Bcl-xL. The addition of interleukin-6 had no effect on
cell death. Glutathione (GSH) has been implicated as an inhibitor of
As2O3-induced cell death either through
conjugating As2O3 or by sequestering reactive
oxygen induced by As2O3. Consistent with this
possibility, increasing GSH levels with N-acetylcysteine attenuated
As2O3 cytotoxicity. Decreases in GSH have been
associated with ascorbic acid (AA) metabolism. Clinically relevant
doses of AA decreased GSH levels and potentiated
As2O3-mediated cell death of all 4 MM cell
lines. Similar results were obtained in freshly isolated human MM
cells. In contrast, normal BM cells displayed little sensitivity to
As2O3 alone or in combination with AA.
Together, these data suggest that As2O3 and AA
may be effective antineoplastic agents in refractory MM and that AA
might be a useful adjuvant in GSH-sensitive therapies.
(Blood. 2001;98:805-813) Multiple myeloma (MM) is an incurable B-cell
malignancy characterized by infiltrating, slow-growing plasma cells in
the bone marrow (BM) that produce monoclonal immunoglobulin molecules
(reviewed in Hallek et al1). MM accounts for 1% of all
cancers and slightly more than 10% of all hematologic cancers, making
it more common than Hodgkin disease and acute leukemia.2
Current therapies for MM include alkylating agents (melphalan) and
steroids (prednisone, dexamethasone) alone or combined with other
antineoplastic agents, including plant alkaloids (vincristine,
vinblastine) and anthracyclines (Adriamycin).2 High-dose
chemotherapy with autologous or allogeneic stem cell transplantation
has resulted in some improvement in survival rates,2 and a
number of newer therapeutics are under investigation, including
thalidomide.3 However, MM has an invariably aggressive
course; essentially all (more than 90%) patients progress to a
chemoresistant or refractory state. Even with treatment, the median
5-year survival rate for patients with MM is less than 25%.2 Thus, it is clear that new therapeutics are needed
for the treatment of refractory MM.
At least 4 distinct mechanisms contribute to the acquisition of the
chemoresistant phenotype in MM that must be overcome or circumvented to
effectively treat refractory disease.4,5 Cellular response
to steroids typically depends on the expression of the glucocorticoid
receptor, and resistance to steroid therapies is classically associated
with down-regulation or loss of glucocorticoid receptor expression in
malignant plasma cells.6,7 The expression drug efflux
pumps, such as mdr gene product P-glycoprotein (PgP), is
also a common trait of chemoresistant MM cells.8-10 Plasma cells from patients with refractory MM and drug-resistant cell lines
overexpress PgP.8-10 Clinically, however, efforts to
overcome PgP with calcium-channel blockers, such as verapamil, or
cyclosporin analogues have yielded disappointing
results.11,12 In addition to limiting the access of
antineoplastic agents to drug targets, chemoresistant cells have been
selected for the ability to defend against mechanisms of drug action.
It is widely held that many classes of chemotherapeutics, including
those used in the treatment of MM, converge on apoptotic pathways to
kill tumor cells. Concordant with this mode of action, changes in
cellular apoptotic threshold are associated with chemoresistance or
advanced disease in a number of solid tumors, leukemias, and
lymphomas.13 Recently, it was reported that expression of
the antiapoptotic protein Bcl-xL is higher in
chemoresistant MM cell lines and in samples from patients with
refractory MM.14 In addition, it is well established that interleukin-6 (IL-6), either in a paracrine or an autocrine fashion, plays an essential role in the malignant progression of MM by regulating the growth and survival of MM cells.15,16
Lastly, chemoresistant MM cells have been reported to metabolize
antineoplastic drugs more efficiently than drug-sensitive
cells.12,17,18 For example, increases in the expression or
activity of glutathione (GSH) and GSH-related enzymes, respectively, in
MM cells confers resistance to alkylating agents such as
melphalan.17,18 Effective treatments for chemorefractory
MM must overcome or bypass the expression of drug efflux pumps,
proliferative and survival signals of IL-6, elevated levels of
antiapoptotic proteins such as Bcl-xL, and increases in
glutathione expression.
