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Keywords:

  • arsenic trioxide;
  • bortezomib;
  • melphalan;
  • multiple myeloma;
  • severe combined immunodeficient mice

Summary

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Arsenic trioxide (ATO) induces apoptosis of malignant plasma cells through multiple mechanisms, including inhibition of DNA binding by nuclear factor kappa-B, a key player in the development of chemoresistance in multiple myeloma (MM). This activity suggests that ATO may be synergistic when combined with other active antimyeloma drugs. To evaluate this, we examined the antimyeloma effects of ATO alone and in combination with bortezomib, melphalan and ascorbic acid (AA) both in vitro and in vivo using a severe combined immunodeficient (SCID)-hu murine myeloma model. Marked synergistic antimyeloma effects were demonstrated when human MM Los Angeles xenograft IgG lambda light chain (LAGλ-1) cells were treated in vitro with ATO and any one of these agents. SCID mice bearing human MM LAGλ-1 tumours were treated with single-agent ATO, bortezomib, melphalan, or AA, or combinations of ATO with either bortezomib or melphalan and AA. Animals treated with any of these drugs alone showed tumour growth and increases in paraprotein levels similar to control mice, whereas animals treated with ATO-containing combinations showed markedly suppressed tumour growth and significantly reduced serum paraprotein levels. These in vitro and in vivo results suggest that addition of ATO to other antimyeloma agents may result in improved outcomes for patients with relapsed or refractory MM.

Multiple myeloma (MM) is a malignancy characterised by the proliferation of a single clone of plasma cells derived from B cells in the bone marrow (Berenson, 2004). The American Cancer Society estimated that approximately 16 570 new cases of myeloma were diagnosed in 2006 in the United States (American Cancer Society, 2006). This disease is associated with serum monoclonal protein or immunoglobulin, development of osteolytic lesions, anaemia and renal failure. At present, the disease is incurable; and although survival has improved, there remains a need for new therapeutic options for patients with this B-cell malignancy.

Arsenic trioxide (ATO) (Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA) is a novel anticancer agent with established activity in relapsed or refractory acute promyelocytic leukaemia (APL) (Niu et al, 1999; Soignet et al, 2001). Although ATO is currently approved for use in relapsed or refractory APL, the mechanism of action underlying the robust antiproliferative activity of ATO supports its use in other haematological malignancies, including MM. ATO has been shown to induce apoptosis of plasma cells through a number of mechanisms, including the downregulation of Bcl-2 expression (Chen et al, 1996; Akao et al, 1998; Zhang et al, 1998) and the inhibition of DNA binding by nuclear factor kappa-B (NF-κB) (Friedman et al, 2002). Established human MM cell lines and freshly isolated primary MM patient cells are sensitive to ATO in vitro at clinically relevant concentrations (Rousselot et al, 1999). Importantly, ATO shows anti-MM activity in the clinical setting. In two separate clinical studies, single-agent ATO, when administered daily, produced clinical responses in 21% and 33% of patients with relapsed or refractory MM (Munshi et al, 2002; Hussein et al, 2004).

Although more widely known for its antioxidant properties, ascorbic acid (AA) or vitamin C (MAC), also possesses pro-oxidant properties. In the plasma, AA is oxidised to dehydroascorbic acid before being transported into the cell, where AA is regenerated through a reaction that converts intracellular free glutathione (GSH) to GSH disulfide (May et al, 2001). This reaction depletes intracellular GSH, the molecule that eliminates reactive oxygen species (ROS), thereby increasing hydrogen peroxide production and sensitising MM cells to chemotherapeutic agents (Grad et al, 2001). As the antitumour activity of ATO is in part dependent upon the generation of ROS, removing GSH with AA enhances the antimyeloma effects of ATO (Grad et al, 2001). Elevated intracellular levels of GSH and GSH-related enzymes in MM cells have been shown to confer drug resistance to alkylating agents such as melphalan (Gupta et al, 1989; Bellamy et al, 1991; Grad et al, 2001). Thus, the combination of melphalan with ATO and MAC may overcome resistance to melphalan in MM cells, which provides the rationale for evaluating MAC in this B-cell malignancy.

In addition to inhibiting DNA binding by NF-κB, ATO has been shown to inhibit NF-κB activation by blocking the degradation of the NF-κB inhibitor IκBα (Mathieu & Besançon, 2006). Because elevated NF-κB activity has been implicated as a key factor involved in resistance of myeloma cells to chemotherapy (Feinman et al, 1999), these mechanisms of ATO suggest that this agent may overcome chemoresistant cancer cells by reducing NF-κB activation (Kapahi et al, 2000).

Bortezomib, a proteasome inhibitor approved for the treatment of patients with relapsed/refractory multiple myeloma, also inhibits the activation of NF-κB, leading to the accumulation of the inhibitor complex IκB. Bortezomib has been shown to overcome chemoresistance in MM cells both in the laboratory (Ma et al, 2003; Hideshima et al, 2005) and in the clinic (Berenson et al, 2006). The mechanism by which bortezomib prevents degradation of IκB differs from that of ATO. Bortezomib inhibits the proteasome, resulting in the accumulation of IκB, whereas ATO prevents phosphorylation of the IκB-complex, blocking its ubiquitinylation and subsequent proteasomal degradation. Thus, these different mechanisms of IκB inhibition provide the rationale for combining these agents to most effectively accomplish the deactivation of NF-κB. Furthermore, other differing mechanisms of action between bortezomib and ATO support the combination of these agents to maximise cytotoxicity against MM cells. Both ATO and bortezomib have been shown to induce their cytotoxic effects on MM cells via caspase-dependent pathways (McCafferty-Grad et al, 2003), including caspase-3, caspase-8 and caspase-9 signaling (Hayashi et al, 2002; Hideshima et al, 2003), and inhibiting activation of signal transducer and activator of transcription 3 (STAT3)- and interleukin-6-induced Janus kinase-STAT signaling; and bortezomib inhibits DNA repair machinery and suppresses adhesion of MM cells to bone marrow stromal cells (Hideshima et al, 2001; Mitsiades et al, 2003).

