Antitumor activity of fludarabine against human multiple myeloma in vitro and in vivo

Fludarabine, a nucleoside analogue, plays a major role in the treatment of B-cell lymphocytic leukemia, hairy cell leukemia, and indolent lymphomas. There is a controversy about antitumor activity of fludarabine in multiple myeloma (MM). The aim of this study was to evaluate the activity of fludarabine against human myeloma cells both in vivo and in vitro. We demonstrated that myeloma cell line RPMI8226 was efficiently inhibited by fludarabine, concomitantly with decreased phosphorylation of Akt, down-regulation of the inhibitor of apoptosis proteins (IAP) family, including XIAP and survivin, and induction of apoptosis related to activation of caspase cascade. Contrary to dexamethasone, the effect of fludarabine on RPMI8226 cells was independent of interleukin-6. Fludarabine also induced cytotoxicity in dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) cells at 48 h with IC50 of 13.48 microg/mL and 33.79 microg/mL, respectively. In contrast, U266 cells were resistant to fludarabine. Moreover, RPMI8226 myeloma xenograft model was established using severe combined immunodeficient mice. The tumors treated with fludarabine at 40 mg/kg increased less than 5-fold in 25 d comparing with approximately 10-fold in the control tumors, demonstrating the antitumor activity of fludarabine in vivo. These results suggest that fludarabine may be an important therapeutic option for MM patients who are resistant to dexamethasone.     

Overexpression of Annexin II affects the proliferation, apoptosis, invasion and production of proangiogenic factors in multiple myeloma

The abnormal expression of Annexin II (AnxA2, A2) has been associated with the development of tumors; however, its expression and function in multiple myeloma (MM) is less known. We compared the expression of AnxA2 in primary myeloma cells from MM patients with that in normal plasma cells from normal subjects and found that myeloma cells from patients had higher expression of AnxA2. Expression of AnxA2 was also significantly higher in MM cell lines U266 and RPMI8226, compared with other hematologic tumor cell lines. Transfecting U266 and RPMI8226 cells with the small interfering RNA (siRNA) that targets human AnxA2 led to significant downregulation of AnxA2 expression, which resulted in the decreased proliferation, invasive potential and increased apoptosis of U266 and RPMI8226 cell lines. Silencing AnxA2 gene by siRNA also inhibited the expression of pro-angiogenic molecules including VEGF-C, VEGF-R2, MMP-2, MMP-9, MT1-MMP and TIMP-2 in the two cell lines. Our data suggested that the AnxA2 is overexpressed in MM patients and myeloma cell lines U266 and RPMI8226, and that AnxA2 overexpression appeared to affect the proliferation, apoptosis, invasive potential and production of pro-angiogenic factors in MM cell lines U266 and RPMI8226.     

Motexafin gadolinium generates reactive oxygen species and induces apoptosis in sensitive and highly resistant multiple myeloma cells

Motexafin gadolinium (MGd), an expanded porphyrin, is a tumor-selective redox-mediator that reacts with many intracellular reducing metabolites. Because redox mechanisms mediate apoptosis in multiple myeloma, we hypothesized that disruption of redox balance by MGd would result in cellular cytotoxicity in myeloma. We examined the effects of MGd on cellular cytotoxicity, apoptosis, reactive oxygen species (ROS) production, and intracellular drug uptake in dexamethasone-sensitive (C2E3), dexamethasone-resistant (1-310 and 1-414) chemotherapy-sensitive (8226-RPMI) and highly chemotherapy-resistant (DOX-10V) myeloma cells. We found complete inhibition of proliferation and cytotoxicity in each sensitive and resistant cell line with 24-hour exposure to clinically relevant concentrations of 50 muM MGd and 50 to 100 microM ascorbate, which was required for the effect. The mechanism of cytotoxicity was related to induction of apoptosis as demonstrated by alteration in mitochondrial membrane potential and elevated annexin V expression. This was accompanied by depletion of intracellular glutathione and increased ROS production. Moreover, catalase substantially abrogated MGd-induced cell death. Using fluorescence microscopy and flow cytometry, we found intracellular uptake of MGd and intracellular ROS production. MGd also induced apoptosis in fresh malignant cells from patients with multiple myeloma. These studies provide a rationale for clinical investigation of this novel redox-mediating agent in patients with multiple myeloma and related disorders.     