In response to recent data that patients with chemorefractory acute
promyelocytic leukemia respond to arsenic trioxide
(As2O3) therapy,19 studies have
been initiated to evaluate the therapeutic potential of
As2O3 in other diseases.20-24
Mechanistic studies of As2O3 action in leukemia
and lymphoid cell lines suggest that antineoplastic activity overcomes
Bcl-2 or Bcl-xL expression.20,22,24,25 Furthermore, it is unlikely that unconjugated
As2O3 would be a substrate for drug efflux
pumps. Thus, As2O3 may be a promising antineoplastic for MM, even in PgP-positive plasma cells with increased
Bcl-xL expression levels. However, the primary determinant of cellular sensitivity to As2O3 appears to be
intracellular GSH levels,23,26-30 and elevated GSH levels
have been associated with the MM chemoresistant
phenotype.12,17,18 Therefore, optimal therapies for
chemoresistant MM should also overcome or bypass increases in GSH
expression. Compounds that decrease intracellular GSH levels are likely
to increase the sensitivity of chemorefractory MM cells to
As2O3. Despite the therapeutic potential of
available GSH-depleting compounds such as ethacrynic acid and
buthionine sulfoximine, toxicities limit widespread
use.31-35 Therefore, new agents that can safely lower GSH
levels must be investigated; one promising candidate is ascorbic acid
(AA), widely heralded as an antioxidant.36 However,
evidence is mounting that AA can also act as an oxidizing agent,
particularly in the presence of compounds that increase the production
of reactive oxygen species (ROS).37,38 The pro-oxidant
effects of AA and the potentiation of cell death induced by free
radicals appear to involve the production of hydrogen peroxide
(H2O2).29,37,38
These data led us to hypothesize that the combination of
As2O3 and AA is likely to be an effective
therapy for chemorefractory MM. Consistent with the finding that GSH
levels dictate As2O3 sensitivity,23,26-30 we report that N-acetylcysteine (NAC)
attenuated As2O3-mediated cell death of MM cell
lines. Using in vitro models of chemosensitive and chemorefractory MM,
we demonstrate that AA depleted intracellular GSH, increased hydrogen
peroxide production, and potentiated As2O3
cytotoxicity. Significantly, AA also increased As2O3-induced cell death of plasma cells
isolated from the bone marrow of patients with MM with little toxicity
in nonplasma cell populations.
Reagents and cell lines
Annexin V-fluorescein isothiocyanate and propidium
iodide staining
Intracellular glutathione measurements Intracellular GSH levels were determined in cell lines and in mononuclear cells from BM aspirates. BM aspirates were obtained from consenting patients with MM, and mononuclear cells were isolated by purification over a Ficoll gradient. BM aspirate (approximately 15 mL) was diluted 1:1 with prewarmed (37°C) PBS and overlaid onto 15 mL prewarmed Ficoll-Hypaque (Amersham, Uppsala, Sweden). After centrifugation at 2000 rpm for 20 minutes at room temperature, the mononuclear cells (including plasma cells) were removed, washed again with PBS, counted on a hemacytometer, and plated. Cells (4 × 106) were treated for 48 hours with AA (100 µM), and intracellular GSH was measured using the Glutathione Assay Kit (Calbiochem, San Diego, CA). Briefly, cells were pelleted, resuspended in 500 µL ice-cold 5% metaphosphoric acid, and homogenized with a Teflon pestle (Wheaton, Millville, NJ) and overhead stirrer. After centrifugation at 4°C and 3000g for 10 minutes, 200 µL supernatant, 700 µL buffer, 50 µL solution R1, and 50 µL solution R2 were combined according to the manufacturer's instructions. Samples were incubated at room temperature for 10 minutes in the dark, and final absorbance was measured