Our animal model of human MM, LAGλ-1, was initially generated from the intramuscular implantation of a fresh bone marrow biopsy from a MM patient (Campbell et al, 2006) and has undergone more than 25 passages during the past 3 years. This tumour retains the morphological and immunological features of the patient's original myeloma cells, including the expression of CD38, CD138 and hIgGλ (Campbell et al, 2006). Furthermore, LAGλ-1 tumour cells implanted in mice grow locally and do not metastasise to the bone marrow. These xenografts also show consistent growth as indicated by increases in both the human paraprotein and the size of the intramuscular tumour. The LAGλ-1 tumour provides an excellent preclinical model to rapidly assess and optimise the development of new therapies to more effectively treat and expand the treatment options for patients with MM. Additionally, because LAGλ-1 maintains the resistance to melphalan that was observed clinically in the patient from which it was derived (Campbell et al, 2006), it is an important tool for preclinical studies to help elucidate the mechanisms that lead to chemoresistance and evaluate novel treatment combinations to overcome drug resistance.

Here, we report our findings on the in vitro and in vivo effects of combining ATO with bortezomib, melphalan and AA on the growth of MM cells. These data provide the rationale for combining ATO with these agents in the treatment of MM patients who have disease that is resistant to these same drugs alone or when used in other combinations.

Methods and materials

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Reagents

Arsenic trioxide (Mallinckrodt Baker, Inc.,) was solubilised in NaOH, diluted to 5 mg/ml with sterile distilled water, and pH- neutralised with HCL. Appropriate concentrations were made using sterile distilled water prior to injection. Bortezomib (Millennium Pharmaceuticals, Cambridge, MA, USA) was mixed with mannitol (in a ratio of 1:10 to increase solubility) and solubilised in 0·9% NaCl at the appropriate concentration prior to injection. Melphalan (Sigma, St Louis, MO, USA) was dissolved in 100 μl acid-EtOH (acid-EtOH: 47 μl concentrated HCl + 1 ml of 100% EtOH) and diluted to 1 ml with phosphate-buffered saline (PBS). Appropriate concentrations were made using sterile distilled water prior to injection. AA (EMD Chemicals Inc., Gibbstown, NJ, USA) was solubilised in sterile distilled water and pH-neutralised with NaOH. Appropriate concentrations were made using sterile distilled water prior to injection.

Cell lines

The human myeloma cell lines RPMI8226 and U266 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium (Omega Scientific, Tarzana, CA, USA) supplemented with 10% fetal bovine serum, 2 mmol/l l-glutamine, 100 IU/ml penicillin, 100 μg/mL streptomycin, and essential amino acids in an atmosphere of 5% carbon dioxide (CO2) at 37°C.

Cell proliferation test

Multiple myeloma cell lines were cultured as described above. Cells were plated in 96-well plates at a concentration of 105 cells/100 μl in each well. Cells were incubated for 24 h prior to treatment with drug. Cells were then treated with no drug or varying concentrations of ATO (0·1–100 μmol/l), bortezomib (0·3–1000 nmol/l), melphalan (0·3–1000 μmol/l), or combinations thereof. MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay]) (Sigma) was added to each well, and absorbance at 575 nm with a reference wavelength of 650 nm was determined on a μQuant microplate spectrophotometer with KC Junior software (Bio-Tek Instruments, Winooski, VT, USA).

Trypan blue exclusion assay

Initially, 5 × 105 RPMI8226 cells were treated with the study drugs. Cell death, recovery, and viability were assayed by the Trypan Blue dye exclusion method. An aliquot of each cell suspension was mixed with an equal volume of Trypan Blue dye and the total number of viable and dead cells was determined using light microscopy. The mean numbers of viable and dead cells were calculated from triplicate experiments.

Apoptosis analysis

Initially, 5 × 105 RPMI8226 cells were treated with the study drugs. After 24 h of treatment, cells were harvested using (where appropriate, for adherent cells) 1 mmol/l ethylene diamine tetra acetic acid in PBS. After incubation with Cytofix/Cytoperm solution (PharMingen BD Biosciences, San Jose, CA, USA) for 30 min at room temperature, the cells were stained with active caspase-3 antibody as previously described (Huerta-Yepez et al, 2004) and subjected to flow cytometry analysis. Fluorescein isothicyanate-conjugated IgG isotype control served as negative control. Analyses were carried out on an Epics XL flow cytometer (Coulter Electronics, Inc., Miami, FL, USA).

