[Cancer Research 64, 8512-8516, December 1, 2004]
© 2004 American Association for Cancer Research
2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl)propionic Acid, a Small Molecule Isocoumarin, Potentiates Dexamethasone-Induced Apoptosis of Human Multiple Myeloma Cells
Naoki Agata1,
Hiroko Nogi2,
Michael Milhollen1,
Surender Kharbanda1 and
Donald Kufe2
1 ILEX Products, Inc., Boston, Massachusetts; and 2 Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
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ABSTRACT
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2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl)propionic acid (NM-3) is a small molecule isocoumarin derivative that has recently entered clinical trials as an orally bioavailable anticancer agent. NM-3 induces lethality of human carcinoma cells by both apoptotic and nonapoptotic mechanisms and potentiates the effects of cytotoxic chemotherapeutic agents. The present studies have evaluated the effects of NM-3 on human multiple myeloma (MM) cells. The results demonstrate that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive MM1.S and dexamethasone-resistant RPMI8226 and U266 MM cells. We show that NM-3 enhances dexamethasone-induced release of the mitochondrial apoptogenic factors cytochrome c and Smac/DIABLO. The results also demonstrate that NM-3 enhances dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 apoptotic pathway. In concert with these results, NM-3 potentiates dexamethasone-induced apoptosis of MM1.S cells. Moreover, NM-3 acts synergistically with dexamethasone in inducing apoptosis of the dexamethasone-resistant RPMI8226 and U266 MM cells. These findings indicate that NM-3 may be effective in combination with dexamethasone in the treatment of MM.
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Introduction
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2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl)propionic acid (NM-3) is an isocoumarin derivative that has recently entered clinical trials for evaluation as an orally bioavailable anticancer agent. The initial demonstration that NM-3 down-regulates expression of the vascular endothelial growth factor indicated that NM-3 could be effective as an inhibitor of angiogenesis. Subsequent work has shown that NM-3 also exhibits direct cytotoxic effects against endothelial cells and carcinoma cells (1
, 2)
. Moreover, NM-3 has been found to potentiate the therapeutic effects of certain chemotherapeutic agents, including 5-fluorouracil, paclitaxel, and cyclophosphamide (3)
. These findings and the favorable toxicity profile in preclinical studies indicated that NM-3 could be given to target both the tumor and its vasculature.
The available evidence indicates that NM-3 increases intracellular levels of reactive oxygen species (2)
. Studies have shown that survival of human umbilical vein endothelial cells is decreased by NM-3 at an IC50 of 1 µg/mL (1)
and that this effect is mediated by reactive oxygen species generation. Human carcinoma cells are also sensitive to the lethal effects of NM-3 but at concentrations 
100-fold higher than that found for endothelial cells (2)
. At an oral dose of 10 mg/kg in mice, plasma levels of NM-3 exceed 100 µg/mL. The demonstration that oral doses of 1 g/kg are well tolerated in animals have indicated that NM-3 plasma levels of >500 µg/mL are achievable, at least in animal models, without acute toxicity. In concert with these findings, plasma NM-3 levels of 170 µg/mL have been achieved in a phase I trial without toxicity (4)
.
The present findings demonstrate that NM-3 induces lethality of human multiple myeloma (MM) cells. We also show that NM-3 potentiates dexamethasone-induced apoptosis of MM cells. These findings indicate that, in addition to having anti-angiogenic activity, NM-3 has direct effects against MM cells at clinically achievable concentrations.
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Materials and Methods
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Cell Culture.
The human multiple myeloma MM1.S, RPMI8226, and U266 cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in an atmosphere of 5% CO2 in air. Cells were treated with NM-3 (ILEX Oncology Inc.), dexamethasone (Sigma Chemical Co., St. Louis, MO), and 30 mmol/L N-acetyl-cysteine (Sigma Chemical Co.).
Cytotoxicity Assay.
Cell survival was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded onto 96-well plates at a density of 3 x 104 (MM1.S) or 6 x 103 (RPMI8226 and U266) cells per well. Cells were incubated with increasing concentrations of NM-3 (50–200 µg/mL) for 2 hours and then increasing concentrations of dexamethasone (10–8-10–5 mol/L) for an additional 24 or 72 hours. After treatment, the cells were incubated with 0.5 mg/mL MTT reagent (Roche Molecular Biomedicals, Mannheim, Germany) for 4 hours and then in 10% SDS/0.01 mol/L HCl overnight at 37°C. The plates were read at 565 nm with 630 nm as a reference using a microplate spectrophotometer (Molecular Devices Corp., Sunnyvale, CA).
