Abstract
Here, we report on the organic arsenical darinaparsin (ZIO-101, S-dimethylarsino-glutathione) and its anti-myeloma activity compared with inorganic arsenic trioxide. Darinaparsin induced apoptosis in multiple myeloma cell lines in a dose-dependent manner, and the addition of N-acetylcysteine, which increases intracellular glutathione (GSH), blocked cytotoxicity of both darinaparsin and arsenic trioxide. In contrast to arsenic trioxide, intracellular GSH does not appear to be important for darinaparsin metabolism, as an inhibitor of GSH synthesis, buthionine sulfoximine, had little effect on drug activity. This discrepancy was resolved when we determined the effects of thiols on drug uptake. The addition of exogenous GSH, l-cysteine, or d-cysteine prevented darinaparsin cellular uptake and cell death but had no effect on the uptake or activity of arsenic trioxide, suggesting a difference in the transport mechanism of these two drugs. In addition, gene expression profiling revealed differences in the signaling of protective responses between darinaparsin and arsenic trioxide. Although both arsenicals induced a transient heat shock response, only arsenic trioxide treatment induced transcription of metal response genes and anti-oxidant genes related to the Nrf2-Keap1 pathway. In contrast to the protective responses, both arsenicals induced up-regulation of BH3-only proteins. Moreover, silencing of BH3-only proteins Noxa, Bim, and Bmf protected myeloma cells from darinaparsin-induced cell death. Finally, treatment of an arsenic trioxide-resistant myeloma cell line with darinaparsin resulted in dose-dependent apoptosis, indicating that cross-resistance does not necessarily develop between these two forms of arsenic in multiple myeloma cell lines. These results suggest darinaparsin may be useful as an alternative treatment in arsenic trioxide-resistant hematologic cancers.[Mol Cancer Ther 2009;8(5):OF1–10]
Introduction
Multiple myeloma is a progressive hematologic malignancy that ultimately results in renal failure, lytic bone lesions, hypercalcemia, and anemia (1). Despite numerous advances in therapy, multiple myeloma remains incurable, with an average survival range from 3 to 6 years (1–3). Standard myeloma treatment traditionally involves high-dose chemotherapy and autologous stem cell transplant (4). However, greater understanding of the biology of myeloma has resulted in the development of targeted drugs, affecting intracellular survival pathways as well as the interaction of myeloma cells with the bone marrow microenvironment (4). These studies resulted in the use of thalidomide and lenalidomide in newly diagnosed patients and bortezomib in previously treated patients. Additionally, bortezomib is also being used in combination with melphalan and prednisone in newly diagnosed patients. Unfortunately, even the most effective treatment eventually fails, resulting in chronic relapse and treatment resistance. Due to this, new therapeutic advances are of the utmost importance.
The success of inorganic arsenic trioxide in the treatment of relapsed promyelocytic leukemia has resulted in resurgence in arsenic treatment of hematologic cancers. The efficacy of both organic and inorganic arsenicals is being evaluated in preclinical studies as well as clinical trials. Treatment of multiple myeloma cell lines and newly isolated patient samples with clinically achievable doses of arsenic trioxide resulted in growth inhibition and apoptosis (2, 5, 6). Similarly, in a phase II trial of refractory or relapsed multiple myeloma patients, arsenic trioxide exhibited modest activity and was well tolerated (7, 8). Interestingly, the cytotoxic effects of arsenic trioxide appear to be strongly related to the intracellular glutathione (GSH) concentration of target cells (9). Indeed, combining arsenic trioxide treatment with GSH-depleting agents, such as buthionine sulfoximine or ascorbic acid, resulted in enhanced growth inhibition and apoptosis and was well tolerated in patients (6, 8, 10). Unfortunately, the limited efficacy of arsenic trioxide as a single agent and the risk of systemic toxicity often make it a drug of last resort.
Organic arsenicals are generally believed to be less toxic and better tolerated than inorganic arsenicals, such as arsenic trioxide (11). The efficacy of the organic arsenical melarsoprol, commonly used to treat African trypanosomiasis, was investigated in hematologic cancers. Treatment of multiple myeloma cell lines and patient samples with pharmacologic concentrations of melarsoprol resulted in dose- and time-dependent growth inhibition and apoptosis (5). Unfortunately, melarsoprol clinical trials in both leukemia and myeloma patients had to be stopped due to central nervous system toxicity and other serious adverse events (7, 12).