In vivo MM tumour model, LAGλ-1

All animal studies were conducted according to protocols approved by the Animal Research Committee under the Office of Research Protection at the University of California at Los Angeles. The LAGλ-1 tumour was developed by surgically implanting a whole core bone marrow biopsy from a patient with an IgGλ-producing myeloma into the hind limb muscle of 6–8 week-old male homozygous CB-17 severe combined immunodeficient (SCID) mice obtained from Harlan Sprague Dawley (Indianapolis, IN, USA) (Campbell et al, 2006). Briefly, SCID mice were anaesthetised with ketamine, xylazine, and isoflurane prior to surgery. A 2·0- to 4·0-mm3 fresh LAGλ-1 tumour was implanted into the superficial gluteal muscle of the left hind limb. Starting on the day of surgery, mice received weekly injections of anti-asialo GM1 rabbit serum (Wako Bioproducts, Richmond, VA, USA) to further suppress immune activity. The LAGλ-1 line has been propagated in vivo by intramuscular implantation passage of tumours obtained intramuscularly from SCID mice. For the experiments described in this report, mice bearing LAGλ-1 tumours received ATO daily for 5 d a week by intraperitoneal injections at 0·05, 0·25, 1·25, or 6·0 mg/kg body weight. Bortezomib (0·25 mg/kg) was administered intravenously twice weekly for the duration of the study. Melphalan (0·6 mg/kg) was administered once weekly via intraperitoneal injection for the duration of the study. AA (100 mg/kg) was administered daily for 5 d a week via intraperitoneal injection for the duration of the study. Each week, technicians who were unaware of the treatment rendered to each mouse measured the size of the tumours with calipers, and tumour volume was determined using the following formula: 4/3π × (width/2)2 × (length/2), which calculates the three-dimensional volume of an ellipse.

Determination of human IgG levels

As the LAGλ-1 tumour produces IgG subclass 1, levels of this human protein in mice were determined weekly by ELISA. Human IgG (hIgG) subclass 1 ELISA kits were purchased from Zymed Laboratories (South San Francisco, CA, USA). Mice bearing LAGλ-1 tumours were bled weekly via retro-orbital bleed. Samples were spun at 13 000 rpm for 30 min and serum was collected. The IgG subclass 1 ELISA kit was prepared according to the manufacturer's specifications. Absorbance at 450 nm with a reference wavelength of 550 nm was determined on a μQuant microplate spectrophotometer with KC Junior software.

Statistical analysis

Tumour growth and hIgG secretion curves were analysed in terms of treatment group means and standard error. The Chou-Talaly method for quantitative analysis of dose-effect relationships was used (Chou & Talalay, 1984). Statistical significance of differences observed in drug-treated mice versus control mice was determined using a Student's t-test. The minimal level of significance was P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Single-agent arsenic trioxide, melphalan or bortezomib inhibits MM cell growth in vitro

First, we determined the effects of single-agent ATO, melphalan and bortezomib on the growth of MM cell lines (RPMI8226 and U266) cultured for 48 h using the MTT assay. These cells were cultured in the presence of ATO (0·1–100 μmol/l) for 48 h (Fig 1A), melphalan (0·3–1000 μmol/l) for 48 h (Fig 1B), or bortezomib (0·3–1000 nmol/l) for 48 h (Fig 1C). For ATO, 50% growth inhibition (IC50) was observed at concentrations of 3 μmol/l for both RPMI8226 and U266. RPMI8226 and U266 cells treated with melphalan showed an approximate IC50 at 30 μmol/l. U266 cells treated with bortezomib alone showed an approximate IC50 between 1 and 3 μmol/l, whereas RPMI8226 was slightly more resistant, with an approximate IC50 between 3 and 10 μmol/l. All MM cells were inhibited in a concentration-dependent manner when treated with ATO, melphalan or bortezomib.

image

Figure 1.  Loss of viability of RPMI8226 and U266 multiple myeloma cell lines after incubation in the presence of increasing concentrations of (A) arsenic trioxide (ATO; 0·1–100 μmol/l) for 48 h, (B) melphalan (0·3–1000 μmol/l) for 48 h or (C) bortezomib (0·3–1000 nmol/l) for 48 h. Cell viability was measured by the MTT assay, and results are expressed as a percentage of the control.

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Arsenic trioxide monotherapy inhibits human MM cell growth in vivo

Having shown that ATO inhibits growth in MM cells in vitro, we next investigated the in vivo effects of ATO in our melphalan-resistant mouse model of human MM LAGλ-1 (Campbell et al, 2006). SCID mice implanted with 2·0- to 4·0-mm3 LAGλ-1 tumour fragments developed measurable tumours and detectable levels of serum hIgG at 19 d postimplantation. Mice were randomly assigned to one of five ATO treatment groups. ATO was administered via intraperitoneal injection daily for 5 d a week at doses of 0·05, 0·25, 1·25 and 6·0 mg/kg. Control mice were given the 0·9% normal saline vehicle only. Mice were bled weekly to quantify hIgG levels, and tumours were measured weekly using calipers.

Animals treated with the lower doses of ATO (0·05, 0·25, and 1·25 mg/kg) showed increases in paraprotein levels (Fig 2A) and tumour growth characteristics (Fig 2B) similar to those animals receiving vehicle. Animals treated with the highest dose of ATO (6·0 mg/kg) showed marked decreases in both hIgG levels (Fig 2A) and tumour growth (Fig 2B) compared with the control group. At the time of euthanasia, only 50% of the mice that received 6·0 mg/kg had measurable tumours compared with 100% of mice treated in all of the other treatment groups. Although mice receiving high-dose ATO therapy showed slight weight loss (<5%), the dose was otherwise well tolerated.

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Figure 2.  Reduction in human IgG levels and tumour volumes in high-dose Arsenic trioxide (ATO)-treated LAGλ-1-bearing severe combined immunodeficient (SCID) mice. Serum level of human IgG (A) and tumour volume (B) were measured in SCID mice after implantation of an LAGλ-1 tumour fragment. Mice received vehicle (bsl00001) or ATO at doses of 0·05 (•), 0·25 (bsl00063), 1·25 (bsl00072) or 6 mg/kg (bsl00066) daily for 5 d a week via intraperitoneal injection.