Immunoblot Analysis.
Cells were harvested, washed with PBS, and lysed using lysis buffer [150 mmol/L NaCl, 10 mmol/L Tris (pH 7.5), 5 mmol/L EDTA, 0.5% Triton X-100, protease inhibitor mixture (Complete; Roche Diagnostics Corp), and 100 µg/mL phenylmethylsulfonyl fluoride]. For the detection of cytochrome c and Smac/DIABLO release from the mitochondria into the cytosol, cells were homogenated in ice-cold buffer A [20 mmol/L HEPES-KOH (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L sodium EDTA, 1 mmol/L sodium EGTA, 1 mmol/L dithiothreitol, and 250 mmol/L sucrose], and cytosolic extracts were isolated by centrifugation. Cell lysates or cytosolic extracts were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immunoblotted with the appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibody. The blots were developed using the ECL kit (Amersham Biosciences, Uppsala, Sweden).
Apoptosis Assay.
Apoptosis was determined by staining cells with annexin-V–FITC and propidium iodide (PI). Cells were washed twice with cold PBS, resuspended in binding buffer [10 mmol/L HEPES/NaOH (pH 7.4), 140 mmol/L NaCl, and 2.5 mmol/L CaCl2], and then stained with annexin-V–FITC (Clontech Laboratories, Palo Alto, CA) and PI (Boehringer Mannheim, Mannheim, Germany). Apoptosis was analyzed by using a flow cytometer (EPICS XL-MCL; Coulter Corp., Fullerton, CA). The cultured cells were also subjected to cell cycle analysis. In brief, cells were washed twice with PBS and fixed with 70% ethanol. Cells were then washed with 0.5 mL of PBS and 1.0 mL of phosphate-citric acid buffer [0.2 mol/L Na2HPO4 and 0.1 mol/L citric acid (pH 7.8)], treated with 200 µg/mL RNase for 15 minutes at 37°C, and stained with 50 µg/mL PI for 30 minutes at room temperature in the dark. Cells were filtered through 35-µm-diameter mesh to remove clumps. Nuclear staining was analyzed by a flow cytometer. Cells with fractional DNA content located on DNA frequency histograms to the left of the G1 peak (sub-G1 cells) were identified as apoptotic cells.
Measurement of Reactive Oxygen Species Levels.
Cells were incubated with 10 µmol/L dichlorodihydrofluorescein diacetate (DCF-DA) (Sigma Chemical Co.) for 15 minutes at 37°C and then treated with NM-3, dexamethasone, or the combination of these agents for 30 minutes at 37°C. Fluorescence of oxidized DCF was measured at an excitation wavelength of 480 nm and an emission wavelength of 525 nm using a flow cytometer (Becton Dickinson, Lincoln Park, NJ).
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Results and Discussion
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NM-3 Potentiates Dexamethasone-Induced Lethality of Dexamethasone-Sensitive and Dexamethasone-Resistant Human Multiple Myeloma Cells.