at 400 nm and compared to a GSH standard curve. To quantitate total protein, the pellet from the above centrifugation was resuspended in 1 N NaOH, and the protein concentration was measured using the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA). Intracellular GSH was normalized to total protein content.Determination of H2O2 production H2O2 production was measured using H2DCFDA (Molecular Probes, Eugene, OR), an H2O2-sensitive fluorescent dye. Briefly, cells (2 × 105) were incubated in the presence or absence of AA (100 µM) for 24 hours. In the last 30 minutes of incubation time, H2DCFDA (0.5 µM) was added to the cell cultures. Cells were washed in PBS, resuspended in FACS buffer (PBS with 1% BSA and 0.01% sodium azide), and analyzed by FACScan flow cytometry.Determination of superoxide production Superoxide production was determined by measuring the conversion of hydroethidine (Molecular Probes) to ethidium. Briefly, cells (2 × 105) were incubated in the presence or absence of As2O3 (2 µM) with or without AA (100 µM) for 24 hours. Cells were washed in PBS, resuspended in 1 mL PBS, and placed on ice. Hydroethidine stock solution (2 mM) was diluted in PBS (25 µL hydroethidine stock into 5 mL PBS) and added (250 µL) to each sample. Samples were immediately vortexed and incubated at 37°C in the dark for 20 minutes. After incubation, cells were stored on ice, protected from light, and analyzed by flow cytometry within 10 minutes.Flow cytometric analysis of mitochondrial membrane potential) Mitochondrial membrane potential was measured using the cationic, lipophilic dye tetramethylrhodamine ethyl ester (TMRE; Molecular Probes). Cells (2 × 105) were incubated in the presence or absence of As2O3 (2 µM) ± AA (100 µM) for 48 hours. Cells were washed in PBS, resuspended in FACS buffer supplemented with TMRE (150 nM), and incubated in the dark at 37°C for 20 minutes. Cells were then washed with PBS supplemented with TMRE (15 nM) and resuspended in FACS buffer supplemented with TMRE (15 nM). Samples were analyzed by flow cytometry.Plasma cell isolation and cell death analysis Bone marrow aspirates were obtained by consent from patients with chemorefractory and newly diagnosed MM. Mononuclear cells (including plasma cells) were isolated from BM biopsies by purification over a Ficoll gradient. BM aspirates (approximately 15 mL) were diluted 1:1 with prewarmed (37°C) PBS and overlaid onto 15 mL prewarmed Ficoll-Hypaque (Amersham). After centrifugation at 2000 rpm for 20 minutes at room temperature, mononuclear cells were removed, washed again with PBS, counted on a hemacytometer, and plated. Cells (4 × 105) were cultured in the presence or absence of As2O3 (2 µM) with or without AA (100 µM) or etoposide (10 µg/mL). After 48 hours, cells were triple stained with a phycoerythrin-conjugated mouse anti-human CD38 antibody (Becton Dickinson), a CyChrome-conjugated mouse anti-human CD45 antibody (Becton Dickinson), and FITC-conjugated Annexin V (Biovision). Controls included unstained cells, control mouse immunoglobulin G (IgG)-stained cells, and single-stained cells. Malignant plasma cells are defined as cells that express high levels of CD38 and no or low levels of CD45 (CD38+/CD45 ).40 Nonplasma cells
were defined as cells with low or no CD38 expression
(CD38). Apoptosis was determined by FACScan analysis
using Cellquest software.
Statistical analyses Data are presented graphically as the mean ± standard deviation (SD) or ± mean standard error (SE). Treatment groups were compared by independent t test or paired t test as appropriate, with P reported in each figure legend. Statistical analyses were performed using Sigmaplot software (Jandel Scientific, Chicago, IL).