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Arsenic trioxide enhances the cytotoxicity of melphalan to inhibit multiple myeloma cell growth in vitro

We examined the synergistic effects of combination treatment with ATO and melphalan against RPMI8226 and U266 MM cells. Experiments were performed in which RPMI8226 and U266 MM cells were cultured for 48 h in the presence of ATO alone (1 μmol/l), melphalan alone at varying concentrations (0·1–10 μmol/l), a constant concentration of ATO (1 μmol/l) and varying concentrations of melphalan administered simultaneously (0·1–10 μmol/l) (Fig 3A), or a constant concentration of melphalan (10 μmol/l) and varying concentrations of ATO (0·1–30 μmol/l) (Fig 3B). Anti-MM effects of these combinations were assessed using MTT assays. RPMI8226 and U266 cells treated with either ATO or melphalan alone showed a concentration-dependent decrease in cell viability. When the concentration of ATO was held constant at 1 μmol/l, a more marked decrease in cell viability was observed with the addition of melphalan at concentrations that ranged from 0·1 to 10 μmol/l than with the use of either drug alone (Fig 3A).

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Figure 3.  Arsenic trioxide (ATO) shows synergistic antimyeloma effects in RPMI8226 and U266 cells when combined with melphalan (Mel). RPMI8226 and U266 cells were incubated for 48 h with (A) ATO (1 μmol/l) alone or in combination with varying concentrations of melphalan (0·1–10 μmol/l) and (B) melphalan (10 μmol/l) alone or in combination with varying concentrations of ATO (0·1–3 μmol/l).

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Similarly, when the melphalan concentration was held constant at 10 μmol/l, a much greater than additive anti-MM effect was observed with the combination of melphalan and ATO at concentrations that varied from 0·1 to 3 μmol/l than with the use of either drug alone (Fig 3B).

Arsenic trioxide combined with melphalan induces cell death in human MM cells

Inhibition of proliferation mediated by ATO alone and in combination with melphalan in RPMI8226 and U266 cells was accompanied by cell death. Determination of apoptosis by activated capsase-3 showed that RPMI8226 cells underwent apoptosis following treatment with the combination of ATO and melphalan (Fig 4C). Total viable cell recovery by the Trypan Blue exclusion assay showed that the treatment with single agent drugs inhibited cell recovery compared with no treatment at all, with melphalan treatment exhibiting the highest inhibition (Fig 4A). Treatment with the combination of AA and melphalan, or AA (10 or 100 μmol/l) and ATO (5 or 10 μmol/l) resulted in no significant difference in cell recovery compared with treatment with each drug alone. However, the combination of ATO (5 or 10 μmol/l) and melphalan resulted in significant inhibition of viable cell recovery compared with control and/or single drug treatment (P < 0·025). Furthermore, the triple combination of melphalan (3 or 30 μmol/l), ATO (5 or 10 μmol/l), and AA (10 or 100 μmol/l) also resulted in significant inhibition compared with control or drug alone (P < 0·025) but not significant compared with the combination of ATO (5 or 10 μmol/l) and melphalan (3 or 30 μmol/l).

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Figure 4.  Combination treatment with melphalan and arsenic trioxide (ATO) inhibits cell growth, contributes to cell death and induces apoptosis in human RPMI8226 multiple myeloma cells treated with ATO (5 or 10 μmol/l), melphalan (3 or 30 μmol/l) or AA (10 or 100 μmol/l) alone or in combination for 24 h. 5 × 105 RPMI8226 cells were cultured in the presence of study drug or media for 24 h. Cells receiving combination drugs were treated simultaneously. (A) Total viable cell recovery by Trypan Blue dye exclusion. (B) Total cell death as measured by Trypan Blue dye exclusion. (C) Determination of apoptosis by active caspase-3 assay. *P-values, combined treatment versus control and/or single drug treatment.

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Total cell death was also determined using the Trypan Blue exclusion method (Fig 4B). All the study drugs exhibited some toxicity (the highest obtained with 3 or 30 μmol/l melphalan) although none were significantly different from that observed in control medium. The combination of ATO (5 or 10 μmol/l) and melphalan (3 or 30 μmol/l) resulted in significant cell death compared with control and/or single drug treatment (P < 0·03). The triple combination of melphalan (3 or 30 μmol/l), ATO (5 or 10 μmol/l), and AA (10 or 100 μmol/l) resulted in significant cell death compared with control or drug alone (P < 0·03) but not significant compared with the combination of ATO and melphalan.

Analysis of activated caspase-3 showed that cell death observed in control cultures could be mainly attributed to apoptosis (Fig 4C), where the cell death observed with all drugs results from the combined effects of apoptosis and some necrosis. The highest, most significant level of apoptosis was observed with the combination of ATO (5 or 10 μmol/l) and melphalan (3 or 30 μmol/l) compared with control and/or single drug treatment (P < 0·04). The triple combination of melphalan (3 or 30 μmol/l), ATO (5 or 10 μmol/l), and AA (10 or 100 μmol/l) resulted in significant differences in apoptosis when compared with control and/or single agent treatment (P < 0·04) but not more than the cell death observed with the ATO–melphalan combination.

Arsenic trioxide, melphalan and ascorbic acid inhibit growth of the melphalan-resistant tumour LAGλ-1

LAGλ-1 was derived from a patient resistant to melphalan-based therapy, and this chemo-resistance was demonstrated through the ability of LAGλ-1to grow in vivo in the presence of high doses of melphalan (Campbell et al, 2006). We sought to determine whether, the addition of ATO at doses that do not show anti-MM activity in vivo could chemosensitise LAGλ-1 to doses of melphalan or AA that also show no activity when administered alone. SCID mice were surgically implanted with LAGλ-1 tumours into the left hind limb muscle as previously described. ATO at 1·25 mg/kg daily for 5 d a week, melphalan weekly at 0·6 mg/kg, and AA at 100 mg/kg daily for 5 d a week were administered intraperitoneally alone or in doublet or triplet combinations. Control mice were administered only 0·9% normal saline.