To determine whether NM-3 affects viability of MM cells, we first studied MM1.S cells that are sensitive to dexamethasone-induced killing. MM1.S cells were exposed to NM-3 for 24 hours and then monitored for viability. Under these experimental conditions, 100 µg/mL NM-3 decreased viability by 10%, and nearly 20% of the MM1.S cells were dead following exposure to 200 µg/mL NM-3 (Fig. 1A)
. MM1.S cells also responded to 10–8 to 10–5 mol/L dexamethasone alone with loss of viability (Fig. 1A)
. Moreover, NM-3 combined with dexamethasone resulted in a greater than additive loss of MM1.S cell viability compared with that found with either agent alone (Fig. 1A)
. Dose effect analysis of the data by CalcuSyn (Biosoft, Inc., Ferguson, MO) demonstrated that the interaction between NM-3 and dexamethasone is synergistic. Similar studies were performed on the dexamethasone-resistant RPMI8226 and U266 MM cells. RPMI8226 cells were treated with 100 and 200 µg/mL NM-3 for 72 hours. Analysis of cell viability demonstrated that approximately 90% of the cells were viable after treatment with 100 µg/mL NM-3 and that 50% were viable at a concentration of 200 µg/mL (Fig. 1B)
. In contrast to MM1.S cells, treatment of the RPMI8226 cells with dexamethasone alone at concentrations of 10–8 to 10–5 mol/L had little if any effect on viability (Fig. 1B)
. However, the combination of NM-3 and dexamethasone resulted in cytotoxicity that was greater than that achieved with either agent alone (Fig. 1B)
. Similar results were obtained with NM-3 treatment of U266 cells. Treatment with 200 µg/mL NM-3 was associated with a 50% decrease in U266 cell viability (Fig. 1C)
. Moreover, whereas dexamethasone alone had little if any effect on U266 cell viability, the combination of NM-3 and dexamethasone was synergistic as determined by dose effect analysis (CalcuSyn) in inducing U266 cell lethality (Fig. 1C)
. These findings indicate that NM-3 modestly induces lethality alone and potentiates the effects of dexamethasone on both dexamethasone-sensitive and dexamethasone-resistant MM cells.
NM-3 Potentiates Dexamethasone-Induced Release of Mitochondrial Apoptogenic Factors and Activation of Caspase-9 and Caspase-3.
To define the mechanisms responsible for the interaction between NM-3 and dexamethasone, we first studied MM1.S cells for release of apoptogenic factors from mitochondria. NM-3 at concentrations of 100 to 400 µg/mL had a minimal effect on release of mitochondrial cytochrome c into the cytosol (Fig. 2A)
. Dexamethasone treatment was associated with a detectable increase in cytosolic cytochrome c levels (Fig. 2A)
. Moreover, the combination of NM-3 and dexamethasone resulted in a more pronounced release of cytochrome c compared with that with either agent alone (Fig. 2A)
. Smac/DIABLO is another mitochondrial protein that induces caspase-dependent cell death by interacting with inhibitor of apoptosis proteins and blocking their caspase inhibitory activity (5
, 6)
. As found for cytochrome c, NM-3 alone had little effect on cytosolic Smac/DIABLO levels (Fig. 2B)
, but clearly potentiated dexamethasone-induced release of Smac/DIABLO from mitochondria (Fig. 2B)
. Release of cytochrome c and Smac/DIABLO contributes to the activation of caspase-9 and caspase-3. Indeed, consistent with the release of apoptogenic factors, NM-3 had no apparent effect on activation of caspase-9 (Fig. 2C)
. Dexamethasone treatment was associated with processing of pro-caspase-9 to the active caspase-9 (Fig. 2C)
, and this response was potentiated by NM-3 (Fig. 2C)
. Similar results were obtained for the activation of caspase-3. NM-3 had no apparent effect, and the combination of NM-3 and dexamethasone was more effective than dexamethasone alone in inducing caspase-3 activation (Fig. 2D)
. These findings indicate that NM-3 potentiates dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 pathway.
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Fig. 2. NM-3 potentiates dexamethasone-induced release of mitochondrial apoptogenic factors and activation of caspase-9 and caspase-3. A and B. MM1.S cells were treated with the indicated concentrations of NM-3 alone, dexamethasone alone, or the combination of these agents for 24 hours. Cytosolic extracts were separated by SDS-PAGE and analyzed by immunoblotting with anti-cytochrome c (A) and anti-Smac/DIABLO (B) antibodies. Positive control lysate from MM1.S cells treated with 20 µmol/L dexamethasone for 16 hours (+ve). C and D. MM1.S cells were treated with the indicated concentrations of NM-3 alone, dexamethasone alone, or the combination of these agents and harvested at the indicated times. Total cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-caspase-9 (C) and anti-caspase-3 (D) antibodies. FL, full length; CF, cleaved fragment.
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NM-3 Potentiates Dexamethasone-Induced Apoptosis.