To determine whether As2O3 has therapeutic
potential in MM, we first investigated the ability of
As2O3 to induce apoptosis in the human MM cell
lines, 8226/S, 8226/Dox40, and U266. The 8226/S cell line is an
IL-6-independent MM line that is sensitive to chemotherapy-induced
apoptosis.10 Selection of 8226/S cells in doxorubicin
resulted in the generation of the 8226/Dox40 line; this cell line
overexpresses both the drug efflux pump, PgP,10 and the
antiapoptotic protein, Bcl-xL.14,39 The
8226/Dox40 cells maintain doxorubicin resistance in the absence of
doxorubicin-containing medium and display a multidrug resistance
phenotype because they are cross-resistant to etoposide (Figure
1A). U266 cells are
dependent on an autocrine IL-6 loop and express high levels of
Bcl-xL compared to the 8226/S cells.39
Cells were treated with increasing concentrations of As2O3, and viability was monitored by Annexin V-PI staining after 48 hours. As seen in Figure 1B, all 3 cell lines were sensitive to As2O3 in a dose-dependent fashion. Published reports indicate that plasma levels of As2O3 peak at 5 to 7 µM and plateau at 1 to 2 µM.19,41 Therefore, the As2O3 cytotoxicity detected between 1 µM and 10 µM occurred at therapeutically achievable doses. Based on the MM cell dose-response curves and the concentration of As2O3 achievable in vivo, we performed all subsequent experiments in this report at a 2 µM As2O3 concentration. IL-6 is the major survival factor for malignant plasma
cells.15,16 For example, IL-6 has been shown to protect
malignant plasma cells from apoptosis from different stimuli, including dexamethasone, CD95/Fas, serum starvation, and
Because 8226/Dox40 cells display multiple mechanisms of chemoresistance
(PgP expression and elevated Bcl-xL levels) and because Bcl-xL expression is emerging as a potentially important
prognostic indicator of chemorefractory MM,14,45 we next
formally determined whether elevated Bcl-xL levels affect
As2O3 sensitivity. First, U266 cells stably
expressing exogenous Bcl-xL (Oshiro et al, manuscript submitted) were analyzed for resistance to different classes of apoptosis-inducing agents and cell death monitored by Annexin V and PI
exclusion. Although there was no difference in the viability of control
cells (U266, 87.2% ± 6.6%; U266/Bcl-xL,
92.3% ± 6.3%), U266/Bcl-xL cells were less sensitive
than U266 cells after etoposide, staurosporine, or Taxol treatment
(P < .001; Figure 2A).
U266/Bcl-xL cells were treated with
As2O3 (2 µM) and assayed for cell viability at the indicated times (Figure 2B). U266/Bcl-xL cells were
initially resistant to As2O3 (73.3% ± 4.3%
viable at 48 hours); however, Bcl-xL could only delay death
induced by As2O3 because the cells were less
than 35% viable at 96 hours. From these studies, we concluded that
As2O3 can overcome or bypass IL-6 signaling,
the drug efflux pump PgP, and elevated Bcl-xL expression in
MM cells.
Consistent with our in vitro data that only approximately 75% cell
death is achieved at a 10 µM dose of As2O3
(Figure 1B), clinical studies have recently shown modest responses with
As2O3 alone in patients with MM.46
Therefore, to optimize MM cell sensitivity to
As2O3, we next initiated studies to determine
the mechanism of As2O3 action in MM cells. In
other cell types, sensitivity to As2O3
correlates with intracellular GSH levels.23,24-30 To determine whether increases in GSH levels affect the sensitivity of MM
cells to As2O3, we measured the ability of NAC
to block As2O3-induced apoptosis. NAC is an
antioxidant that functions by donating a cysteine to the de novo
synthesis of GSH. Consistent with a crucial role for GSH in determining
cellular sensitivity to As2O3, NAC abrogated
As2O3-mediated cell death (Figure
3).