LAGλ-1-bearing mice treated with single-agent ATO, melphalan or AA, and the combination of ATO and AA showed no significant differences in tumour volume or human paraprotein levels when compared with control mice (Figs 5A and B). After 3 weeks of treatment with melphalan and ATO, we found significant anti-MM effects on both paraprotein secretion and tumour growth compared with mice receiving single-agent treatment or vehicle alone (Figs 5A and B). Notably, the most dramatic anti-MM effects were observed in mice that were treated with all three agents together (Figs 5A and B); paraprotein levels (P < 0·001) and tumour volume (P < 0·003) were significantly reduced, compared with control mice. Interestingly, the mice that received the combination of AA and melphalan exhibited significant reduction in both human paraprotein levels and tumour volume compared with animals treated with single-agent AA, melphalan or vehicle. These latter findings support previous studies demonstrating the importance of GSH in melphalan resistance (Bellamy et al, 1991; Grad et al, 2001), because lowering free GSH with AA enhances the anti-MM effects of melphalan in the LAGλ-1 model. In contrast to the results from the in vitro studies, the combination of ATO and AA at these doses showed no significant anti-MM effects in the LAGλ-1-bearing mice. Importantly, mice receiving either the ATO and melphalan, AA and melphalan, or the MAC combination treatment tolerated the therapy well without signs of toxicity.

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Figure 5.  Reduction in human IgG levels and tumour volumes in LAGλ-1-bearing severe combined immunodeficient (SCID) mice treated with a combination of melphalan, arsenic trioxide (ATO), and vitamin C (MAC). Serum levels of human IgG (A) and tumour volume (B) were measured in SCID mice after implantation with a LAGλ-1 tumour fragment. Mice received vehicle (bsl00001) or AA (bsl00066, 100 mg/kg), melphalan (bsl00072, 0·6 mg/kg), ATO (bsl00063, 1·25 mg/kg), ascorbic acid + melphalan (•), ATO + ascorbic acid (bsl00000), ATO + melphalan (Δ), or melphalan + ATO + ascorbic acid (bsl00083, MAC). *P-values, MAC versus vehicle.

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Arsenic trioxide enhances the cytotoxicity of bortezomib to inhibit multiple myeloma cell growth in vitro

We examined the synergistic effects of combination treatment with ATO and bortezomib against RPMI8226 and U266 cells. Experiments were performed in which each MM cell line was cultured for 48 h in the presence of ATO alone (1 μmol/l), bortezomib alone at varying concentrations (0·03–3 nmol/l), a constant concentration of ATO (1 μmol/l) and varying concentrations of bortezomib administered simultaneously (0·03–3 nmol/l) (Fig 6A), or a constant concentration of bortezomib (3 nmol/l RPMI8226 and 1 nmol/l U266) and varying concentrations of ATO (0·01–3 μmol/l) (Fig 6B). Anti-MM effects of these combinations were assessed using MTT assays. Both RPMI8226 and U266 cells treated with either ATO or bortezomib alone showed a concentration-dependent decrease in cell viability. When the concentration of ATO was held constant at 1 μmol/l, a more marked decrease in cell viability was observed with the addition of bortezomib, at concentrations that ranged from 0·03 to 3 nmol/l, than the effects of either drug alone (Fig 6A).

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Figure 6.  Arsenic trioxide (ATO) shows synergistic anti multiple myeloma effects in RPMI8226 and U266 cells when combined with bortezomib (Bort). RPMI8226 and U266 cells were incubated for 48 h with (A) ATO (1 μmol/l) alone or in combination with varying concentrations of bortezomib (0·1–3 nmol/l, RPMI8226; 0·03–1 nmol/l, U266), and (B) bortezomib alone (3 nmol/l, RPMI8226; 1 nmol/l, U266) or in combination with varying concentrations of ATO (0·01–3 μmol/l).

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Similarly, when the bortezomib concentration was held constant at 1 nmol/l (U266) or 3 nmol/l (RPMI8226), a much greater than additive anti-MM effect was observed with the combination of bortezomib and ATO, at concentrations that varied from 0·01 to 3 μmol/l, than with the use of either drug alone (Fig 6B).

Arsenic trioxide enhances the cytotoxicity of bortezomib in vivo

Severe combined immunodeficient mice surgically implanted with 2·0–4·0 mm3 LAGλ-1 tumour fragments developed measurable tumours and detectable levels of serum hIgG. Twenty-six days following implantation of the tumour, hIgG levels were measured in the mouse serum, and mice were blindly assigned to one of four treatment groups: ATO alone (1·25 mg/kg) administered daily for 5 d a week for the duration of the study via intraperitoneal injection; bortezomib alone (0·25 mg/kg) administered twice weekly via intravenous injection; the combination of ATO and bortezomib; and 0·9% normal saline as vehicle. Animals treated with either ATO or bortezomib alone showed paraprotein secretion (Fig 7A) and tumour growth characteristics (Fig 7B) similar to those animals receiving vehicle. compared with mice treated with control and/or single-agent therapy, the animals receiving the combination of ATO and bortezomib showed significant inhibition of LAGλ-1, as demonstrated by both significantly reduced tumour growth (P < 0·003, Fig 7B) and decreased hIgG secretion (P < 0·001; Fig 7A).

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Figure 7.  Reduction in human IgG levels and tumour volumes in LAGλ-1-bearing severe combined immunodeficient (SCID) mice treated with a combination of arsenic trioxide (ATO) and bortezomib. Serum levels of human IgG (A) and tumour volume (B) were measured in SCID mice after implantation with a LAGλ-1 tumour fragment. Mice received vehicle (bsl00001) or bortezomib (bsl00066, 0·25 mg/kg), ATO (bsl00072, 1·25 mg/kg), or ATO + bortezomib (bsl00063). *P-values, ATO + bortezomib versus control and/or single agent.