To assess the effects of NM-3 on the induction of apoptosis, MM1.S cells were treated with NM-3 in the absence and presence of 1 µmol/L dexamethasone. Flow cytometric analysis demonstrated that MM1.S cells with sub-G1 DNA was increased to 8% after exposure to 200 µg/mL NM-3, whereas treatment with 50 and 100 µg/mL NM-3 had no apparent effect (Fig. 3A)
. Treatment with dexamethasone alone induced 20% apoptosis, and this response was increased by NM-3 in a concentration-dependent manner (Fig. 3A)
. Thus, the induction of apoptosis by 200 µg/mL NM-3 and dexamethasone was greater than that obtained with either agent alone (Fig. 3A)
. To extend these results, the MM1.S cells were stained with annexin-V–FITC and PI. Exposure to NM-3 alone was associated with little effect on annexin-V staining (Fig. 3B)
. Treatment with dexamethasone alone was associated with 19% of cells staining with annexin-V (Fig. 3B)
. Moreover, dexamethasone-induced annexin-V staining was enhanced by NM-3 in a concentration-dependent manner (Fig. 3B)
. To assess the effects of NM-3 on dexamethasone-resistant MM cells, RPMI8226 cells were treated with 100 µg/mL NM-3 for 72 hours and then assayed for annexin-V and PI staining. The results demonstrate that, compared with untreated cells, treatment with 100 µg/mL NM-3 had no apparent effect on the percentage of apoptotic cells (Fig. 3C)
. Consistent with the resistance of RPMI8226 cells to dexamethasone, there was also no induction of apoptosis when these cells were treated with dexamethasone alone (Fig. 3C)
. By contrast, treatment with NM-3 and dexamethasone was associated with nearly 20% annexin-V–positive, PI-negative apoptotic cells (Fig. 3C)
. Moreover, the NM-3/dexamethasone combination resulted in more than 10% annexin-V–positive, PI-positive late apoptotic/necrotic cells (Fig. 3C)
. Similar results were obtained with the U266 cells. Treatment with NM-3 alone or dexamethasone alone had no apparent effect on induction of apoptosis (Fig. 3D)
. However, the combination of NM-3 and dexamethasone was associated with an increase in the percentage of apoptotic U266 cells (Fig. 3D)
. The combination also resulted in increases in the percentage of annexin-V–positive, PI-positive cells, indicating the induction of late apoptosis or necrosis. These findings indicate that NM-3 induces death of dexamethasone-resistant MM cells by apoptotic mechanisms.
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Fig. 3. NM-3 enhances dexamethasone-induced apoptosis of MM cells. A and B. MM1.S cells were treated with the indicated concentrations of NM-3 alone or in combination with 1 µmol/L dexamethasone for 24 hours. Cells with sub-G1 DNA were determined by flow cytometry (A) or annexin-V–FITC and PI staining (B). RPMI8226 (C) and U266 (D) cells were treated with the indicated concentrations of NM-3 alone, 1 µmol/L dexamethasone alone, or the combination of both agents for 72 hours and analyzed by annexin-V–FITC and PI staining.
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NM-3 Potentiates Dexamethasone-Induced Apoptosis in Part by Increasing Reactive Oxygen Species Levels.
Treatment of human carcinoma cells with NM-3 is associated with increases in intracellular reactive oxygen species levels (2)
. To determine whether NM-3 has similar effects on MM cells, we incubated MM1.S cells with DCF-DA and assessed reactive oxygen species-mediated conversion of DCF-DA to the fluorescent compound DCF. The results show that NM-3 increases reactive oxygen species levels compared with that in control MM1.S cells (Fig. 4A)
. Dexamethasone had little effect on reactive oxygen species levels when used alone or in combination with NM-3 (Fig. 4A)
. Similar results were obtained with RPMI8226 cells (Fig. 4B)
. When assaying U266 cells, NM-3 alone and in combination with dexamethasone also increased reactive oxygen species levels, whereas exposure to dexamethasone alone resulted in a decrease (Fig. 4C)
. To determine whether NM-3–induced increases in reactive oxygen species are responsible for potentiating dexamethasone, we treated MM1.S cells in the absence and presence of the antioxidant N-acetyl-cysteine. The results show that N-acetyl-cysteine significantly attenuates but does not completely block the NM-3-induced potentiation of dexamethasone (Fig. 4D)
. Similar effects were observed with RPMI8226 and U266 cells (data not shown). These findings indicate that NM-3–induced increases in reactive oxygen species levels contribute in part to enhancing sensitivity of MM cells to dexamethasone.