It follows that if increased GSH synthesis attenuated As2O3-induced cell death, compounds that decrease MM cellular GSH levels are likely to potentiate As2O3-mediated apoptosis. In addition to its well-documented antioxidant effects,36 AA has been shown to have pro-oxidant properties.37,38 Auto-oxidation of AA to dehydroascorbate can result in the production of H2O2.38,47,48 Dehydroascorbate is then rapidly reduced back to AA by glutaredoxin in a GSH-dependent manner. This reduction of dehydroascorbate to AA results in a decrease of reduced intracellular GSH.38,47,48 Because AA is well tolerated in vivo,49 it is possible that the combination of AA and As2O3 would be a more efficient therapy in chemoresistant MM than As2O3 alone. Therefore, we tested the ability of AA to deplete GSH and to augment As2O3 cytotoxicity in MM cell lines. First, we determined whether AA treatment depletes GSH in MM cell
lines. As seen in Figure 4A, incubation
with AA at a concentration achievable in vivo with a vitamin C
supplement (100 µM)49 results in the diminution (more
than 2-fold) of GSH levels in all 4 cell lines. Consistent with its
proposed pro-oxidant activity, the AA-mediated decrease in
intracellular GSH levels correlated functionally with an increase in
H2O2 production (Figure 4B). However, AA alone had no effect on cell viability (Figure 4B), suggesting that AA does
not produce a sufficient level of H2O2 to
initiate oxidative damage. Rather, AA treatment increases basal levels
of cellular H2O2.
The depletion of GSH and the subsequent elevation of basal
H2O2 by AA should render the cell more
susceptible to damage from compounds that are metabolized by GSH
conjugation. Additionally, AA-mediated decreases in GSH could impair
the ability of the cell to recover from oxidative insults. To determine
whether AA-mediated decreases in GSH are sufficient to increase MM cell
sensitivity to As2O3, MM cells were treated
with As2O3 in the presence or absence of AA,
and cell viability was measured by flow cytometry. The 8226/S,
8226/Dox40, and U266 cell lines were analyzed after 48-hour incubation;
the U266/Bcl-xL cells were assayed after 72 hours of
treatment. As2O3 alone induced cell death
(between 30% and 60%) in each cell line, as indicated in Figure
5. However, the combination of AA and
As2O3 resulted in a significant increase in
apoptosis compared to As2O3 treatment alone
(approximately 30% increase) in all 4 cell lines (Figure 5).
Taken together, results of the NAC and AA studies are consistent with those from a model in which GSH is a critical determinant of As2O3 sensitivity in MM cells. There is evidence that GSH can conjugate arsenicals, resulting in a complex that is easily excreted by the MRP2/cMOAT transporter.28,50 Therefore, depletion of GSH levels could decrease As2O3 efflux, thereby causing an intracellular accumulation of As2O3 and increasing cytotoxicity. However, because neither 8226/S nor 8226/Dox40 cells express MRP2/cMOAT,51-55 it is unlikely that GSH-dependent efflux mechanisms underlie the AA-mediated increase in cell death observed in MM cells. Alternatively, GSH depletion and consequent elevation in basal
H2O2 levels could render the cell more
sensitive to oxidative damage. Because AA potentiated
As2O3-mediated cell death, it is possible that
As2O3 treatment increased ROS production. To
directly assess the effects of As2O3 on ROS
production, we used flow cytometric techniques to measure superoxide
production in MM cell lines after 24 hours of
As2O3 treatment (Figure
6). The production of superoxide coincided with the initial detection of apoptotic cells (Figure 6). AA
alone had no effect on superoxide production (P > .05), but the combination of AA and As2O3 resulted in
higher levels of superoxide than did As2O3
alone (Figure 6; P < .0003 for 8226/S, 8226/Dox40, U266
cells; P < .0005 for U266/Bcl-xL cells).
Superoxide is predominantly produced in the mitochondria and can result
in free radical attack of membrane phospholipids, leading to a loss of
mitochondrial membrane potential. Therefore, we next determined whether
mitochondrial membrane integrity was affected by
As2O3 and whether AA could augment an
As2O3-mediated loss of integrity. Using flow
cytometric techniques, we measured the ability of MM cell mitochondria
to maintain a membrane potential in the presence and absence of
As2O3. As2O3 treatment
resulted in a depolarization of the inner mitochondrial membrane
(Figure 7). AA alone had no effect on
mitochondrial membrane potential, but the combination of
As2O3 and AA resulted in a greater percentage
of cells displaying decreased mitochondrial membrane potential than
with As2O3 alone (Figure 7). These findings are
consistent with As2O3 exerting cytotoxic
effects through the generation of ROS, resulting in free radical
cellular damage.