All‐trans retinoic acid can intensify the growth inhibition and differentiation induction effect of rosiglitazone on multiple myeloma cells

Objective: Activation of PPARγ by its ligands has shown potential anti‐neoplastic effects in solid tumors. In this study, we investigate the effects of rosiglitazone (RGZ) alone as well as in combination with all trans‐retinoic acid (ATRA) on human myeloma cell lines and try to address its potential mechanism. Methods: U266, RPMI‐8226 and primary myeloma cells from patients were treated with different concentrations of RGZ in the presence or absence of ATRA and various biological responses were studied by the methods of [³H] thymidine incorporation, MTT, cell cycle analysis, Annexin V‐PI staining, Wright‐Giemsa staining, CD49e expression assay, light chain protein detection, RT‐PCR and caspase‐3 activity assay. Results: We report that exposure to RGZ induced proliferation inhibition and viability reduction in a dose‐dependent manner in both U266 and RPMI‐8226 cells. A similar exposure to RGZ also induced cell cycle arrest and cell apoptosis of myeloma cells. A combination of RGZ with ATRA enhanced the effects of RGZ and induced cell cycle arrest and apoptosis more profoundly in both cell lines. RGZ treated cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. Similar cell apoptosis and differentiation were observed when primary CD138⁺ myeloma cells were treated with RGZ and ATRA. The mRNA expressions of FLIP, XIAP and survivin were detected in both cell lines and the levels decreased significantly after culture with RGZ. The addition of ATRA in culture medium made these changes more apparently. Caspase‐3 activity was increased upon exposure to RGZ in both U266 and RPMI‐8226 cells while combination of RGZ and ATRA brought out more effective activation of caspase‐3. Similar apoptosis and cell differentiation induced by RGZ and ATRA can also be observed in primary CD138⁺ cells from myeloma patients. Conclusion: Concomitant RXRα activation by ATRA enhanced the inhibitory effects of RGZ on myeloma cell proliferation, cell cycle, apoptosis and differentiation. Combination of RGZ and ATRA may be a useful therapy for human multiple myeloma.     

Non-steroidal anti-inflammatory agent ibuprofen-induced apoptosis, cell necrosis and cell cycle alterations in human leukemic cells in vitro

The cytotoxic activity of the non-steroidal anti-inflammatory agent ibuprofen to human promyelocytic leukemia cell line HL-60, its multidrug-resistant subline HL-60/VCR (MDR-1 gene coded P-glycoprotein), as well as myeloma U266 and B-lymphoblastoid ARH-77 cell lines was demonstrated with the aid of flow cytometric analysis. Ibuprofen inhibited proliferation and induced apoptosis (detected as sub-G0 nuclei, fluorescein diacetate staining, Annexin-V binding cells and agarose electrophoretic detection of nucleosomal DNA fragmentation) in promyelocytic cells and, to a lesser extent, in U266 and ARH-77 cells.     

Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells

Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity. It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM. The development of chemoresistance is a common feature in patients with MM. In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein. In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation. Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12–48 hr. Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis. Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene. HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.     

Analysis of histone deacetylase inhibitor, depsipeptide (FR901228), effect on multiple myeloma