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Discussion

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

During the course of their disease, MM patients’ tumour cells inevitably develop a chemoresistant phenotype when treated with chemotherapeutic agents. This is important to consider when developing new therapies and regimens that must overcome the chemoresistant threshold to effectively improve overall treatment. Here, the results from these studies involving human myeloma cells in vitro and in vivo in our SCID-hu animal model of myeloma, LAGλ-1, provide the rationale for combining ATO with several currently active antimyeloma drugs for patients with this incurable B-cell malignancy.

Multiple myeloma cells cultured in vitro in the presence of clinically achievable concentrations of ATO (0·01–3 μmol/l) were found to show significant inhibition of cell growth, which suggests a favourable therapeutic index for the use of this drug in myeloma patients. These in vitro results prompted the evaluation of ATO in vivo in our SCID-hu animal model of myeloma, LAGλ-1. SCID mice implanted intramuscularly with this tumour showed marked anti-MM effects at the highest dose administered (6·0 mg/kg) daily for 5 d a week but not at several lower doses. The dose that produced the anti-MM effect was comparable with other in vivo preclinical xenograft studies using ATO (Kito et al, 2003; Rousselot et al, 2004).

ATO has previously been shown to induce apoptosis in vitro (Friedman et al, 2002; Han et al, 2005). Although RPMI8226 cells cultured with either media or AA alone showed no significant apoptotic effects in our study, cells cultured with either melphalan or ATO showed an increase in cells undergoing apoptosis. However, a significant increase in caspase-3 activation was observed in cells treated with the combination of ATO and melphalan as compared with control and/or single agent treatment.

We have previously shown that LAGλ-1 in SCID mice responds to high doses (0·5 mg/kg) of the proteasome inhibitor bortezomib (Campbell et al, 2006). Both ATO and bortezomib have been shown to reduce NF-κB activation in tumour cells (Friedman et al, 2002; Mitsiades et al, 2002). Although ATO and bortezomib share some similar potential mechanisms by which they inhibit myeloma (Miller et al, 2002; Chauhan et al, 2005), these agents differ in the way they inhibit NF-κB and functionally modulate diverse signaling pathways, such as those involving mitogen-activated protein kinase and phosphoinositide-3 kinase/Akt (Cavigelli et al, 1996; Chauhan et al, 2005), and affect Bcl-2 levels (Chen et al, 1996; Akao et al, 1998). Thus, it is possible that combinations of these two agents may be more effective than either alone for the treatment of myeloma.

Exposure of RPMI8226 and U266 cells to low concentrations of ATO and bortezomib produced significantly more growth inhibition than treatment with either of these agents alone. These results suggest that treatment with this combination is synergistic in its antimyeloma effects and that some of the anti-MM effects of these two agents are different from each other. In support of these in vitro findings, when we combined doses of ATO (1·25 mg/kg) and bortezomib (0·25 mg/kg) that showed no anti-MM effects when each was administered alone to the LAGλ-1-bearing animals, significant tumour growth inhibition and decreased serum paraprotein levels were observed. Such a low-dose combination may have the potential to limit toxicity for patients who are elderly or have other comorbidities that preclude higher-dose or other aggressive chemotherapy-based regimens. Indeed, results from a recent multicenter, open-label, phase I dose-escalation study have shown that an ATO/bortezomib/AA regimen was well tolerated and produced significant clinical benefits in a heavily pretreated study population even among patients receiving lower doses of these drugs (Berenson et al, 2007).

We investigated, whether low doses of ATO were capable of chemosensitising LAGλ-1 to melphalan. Our studies showed that the addition of ATO could overcome the melphalan resistance of LAGλ-1, as demonstrated by the nearly 50% reduction in tumour volume in the LAGλ-1-bearing mice treated with this combination compared with those animals treated with ATO, melphalan, or vehicle alone. Interestingly, a reduction in tumour volume and levels of serum paraprotein was also observed among mice treated with the combination of AA and melphalan. As noted above, this observation supports the concept that AA can enhance the antimyeloma effects of melphalan, perhaps by depleting intracellular GSH. It is important to note that the most profound anti-MM effects on LAGλ-1 growth in vivo were produced when the mice were treated with all three agents (MAC) together. Indeed, in a clinical study of the MAC combination in patients with relapsed or refractory MM, objective responses were observed in 48% of patients, including those who had previously been treated with melphalan and bortezomib (Berenson et al, 2006).

The data described here, provide the preclinical rationale for combining ATO with other available antimyeloma agents. These preclinical results have recently been translated into clinical trials and show the efficacy of some of these ATO-containing combinations for the treatment of patients with relapsed or refractory MM (Berenson et al, 2006; Berenson et al, 2007). In addition to the promising results of the phase I trial combining bortezomib and AA with ATO (Berenson et al, 2007), recent results from a large multicenter phase II study showed that nearly half of 65 heavily pretreated MM patients showed responses to the MAC combination, with a median duration of response of 12 months, and that this regimen was well tolerated (Berenson et al, 2006). Clearly, there is much to learn about combination therapy in MM as well as dosing and scheduling of multiple agents. For example, recent studies from our laboratory show that more frequent administration of lower doses of liposomal doxorubicin produces a much greater anti-MM effect on LAGλ-1 and is better tolerated by the mice than are less frequent higher doses of this drug (Campbell et al, 2006). Future efforts will be directed toward optimising the dose and schedule of these combination treatments for patients with MM. In addition, other agents known to be active in MM patients will also be evaluated with ATO, including anthracyclines, cyclophosphamide, thalidomide and lenalidomide. These studies should rapidly provide support for clinical trials evaluating the most promising ATO-containing regimens from the results of these preclinical studies. Ultimately, these efforts should help to increase the number of therapeutic options for patients with this incurable B-cell malignancy.