Based on our finding that As2O3 and AA induced
apoptosis in drug-sensitive and drug-resistant MM cell lines, we
expanded our studies to test the cytotoxicity of
As2O3 and the combination of
As2O3 and AA in samples from patients with MM.
First, to determine whether AA can deplete GSH in freshly isolated
cells, mononuclear cells (including plasma cells) isolated from BM
aspirates from 2 patients were incubated in the presence or absence of
a clinically achievable concentration of AA (100 µM) for 48 hours.
Intracellular GSH levels were measured as described in "Materials and
methods." Because of the small number of total cells isolated from
the BM aspirates, it was not possible to sort plasma cells
(CD38+/CD45
Additionally, viability studies were performed with mononuclear cells isolated by consent from patients with newly diagnosed (n = 9) and chemorefractory (n = 14) MM. Mononuclear cells from BM aspirates were isolated as described in "Materials and methods." Cells were cultured in the presence or absence of As2O3 with or without AA or etoposide for 48 hours and were stained with anti-CD38 and anti-CD45 antibodies and with Annexin V, and the viability of triple-stained cells was monitored by flow cytometry. Etoposide was included in these studies for 2 reasons. First, it allowed for a comparison to an apoptosis-inducing agent sensitive to drug efflux pumps. Second, because patients with chemorefractory MM were not treated with the same therapeutic protocols, we wanted to use an agent that no patient had been treated with to eliminate any specific drug resistance that might confound our analyses. These studies revealed that As2O3 selectively
induced apoptosis of the plasma cell population
(CD38+CD45
MM cells ultimately acquire a chemoresistant phenotype that includes expression of drug efflux pumps, changes in apoptotic threshold, and increased ability to detoxify or metabolize chemotherapeutic agents. Effective therapies for refractory MM must be able to overcome or bypass these adaptive cellular changes. In this report we show that As2O3 is not sensitive to drug efflux pump mechanisms of resistance in MM. The PgP-positive cell line 8226/Dox40 undergoes apoptosis in response to As2O3 in vitro, similar to the drug-sensitive cell line 8226/S (Figure 1B). IL-6 is unable to attenuate As2O3-induced cell death (Figure 1C). Similarly, increased expression of the antiapoptotic protein Bcl-xL does not confer resistance to As2O3 because MM cells that express exogenous Bcl-xL are sensitive to As2O3 (Figure 2B). These data are consistent with those from recent studies demonstrating that As2O3 induces apoptosis in leukemic cell lines that overexpress Bcl-2 or Bcl-xL.20,22,24,25 Others have shown that As2O3 overcomes Bcl-2 by down-regulating gene expression22,25; however, we did not detect any changes in Bcl-xL expression after As2O3 treatment (J.M.G., N.J.B., and L.H.B., unpublished observation, March 2000). Furthermore, As2O3 treatment induced apoptosis in plasma cells from patients for whom repeated rounds of VAD and melphalan-prednisone therapy failed (Figure 9B). These data indicate that As2O3 is likely to overcome drug efflux mechanisms and the antiapoptotic effects of IL-6 and Bcl-xL in chemorefractory MM. Our data indicate that ROS production is a critical contributor to As2O3-induced MM cell death. First, As2O3-mediated cell death was abrogated in the presence of NAC (Figure 3). It is relevant to note that As2O3 has been reported to bind to vicinal thiol groups56 and that NAC contains 2 such thiol groups. However, the protective effects of NAC are likely due to increases in GSH levels and not to NAC sequestering of As2O3 because the addition of dithiothreitol, at a concentration 5 times higher than that of NAC used in these studies (50 µM dithiothreitol versus 10 µM NAC), could only partially protect against As2O3-induced cell death in MM cells (J.M.G., N.J.B., and L.H.B., unpublished observation, November 1999). Additional evidence supporting a role for oxidative pathways in As2O3-mediated cell death stems from the relation between AA, GSH, and As2O3 cytotoxicity. AA has been shown to decrease GSH levels and to potentiate As2O3-mediated cell death in lymphoid and myeloid cells.29 The GSH redox system is known to affect the activity of arsenicals.23,26-30 First, As2O3 sensitivity correlates with intracellular GSH levels in other model systems.23 Cells expressing higher levels of GSH23,27 or GSH-associated enzymes26,27 are less sensitive to As2O3 than cells expressing lower levels of these molecules.27,57-59 Moreover, cells with increased GSH levels can be sensitized to As2O3 by agents that deplete intracellular GSH, such as buthionine sulfoximine or