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Fig. 4. NM-3 potentiates dexamethasone-induced apoptosis in part by increasing reactive oxygen species levels. MM1.S (A), RPMI8226 (B), and U266 (C) cells were preincubated with 10 µmol/L DCF-DA for 15 minutes and then treated with 100 µg/mL NM-3, 1 µmol/L dexamethasone, or both agents for 30 minutes. Reactive oxygen species-mediated oxidation of DCF-DA to DCF was assayed by flow cytometry. Open histograms, control cells. Solid histograms, treated cells. D. MM1.S cells were exposed to 200 µg/mL NM-3, 1 µmol/L dexamethasone, or both agents in the absence and presence of 30 mmol/L N-acetyl-cysteine for 24 hours. Cells with sub-G1 DNA were determined by flow cytometry. The results are expressed as the percentage (mean ± SD of three experiments) of apoptotic cells.
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NM-3 as a Potential Anti-Multiple Myeloma Agent.
NM-3 has completed phase I clinical evaluation of administration orally twice a day at doses of 1,000 and 1,500 mg/m2. Plasma NM-3 levels of >100 µg/mL have been achieved without toxicity (4)
. The present results demonstrate that NM-3 alone induces modest killing of MM cells at concentrations of 100 µg/mL. We have also found that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive and dexamethasone-resistant MM cells. The results indicate that NM-3 increases the apoptotic response to dexamethasone by sensitizing cells to activation of the intrinsic apoptotic pathway. NM-3 increases reactive oxygen species levels in diverse cell types (2)
and thereby activates p53 and the sensitivity of cells to pro-apoptotic agents. In this regard, NM-3 has been shown to increase the effectiveness of irradiation, 5-fluorouracil, paclitaxel, and cyclophosphamide in the treatment of syngeneic and human tumor xenograft models in mice (1
, 3)
. NM-3 also induces killing of endothelial cells and thus may act by targeting both the tumor and its vasculature (1)
. The present studies extend these findings by demonstrating that NM-3 can potentiate the lethal effects of dexamethasone on MM cells. Moreover, this potentiation is mediated in part by NM-3–induced increases in reactive oxygen species levels. Our data indicate that NM-3 may be effective in combination with dexamethasone in the treatment of patients with dexamethasone-sensitive or dexamethasone-resistant myeloma. A phase II trial of NM-3 in patients with myeloma is under way.
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ACKNOWLEDGMENTS
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We acknowledge the technical assistance of Kamal Chauhan.
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FOOTNOTES
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Grant support: National Cancer Institute grants CA100707 and CA42802.
Note: D. Kufe has a financial interest in ILEX.
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.
Requests for reprints: Donald Kufe, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115. E-mail: donald_kufe{at}dfci.harvard.edu
Received 7/22/04.
Revised 9/14/04.
Accepted 10/12/04.
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- Yin L, Ohno T, Weichselbaum R, Kharbanda S, Kufe D The novel isocoumarin NM-3 induces lethality of human carcinoma cells by generation of reactive oxygen species. Mol Cancer Ther 2001;1:43-8.[Abstract/Free Full Text]
- Reimer C, Agata N, Tammam J, et al Antineoplastic effects of chemotherapeutic agents are potentiated by NM-3, an inhibitor of angiogenesis. Cancer Res 2002;62:789-95.[Abstract/Free Full Text]
- Bonate PL, Eder JP, Soulie P, et al Pharmacokinetics of a new antiangiogenic isocoumarin derivative, NM-3, in two oral once a day-dose schedules with escalating doses. Proc Am Soc Clin Oncol 2003;22:134(abstract 537).
- Du C, Fang M, Li Y, Li L, Wang X Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000;102:33-42.[CrossRef][Medline]
- Verhagen AM, Ekert PG, Pakusch M, et al Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000;102:43-53.[CrossRef][Medline]
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ABSTRACT
Introduction
), 50 (
), 100 (
), or 200 (x) µg/mL NM-3 for 2 hours, and then the indicated concentrations of dexamethasone were added for an additional 24 hours. The number (mean ± SE of three replicates) of viable cells was determined at 26 hours. RPMI8226 (B) and U266 (C) cells were treated with 100 (