Darinaparsin is a novel organic arsenical consisting of GSH-conjugated dimethylarsenic. Initial studies indicate this arsenical is active against xenograft tumors and has a maximum tolerated dose that is 35-fold higher than arsenic trioxide in mice (13). Furthermore, phase I studies have indicated its safety in cancer patients and various phase II clinical studies with oral and i.v. administration are under way (14). However, the underlying mechanism by which darinaparsin exerts its apoptotic effect has yet to be determined.
In this study, we used gene expression profiling to investigate differences in myeloma cell response to arsenic trioxide and darinaparsin to determine the likely usefulness of darinaparsin in arsenic trioxide-resistant myeloma. The mechanism of cellular uptake, metabolism, and signaling of protective responses appears to differ from that of arsenic trioxide, suggesting that darinaparsin may be an effective treatment for arsenic trioxide-resistant multiple myeloma.
Materials and Methods
Cell Lines
Multiple myeloma cell lines U266 and 8226/S were purchased from the American Type Culture Collection. MM.1s cell line was obtained from Dr. Steven Rosen (Northwestern University), and KMS11 cell line was provided by Dr. P. Leif Bergsagel (Mayo Clinic). Cells were maintained on supplemented RPMI 1640 as described previously (15). 8226/S-ATOR05 was maintained on supplemented RPMI 1640 containing 1 μmol/L arsenic trioxide.
Reagents
Buthionine sulfoximine, GSH, L-cysteine, D-cysteine, propidium iodide, and Igepal CA-360 were purchased from Sigma-Aldrich. Nitric acid, trace metal grade, and hydrogen peroxide, ACS grade, were purchased from Fisher Scientific. Standards (1,000 mg/L) of arsenic and yttrium in 2% nitric acid, ICP-MS grade, were purchased from GFS Chemicals. Annexin V- FITC was purchased from Biovision. N-acetylcysteine (NAC) was purchased from Bedford Laboratories. Darinaparsin (ZIO-101), S-dimethylarsino-glutathione, was provided by Ziopharm Oncology. Arsenic trioxide was provided by Cephalon.
Cellular Assays
Cells were cultured at a concentration of 2.5 × 105/mL in supplemented RPMI 1640 as described previously (15) and incubated with the indicated concentrations of darinaparsin or arsenic trioxide and/or 100 μmol/L buthionine sulfoximine, 10 mmol/L NAC, 5 mmol/L GSH, 5 mmol/L L-cysteine, or 5 mmol/L D-cysteine.
Viability Assays
Cell viability was measured by Annexin V-FITC and propidium iodide staining as described previously (6). Data were acquired on a FACScan flow cytometer (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson).
Small Interfering RNA Assays
ON-TARGETplus SMART pool small interfering RNAs (siRNA) were purchased from Dharmacon RNA Technologies. siCONTROL nontargeting siRNA [si(-)], siBmf (Bmf and L-004393), siNoxa (PMAIP1 and L-005275), and siBim (BCL2L11 and L-004383) were used. siRNAs were electroporated into 5 × 106 cells using Amaxa Program G-015 (Amaxa) following the manufacturer's instructions as described previously (15). Transfected cells were placed in 6-well plates with 3 mL medium for 16 h at 37°C and subsequently treated with 2 μmol/L darinaparsin. Cells were collected at 6 and 24 h for protein expression analysis and at 24 and 48 h for apoptosis determination.
Real-time PCR
Total RNA was isolated from si(-) control and siBmf-transfected samples at 6 and 24 h following treatment using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from 1 μg total RNA using the MuLV reverse transcriptase and random hexamer primers from the GeneAmp RNA PCR Kit (Applied Biosystems). Subsequent cDNA was amplified using the 20× human Bmf Mix (Hs00372937_m1, 204408) and the TaqMan Gene Expression Assay (Applied Biosystems) on the 7700 Sequence Detection System following the manufacturer's protocol. TaqMan human GAPDH (402869) was used as internal control and Bmf mRNA expression was calculated relative to GAPDH mRNA expression.