Acknowledgement

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

The authors gratefully acknowledge Anna Lau, PhD and Christine Pan James for helping develop and edit the manuscript.

References

  1. Top of page
  2. Summary
  3. Methods and materials
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  • Akao, Y., Mizoguchi, H., Kojima, S., Naoe, T., Ohishi, N. & Yagi, K. (1998) Arsenic induces apoptosis in B-cell leukaemic cell lines in vitro: activation of caspases and down-regulation of Bcl-2 protein. British Journal of Haematology, 102, 10551060.
  • American Cancer Society. (2006) Cancer Facts and Figures 2006. American Cancer Society, Atlanta.
  • Bellamy, W.T., Dalton, W.S., Gleason, M.C., Grogan, T.M. & Trent, J.M. (1991) Development and characterization of a melphalan resistant human multiple myeloma cell line. Cancer Research, 51, 9951002.
  • Berenson, J.R. (2004) Biology and Treatment of Multiple Myeloma. Humana Press, Totowa, NJ.
  • Berenson, J.R., Boccia, R., Siegel, D., Bozdech, M., Bessudo, A., Stadtmauer, E., Pomeroy, J.T., Steis, R., Flam, M., Lutzky, J., Jilani, S., Volk, J., Wong, S.F., Moss, R., Patel, R., Ferretti, D., Russell, K., Louie, R., Yeh, H.S. & Swift, R.A. (2006) Efficacy and safety of melphalan, arsenic trioxide and ascorbic acid combination therapy in patients with relapsed or refractory multiple myeloma: a prospective, multicentre, phase II, single-arm study. British Journal of Haematology, 135, 174183.
  • Berenson, J.R., Matous, J., Swift, R.A., Mapes, R., Morrison, B. & Yeh, H.S. (2007) A phase I/II study of arsenic trioxide/bortezomib/ascorbic acid (ABC) combination therapy for the treatment of relapsed or refractory multiple myeloma. Clinical Cancer Research, 13, 17621768.
  • Campbell, R.A., Manyak, S.J., Yang, H.H., Sjak-Shie, N.N., Chen, H., Gui, D., Popoviciu, L., Wang, C., Gordon, M., Pang, S., Bonavida, B., Said, J. & Berenson, J.R. (2006) LAGlambda-1: a clinically relevant drug resistant human multiple myeloma tumor murine model that enables rapid evaluation of treatments for multiple myeloma. International Journal of Oncology, 6, 14091417.
  • Cavigelli, M., Li, W.W., Lin, A., Su, B., Yoshioka, K. & Karin, M. (1996) The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. The EMBO Journal, 15, 62696279.
  • Chauhan, D., Hideshima, T., Mitsiades, C., Richardson, P. & Anderson, K.C. (2005) Proteasome inhibitor therapy in multiple myeloma. Molecular Cancer Therapeutics, 4, 686692.
  • Chen, G.Q., Zhu, J., Shi, X.G., Ni, J.H., Zhong, H.J., Si, G.Y., Jin, X.L., Tang, W., Li, X.S., Xong, S.M., Shen, Z.X., Sun, G.L., Ma, J., Zhang, P., Zhang, T.D., Gazin, C., Naoe, T., Chen, S.J., Wang, Z.Y. & Chen, Z. (1996) In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood, 88, 10521061.
  • Chou, T.C. & Talalay, P. (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 2755.
  • Feinman, R., Koury, J., Thames, M., Barlogie, B., Epstein, J. & Siegel, D.S. (1999) Role of NF-kappaB in the rescue of multiple myeloma cells from glucocorticoid-induced apoptosis by Bcl-2. Blood, 93, 30443052.
  • Friedman, J.M., Ma, M.H., Manyak, S.J., Borad, M.J., Altamirano, C.V., Tang, Y.M., Neeser, J.A., Okopnik, J., Roussos, E., Yang, H., Vescio, R. & Berenson, J.R. (2002) Arsenic trioxide causes apoptosis, growth inhibition and increased sensitivity to chemotherapeutic agents in multiple myeloma cells through inhibition of (NF)-kB activity. Proceedings of the American Association of Cancer Research, 43, 925 (Abstract 4585).
  • Grad, J., Bahlis, N., Reis, I., Oshiro, M., Dalton, W. & Boise, L. (2001) Ascorbic acid enhances arsenic trioxide-induced cytotoxicity in multiple myeloma cells. Blood, 98, 805813.
  • Gupta, V., Singh, S.V., Ahmad, H., Medh, R.D. & Awasthi, Y.C. (1989) Glutathione and glutathione S-transferases in a human plasma cell line selected for resistance to melphalan. Biochemical Pharmacology, 38, 19932000.
  • Han, S.S., Kim, K., Hahm, E.R., Park, C.H., Kimler, B.F., Lee, S.J., Lee, S.H., Kim, W.S., Jung, C.W., Park, K., Kim, J., Yoon, S.S., Lee, J.H. & Park, S. (2005) Arsenic trioxide represses constitutive activation of NF-kappaB and COX-2 expression in human acute myeloid leukemia, HL-60. Journal of Cell Biochemistry, 94, 695707.
  • Hayashi, T., Hideshima, T., Akiyama, M., Richardson, P., Schlossman, R.L., Chauhan, D., Munshi, N.C., Waxman, S. & Anderson, K.C. (2002) Arsenic trioxide inhibits growth of human multiple myeloma cells in the bone marrow microenvironment. Molecular Cancer Therapeutics, 1, 851860.
  • Hideshima, T., Richardson, P., Chauhan, D., Palombella, V.J., Elliott, P.J., Adams, J. & Anderson, K.C. (2001) The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Research, 61, 30713076.