Multiple myeloma (MM) is a neoplastic proliferation of plasma cells and remains an incurable disease because of the development of drug resistance. Histone deacytylase (HDAC) inhibitors are a new class of chemotherapeutic reagents that cause growth arrest and apoptosis of neoplastic cells. Depsipeptide, a new member of the HDAC inhibitors, was found to be safe in humans and has been shown to induce apoptosis in various cancers. In order to evaluate the effects of depsipeptide, a MM cell line, U266 [interleukin (IL)-6 dependent], was analysed for viability and apoptosis. The combined effect of depsipeptide with melphalan and changes in BCL-2 family proteins (BCL-2, BCL-XL, BAX and MCL-1) were also investigated. In addition, the RPMI 8226 cell line (IL-6 independent), and primary patient myeloma cells were also analysed for apoptosis after depsipeptide treatment. Depsipeptide induced apoptosis in both U266 and RPMI 8226 cell lines in a time- and dose-dependent fashion, and in primary patient myeloma cells. We also demonstrated that depsipeptide had an additive effect with melphalan (10 micromol/l). BCL-2, BCL-XL and MCL-1 showed decreased expression in depsipeptide-treated samples. Based on recent clinical trials demonstrating minimal clinical toxicity, our study supports the future clinical utilization of depsipeptide in the management of MM.     

Elimination of clonogenic stem cells from human multiple myeloma cell lines by a plasma cell-reactive monoclonal antibody and complement

The monoclonal antibody (MoAb) MM4 reacts with human multiple myeloma (MM) cell lines and bone marrow from patients with plasma cell dyscrasias but not with normal peripheral blood or bone marrow cells. Treatment with MM4 and rabbit complement (C') was cytotoxic to the plasma cell-derived cell lines GM 1312, RPMI 8226, and ARH-77, as demonstrated by chromium release microcytotoxicity and trypan blue exclusion assays. The same treatment eliminated greater than 99% of clonogenic myeloma stem cell colony formation of these cell lines, with less than 20% inhibition of normal human bone marrow pleuripotent progenitor colony formation in vitro. As an experimental model to explore the efficacy of MM4 + C' in purging MM-involved bone marrow, normal marrow cells were mixed with RPMI 8226 or GM 1312 cells in the ratio of 90:10 or 50:50 (marrow:myeloma cells). Colony growth assays indicated that MM4 + C' eliminated at least 2 logs of clonogenic myeloma stem cells in both 90:10 and 50:50 preparations, while sparing the majority of normal marrow progenitors (inhibition of CFU-C:10% to 13%; BFU-E:0%). The selectivity of MM4-mediated cytotoxicity may be useful for eliminating myeloma clonogenic stem cells from bone marrow of patients with multiple myeloma.     

Growth inhibition of RPMI 8226 human myeloma cells by peripheral blood lymphocytes.

To clarify the components of cellular immunity responsible for defense against the clonal development of myeloma cells, we tested the capacity of human peripheral blood lymphocytes (PBLs) to inhibit the growth of 3 human myeloma cell lines (RPMI 8226, OPM-1, and OPM-2). RPMI 8226 was found to be sensitive to PBLs, showing almost complete growth arrest when cultured with PBLs for 72 h. Inhibition of the growth of RPMI 8226 cells required direct cell-to-cell contact but not presensitization of the PBLs to the target cells, and did not depend on the generation of soluble factors. CD3+, CD4-, CD8- and CD16- cells were found to be the major subset contributing to inhibition of the growth of RPMI 8226 cells, and this growth inhibition was cytostatic rather than cytotoxic. These characteristics distinguished it from growth inhibition mediated by the natural killer system. Impaired PBL-mediated growth inhibition of RPMI 8226 cells was found in patients with various hematologic diseases, including myeloma. It therefore appears that the CD3+, CD4-, CD8- and CD16- cell subset might be involved in tumor immunity in myeloma.     

A human granulocyte-specific antigen characterized by use of monoclonal antibodies

Hybridoma cell lines secreting monoclonal antibodies that bind to a surface antigen of human neutrophils have been prepared by fusion of mouse myeloma cells with spleen cells from mice immunized with human neutrophils. Several of the monoclonal antibodies (AHN 1-6) were specific for a neutrophil surface antigen and did not bind lymphocytes, monocytes, red blood cells, platelets, or basophils. All of the granulocyte-specific antibodies immunoprecipitated a polypeptide of 145,000 daltons and an isoelectric point of about 4.5 and other heterogeneous polypeptides of 105,000 daltons. These same components were the major lactoperoxidase-labeled proteins precipitated by hyperimmune mouse serum. The antibodies were further characterized for binding to several human myeloid leukemia cell lines and cells from patients with myeloid or lymphoid leukemia. All antibodies bound the HL- 60, ML1, ML2, ML3, K562, and U937 myeloid leukemia cell lines. None of the antibodies bound the RPMI 6410 Raji, RPMI 8226, MOLT 4, or Daudi lymphoid cell lines. All of the hybridoma cell lines (AHN 1-6) produced IgM antibodies that were cytotoxic.     