ethacrynic acid.29 GSH exerts antioxidant effects by a variety of mechanisms. It can conjugate and thereby inactivate molecules that generate free radicals. In a cell-free system, trivalent arsenic can complex with GSH, forming a transient As(GS)3 molecule.30 Additionally, GSH conjugation can render a molecule more easily excreted through drug efflux pumps.28 Kala et al28 recently demonstrated that biliary excretion of sodium arsenite requires GSH and the MRP2/cMOAT transporter. However, because MRP2/cMOAT expression is not detected in myeloma cells, including 8226/S and 8226/Dox40,51-55 and because primary MM samples express comparable levels of MRP as normal plasma cells,60,61 this mechanism is not likely to be relevant in MM cells. It is important to note that though our studies indicate that a GSH-dependent efflux of As2O3 or its metabolites is unlikely in MM cells, the possibility remains that GSH conjugation is involved in regulating As2O3 sensitivity. GSH can also become oxidized and provide electrons for enzymes such as GSH peroxidase, which reduce H2O2 to H2O. Clearly, the direct conjugation of As2O3 and the inhibition of As2O3-induced ROS production through GSH peroxidase are not mutually exclusive mechanisms because both can contribute to As2O3 detoxification. The relevant mode(s) of action for GSH inhibition of As2O3-induced MM cell death remains to be determined. The precise determinants of whether AA exerts pro- or antioxidant effects are unclear, but they appear to be temporal.38 Cotreatment of AS52 cells with AA (50 µM) and RGS resulted in increased cell death compared to treatment with RGS alone.38 However, when AS52 cells were pretreated for 24 hours with AA and then challenged with RGS, the cells were protected. Therefore, the timing of AA treatment may have an impact on whether AA acts to potentiate or dampen cytotoxicity from oxidative damage.38 Data from our studies with MM cell lines led us to postulate that the combination of As2O3 and AA might be a feasible therapy for patients with refractory and plateau MM. To begin to address this hypothesis, we expanded our in vitro work to include studies using plasma cells from patients with newly diagnosed and refractory MM. Similar to what was determined with the MM cell lines, AA treatment depleted GSH levels in mononuclear cells from the BM of patients with MM. Freshly isolated MM plasma cells were sensitive to As2O3, and AA potentiated the cytotoxicity in refractory patient samples; nonplasma cell populations were relatively unaffected (Figure 9A-B). MM cultures contained a mixed population of mononuclear cells, including BM stromal cells (BMSCs), and the presence of BMSCs in the MM patient cultures might have affected the therapeutic efficacy of As2O3. Interaction with BMSCs has been reported to confer cytoprotection against certain antineoplastic agents and to provide key mitogenic and antiapoptotic growth factors and cytokines.62,63 Additionally, cell adhesion-mediated drug resistance is emerging as a potentially BMSC-related mechanism whereby malignant cells may escape apoptotic signals and allow for the development of more classical drug resistance phenotypes (efflux pumps and so on).64 Taken together, our data with MM cell lines and in MM patient samples suggest that the combination of As2O3 and AA is a strong candidate as a therapy for MM. To determine the safety and efficacy of the combination therapy in patients with refractory and plateau MM, a National Cancer Institute-sponsored phase I/II clinical trial has been opened at the University of Miami. Because most patients with MM will ultimately progress to a refractory state and succumb to their cancer, it is critical to identify new therapeutics capable of overcoming or circumventing chemoresistance. As2O3 is emerging as an effective agent in the treatment of refractory acute promyelocytic leukemia.19,41,65 Significantly, the safety and efficacy of As2O3 in the treatment of refractory acute promyelocytic leukemia has been demonstrated.19,41,65 The AA and As2O3 concentrations used in this report are well within clinically achievable serum levels and are reportedly well tolerated independently.19,41,49,65 In summary, As2O3 is an exciting new addition to the antineoplastic arsenal and warrants serious investigation as a therapeutic for MM. The mechanism of As2O3-mediated cell death appears to be multifactorial and cell specific.20-22,24,25,56,66 Our studies support a role for GSH and ROS production as critical mediators of As2O3-induced cell death in MM cells. The addition of AA to As2O3-based and other GSH-sensitive regimes can potentially optimize the cytotoxic effects of specific chemotherapeutics with little toxicity.