  • Hideshima, T., Mitsiades, C., Akiyama, M., Hayashi, T., Chauhan, D., Richardson, P., Schlossman, R., Podar, K., Munshi, N.C., Mitsiades, N. & Anderson, K.C. (2003) Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341. Blood, 101, 15301534.
  • Hideshima, T., Bradner, J.E., Wong, J., Chauhan, D., Richardson, P., Schreiber, S.L. & Anderson, K.C. (2005) Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma. Proceedings of the National Academy of Sciences of the United States of America, 102, 85678572.
  • Huerta-Yepez, S., Vega, M., Jazirehi, A., Garban, H., Hongo, F., Cheng, G. & Bonavida, B. (2004) Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-kappa B and inhibition of Bcl-xl expression. Oncogene, 23, 49935003.
  • Hussein, M.A., Saleh, M., Ravandi, F., Mason, J., Rifkin, R.M. & Ellison, R. (2004) Phase 2 study of arsenic trioxide in patients with relapsed or refractory multiple myeloma. British Journal of Haematology, 125, 470476.
  • Kapahi, P., Takahashi, T., Natoli, G., Adams, S.R., Chen, Y., Tsien, R.Y. & Karin, M. (2000) Inhibition of NF-kappa B activation by arsenite through reaction with a critical cysteine in the activation loop of Ikappa B kinase. Journal of Biological Chemistry, 275, 3606236066.
  • Kito, M., Matsumoto, K., Wada, N., Sera, K., Futatsugawa, S., Naoe, T., Nozawa, Y. & Akao, Y. (2003) Antitumor effect of arsenic trioxide in murine xenograft model. Cancer Science, 94, 10101014.
  • Ma, M.H., Yang, H.H., Parker, K., Manyak, S., Friedman, JM, Altamirano, C., Wu, Z.Q., Borad, M.J., Frantzen, M., Roussos, E., Neeser, J., Mikail, A., Adams, J., Sjak-Shie, N., Vescio, R.A. & Berenson, J.R. (2003) The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents. Clinical Cancer Research, 9, 11361144.
  • Mathieu, J. & Besançon, F. (2006) Clinically tolerable concentrations of arsenic trioxide induce p53-independent cell death and repress NF-kappa B activation in Ewing sarcoma cells. International Journal of Cancer, 119, 17231727.
  • May, J.M., Qu, Z., Li, X. (2001) Requirement for GSH in recycling of ascorbic acid in endothelial cells. Biochemical Pharmacology, 62, 873881.
  • Miller, W.H. Jr, Schipper, H.M., Lee, J.S., Singer, J. & Waxman, S. (2002) Mechanisms of action of arsenic trioxide. Cancer Research, 62, 38933903.
  • Mitsiades, N., Mitsiades, C.S., Poulaki, V., Chauhan, D., Richardson, P.G., Hideshima, T., Munshi, N., Treon, S.P. & Anderson, K.C. (2002) Biologic sequelae of nuclear factor-kappaB blockade in multiple myeloma: therapeutic applications. Blood, 99, 40794086.
  • Mitsiades, N., Mitsiades, C.S., Richardson, P.G., Poulaki, V., Tai, Y.T., Chauhan, D., Fanourakis, G., Gu, X., Bailey, C., Joseph, M., Libermann, T.A., Schlossman, R., Munshi, N.C., Hideshima, T. & Anderson, K.C. (2003) The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood, 101, 23772380.
  • Munshi, N.C., Tricot, G., Desikan, R., Badros, A., Zangari, M., Toor, A., Morris, C., Anaissie, E. & Barlogie, B. (2002) Clinical activity of arsenic trioxide for the treatment of multiple myeloma. Leukemia, 16, 18351837.
  • Niu, C., Yan, H., Yu, T., Sun, H.P., Liu, J.X., Li, X.S., Wu, W., Zhang, F.Q., Chen, Y., Zhou, L., Li, J.M., Zeng, X.Y., Yang, R.R., Yuan, M.M., Ren, M.Y., Gu, F.Y., Cao, Q., Gu, B.W., Su, X.Y., Chen, G.Q., Xiong, S.M., Zhang, T.D., Waxman, S., Wang, Z.Y., Chen, Z., Hu, J., Shen, Z.X. & Chen, S.J. (1999) Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood, 94, 33153324.
  • Rousselot, P., Labaume, S., Marolleau, J.P., Larghero, J., Nogeura, M.H., Brouet, J.C. & Fermand, J.P. (1999) Arsenic trioxide and melarsoprol induce apoptosis in plasma cell lines and in plasma cells from myeloma patients. Cancer Research, 59, 10411048.
  • Rousselot, P., Larghero, J., Labaume, S., Poupon, J., Chopin, M., Dosquet, C., Marolleau, J.P., Janin, A., Brouet, J.C. & Fermand, J.P. (2004) Arsenic trioxide is effective in the treatment of multiple myeloma in SCID mice. European Journal of Haematology, 72, 166171.
  • Soignet, S.L., Frankel, S.R., Douer, D., Tallman, M.S., Kantarjian, H., Calleja, E., Stone, R.M., Kalaycio, M., Scheinberg, D.A., Steinherz, P., Sievers, E.L., Coutre, S., Dahlberg, S., Ellison, R. & Warrell, R.P. Jr. (2001) United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. Journal of Clinical Oncology, 19, 38523860.
  • Zhang, W., Ohnishi, K., Shigeno, K., Fujisawa, S., Naito, K., Nakamura, S., Takeshita, K., Takeshita, A. & Ohno, R. (1998) The induction of apoptosis and cell cycle arrest by arsenic trioxide in lymphoid neoplasms. Leukemia, 12, 13831391.