The effects of thalidomide on chemotactic migration of multiple myeloma cell lines

We examined the effect of thalidomide and dexamethasone on the migration of multiple myeloma (MM) cell lines, U266, RPMI8226, and NCI-H929, using chemotaxis chamber plates. U266 underwent chemotactic migration in response to stromal-cell derived factor-1 alpha (SDF-1alpha), and other cell lines underwent random migration in response to SDF-1alpha or monocyte chemotactic protein-1 alpha. Following preincubation with 1 mug/ml thalidomide, the cell lines showed reduced migratory capacity in response to SDF-1alpha. Concerning the corresponding receptors, CXC chemokine receptor 4 was detected only on the surface of U266, by flow cytometry, whereas chemokine (C-C motif) receptor 2 was not detected on all three cell lines. Moreover, decreased migration by thalidomide was not accompanied by altered expression of the corresponding receptors of each cell line. This is the first report to show the effects of thalidomide on the migration of MM cell lines. The results suggest that the inhibition of chemotactic migration might be one of the mechanisms of the success of thalidomide in controlling MM.     

In vitro treatment with retinoids decreases bcl-2 protein expression and enhances dexamethasone-induced cytotoxicity and apoptosis in multiple myeloma cells

All-trans retinoic acid (ATRA) has been shown to inhibit in vitro growth of multiple myeloma (MM) cells, and this effect can be further potentiated by the addition of Dexamethasone (DEX). We here extended this study by testing the activity of 9-cis retinoic acid (9-cis RA) and 13-cis retinoic acid (13-cis RA), both alone and in combination with DEX, in two MM cell lines, U266 and RPMI 8226. Furthermore, we aimed at investigating the mechanisms involved in the interactions of retinoids and DEX in this setting. 9-cis RA appeared to be the most active agent in U266 cell line (IC50 = 1.2 mumol/l vs 10.5 and 9.8 mumol/l obtained with ATRA and 13-cis RA, respectively) while, in RPMI 8226 cell line, 9-cis RA and 13-cis RA were almost equally cytotoxic (IC50 = 1 and 0.8 mumol/l) and ATRA was less effective. Co-incubation with DEX resulted in a synergistic cytotoxic activity in both the cell lines except for the combinations DEX + 9-cis RA in U266 cell line and DEX + 13-cis RA in RPMI 8226 cell line, where the effect was merely additive. A synergistic cytotoxic effect of retinoids and DEX was also observed on fresh MM cells obtained from 7 patients. Both retinoids and DEX are known to be inducers of apoptosis; we verified that the combined inhibitory activity of retinoids and DEX could be attributed to an increased induction of apoptosis. This effect may be mediated by a reduced intracellular expression of BCL-2 protein, which indeed observed after prolonged in vitro treatment with retinoids. It has been described recently that an enhanced expression of BCL-2 protein can contribute to the occurrence of early chemoresistance; the downregulation of BCL-2 protein induced by retinoids could thus be exploited, by means of novel chemotherapy plus retinoids combinations, in order to improve the efficacy of conventional chemotherapy in MM.     

Synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with histone deacetylase inhibitors,melphalan, or doxorubicin in multiple myeloma

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP‐AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM‐2, RPMI‐8226, U‐266, LP‐1, MM1.S, and primary myeloma cells were exposed to NVP‐AUY922 and one of the combination partners histone deacetylase inhibitor NVP‐LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP‐AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP‐AUY922 with SAHA, NVP‐LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI‐8226 cell line and relative resistance of some primary myeloma cells against NVP‐AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with melphalan, doxorubicin, NVP‐LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.     

beta-lapachone,a novel plant product,overcomes drug resistance in human multiple myeloma cells

OBJECTIVE: To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). MATERIALS AND METHODS: Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. CONCLUSION: These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.     