We thank Kapil Bhalla, Kelvin Lee, Enrique Cepero, and Bryan Johnson for their insight and their helpful comments.
Submitted November 22, 2000; accepted March 26, 2001.
Supported in part by National Institutes of Health grant R29CA77837 (L.H.B.), a seed grant from the Viral Oncology Program of the Sylvester Cancer Center (L.H.B.), an ASCO Young Investigator Award (N.J.B.), and an AICR postdoctoral fellowship (J.M.G.).
J.M.G. and N.J.B. contributed equally to this article.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Lawrence Boise, Department of Microbiology and Immunology, University of Miami School of Medicine, PO Box 016960 (R-138), Miami, FL 33101; e-mail: lboise{at}med.miami.edu.
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Y. Dai, T. H. Landowski, S. T. Rosen, P. Dent, and S. Grant Combined treatment with the checkpoint abrogator UCN-01 and MEK1/2 inhibitors potently induces apoptosis in drug-sensitive and -resistant myeloma cells through an IL-6-independent mechanism Blood, October 16, 2002; 100(9): 3333 - 3343. [Abstract] [Full Text] [PDF]
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W. H. Miller Jr., H. M. Schipper, J. S. Lee, J. Singer, and S. Waxman Mechanisms of Action of Arsenic Trioxide Cancer Res., July 15, 2002; 62(14): 3893 - 3903. [Abstract] [Full Text] [PDF]
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 W. H. Miller Jr. Molecular Targets of Arsenic Trioxide in Malignant Cells Oncologist, April 1, 2002; 7(90001): 14 - 19. [Abstract] [Full Text] [PDF]
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M. A. Hussein Nontraditional Cytotoxic Therapies for Relapsed/Refractory Multiple Myeloma Oncologist, April 1, 2002; 7(90001): 20 - 29. [Abstract] [Full Text] [PDF]
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R. B. Gartenhaus, S. N. Prachand, M. Paniaqua, Y. Li, and L. I. Gordon Arsenic Trioxide Cytotoxicity in Steroid and Chemotherapy-resistant Myeloma Cell Lines: Enhancement of Apoptosis by Manipulation of Cellular Redox State Clin. Cancer Res., February 1, 2002; 8(2): 566 - 572. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) or the presence of etoposide (10 µg/mL, 
) or doxorubicin (200 nM, 
) for 48 hours. Viability was assessed as described in
"Materials and methods." Data are presented as the mean ± SD
of at least 3 experiments. *Means for the treatment groups were
significantly lower than for the control group
(P < .0001). (B) Cells were incubated in the presence or
absence of the indicated concentration of As2O3
(1-100 µM) for 48 hours. Viability was assessed as described in
"Materials and methods." Data are presented as the mean ± SD
of 5 experiments with 8226/S (
) cells and of 3 experiments with U266 cells (
). (C) Cells were treated with (
) or
without IL-6 (
-irradiation.
) or absence (
in an arsenic-resistant Chinese hamster ovary cell line.
Biochem J.
1992;288:977-982.