Proteasome inhibitors induce growth inhibition and apoptosis in myeloma cell lines and in human bone marrow myeloma cells irrespective of chromosome 13 deletion

Purpose In this study, we investigated the effects of cell-permeable proteasome inhibitors MG-132, MG-262, PSI, and lactacystin on multiple myeloma cell lines OPM-2, U266, RPMI 8226-S, freshly isolated plasma cells with or without deletion of chromosome 13 from patients with multiple myeloma and plasma cell leukemia, and CD34+ human hematopoietic stem cells. The effects of proteasome inhibitors on cell cycle progression, cell growth, and apoptosis were determined.Methods MTT-assay was used to examine the cytotoxicity, and annexin-V staining to quantify apoptosis. Cell cycle analyses were performed using 7-ADD and Ki-67 staining by flow cytometry.Results PSI was the most potent proteasome inhibitor among those tested with a half maximal cytotoxicity (IC₅₀) of 5.7 nM, followed by MG-262, MG-132, and lactacystin. Growth inhibition occurred irrespective of chromosome 13 status. Cell cycle arrest occurred in a dose- and time-dependent manner. Low, subapoptotic dosages led to a partial loss of Ki-67 antigen, whereas apoptotic dosages led to reduced Ki-67 levels. Apoptosis was partially dependent on activation of caspase-3, since Ac-DEVD-cho, a caspase-3 inhibitor, could reduce apoptosis significantly. The cytotoxicity of the four proteasome inhibitors tested was significantly lower in human hematopoietic stem cells than in myeloma cells.Conclusions Our results show that proteasome inhibitors induce time- and dose-dependent cell cycle alterations, growth inhibition, and apoptosis in human myeloma cells irrespective of chromosome 13 deletion.     

Myeloma cells express Fas antigen/APO-1 (CD95) but only some are sensitive to anti-Fas antibody resulting in apoptosis

To find out which cytokines are involved in the pathogenesis of multiple myeloma, we investigated cytokine receptor expression on myeloma cells using a panel of monoclonal antibodies (MoAbs). Flow cytometric analysis of five myeloma cell lines (RPMI8226, ARH77, KMM-1, U266, and Hs) and myeloma cells freshly isolated from eight patients showed that interleukin-1 receptor (IL-1R) type I and type II, IL-2R alpha and beta chains, IL-4R, IL-6R, IL-7R, IL-8R, granulocyte macrophage colony-stimulating factor receptor (GM-CSFR), c-kit (stem cell factor receptor [SCFR]), membrane bound stem cell factor (MBSCF), and tumor necrosis factor (TNF) receptors type I and type II were not always detected on the myeloma cells. However, interferon-gamma receptor, gp130, and Fas antigen were constitutively expressed, except one sample. To determine the role of Fas antigen on myeloma cells, these cells were cultured with anti-Fas MoAb. Apoptotic changes characterized by loss of cell volume, membrane blebbing, fragmentation of nuclei, and condensed chromatin were observed in three of five myeloma cell lines. When bcl-2 expression was examined, it was seen in all the cell lines regardless of the sensitivity to anti-Fas MoAb. Furthermore, anti-Fas MoAb not only induced apoptosis of freshly isolated myeloma cells but also inhibited the DNA synthesis, although such effects varied from patient to patient. The data indicate that only some myeloma cells undergo apoptosis in response to the signal mediated by the Fas antigen.     

Ascorbic acid enhances arsenic trioxide-induced cytotoxicity in multiple myeloma cells

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 (As(2)O(3)) 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 As(2)O(3). As(2)O(3) induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-x(L). The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As(2)O(3)-induced cell death either through conjugating As(2)O(3) or by sequestering reactive oxygen induced by As(2)O(3). Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As(2)O(3) cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As(2)O(3)-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 As(2)O(3) alone or in combination with AA. Together, these data suggest that As(2)O(3) 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)