Western Blot analysis
Western blot analysis was done as described previously (16). Briefly, cells were washed once with PBS buffer and lysed in radioimmunoprecipitation assay buffer containing protease inhibitors and protein concentrations were determined using the BCA Protein Assay (Pierce Biotechnology). Total protein (20 μg) was subjected to SDS-PAGE and transferred to nitrocellulose membranes (Protan Nitrocellulose Transfer Membrane; Whatman).
Antibodies
The following antibodies were used for Western blot analysis: primary, mouse anti-Noxa (Abcam), rabbit anti-Bim (Chemicon), rabbit anti-heme oxygenase-1 (Santa Cruz Biotechnology), mouse anti-NAD(P)H quinone oxidoreductase 1 (Cell Signaling Technology), and rabbit anti-actin (Sigma-Aldrich); secondary, anti-mouse IgG1 horseradish peroxidase conjugate (Roche Applied Science) and ECL rabbit IgG horseradish peroxidase-linked whole antibody (GE Healthcare).
Gene Expression Profiling
8226/S and KMS11 cell lines were treated for 0, 6, and 24 h with 2 μmol/L arsenic trioxide or 2 μmol/L (8226/S) or 3 μmol/L (KMS11) darinaparsin, and total RNA was subsequently isolated using the RNeasy Mini Kit (Qiagen). Hybridization and initial data analysis were done by Expression Analysis, probing Affymetrix Hu133 2.0 Plus Chips as described previously (15). These data have been deposited in National Center for Biotechnology Information's Gene Expression Omnibus and are accessible by GEO Series accession no. GSE14519.
Luciferase Assay
Reporter constructs pGL3-promoter, pGL3-ARE-Luc, and pGL3-HSE-Luc were kindly provided by Dr. Craig Logsdon (M. D. Anderson Cancer Center; ref. 17). Each construct (3 μg) was cotransfected with 0.3 μg of the pRL reporter plasmid (Promega) by electroporation (Amaxa) following the manufacturer's instructions. Briefly, 4 × 106 cells were electroporated in 100 μL Nucleofector solution (Amaxa reagent V) with the appropriate volume of construct DNA using the preselected Amaxa Program G-015. Electroporated cells were placed into 6-well plates with 3 mL medium for 16 h at 37°C. Cells were treated with 2 μmol/L darinaparsin or arsenic trioxide and samples were harvested at 6 h for luciferase activity determination using the Dual Luciferase Assay Kit from Promega following the manufacturer's protocol.
Total Arsenic Determination
Cell Digestion. Five hundred microliters of 0.5% (v/v) Igepal were added to the previously washed cell pellets. With the use of an ultrasonic probe (Fisher Scientific), the cells were lysed and then homogenized. Five microliters of the solution were removed for protein analysis (BCA Assay; Pierce Biotechnology) and 500 μL concentrated nitric acid was added to the remaining sample. The samples were incubated for 1 h, and 30% hydrogen peroxide (250 μL) was added.
Standard and Sample Preparation. One to 40 μg/L of arsenic standards were prepared from a 1 mg/L stock, previously prepared from the 1,000 mg/L standard in 2% nitric acid. Yttrium was used as an internal standard at a concentration of 20 μg/L. For sample analysis, 250 μL of the cell digest was added to the internal standard and diluted to 5 mL with DDI water.
Instrument: ICP-MS. An Elan DRC-e (Perkin-Elmer) was used as an element specific detector. The ICP-MS was equipped with an autosampler, a cyclonic spray chamber, and a Meinhard nebulizer. The following signals were monitored: 75 for arsenic, 89 for yttrium, 77 for ArCl, and 82 to monitor, and correct for, any polyatomic interferences resulting from the presence of chloride in the samples. Data were treated with Elan software version 3.4 (Perkin-Elmer Sciex).
Statistical Analysis
All data are presented as mean ± SE of at least three independent experiments. GraphPad QuickCalcs online t test calculator was used for determination of statistical significance.
Results
Multiple Myeloma Cell Lines Display a Sensitivity Pattern to Darinaparsin That Is Distinct from Arsenic Trioxide
To determine the sensitivity of multiple myeloma cell lines to darinaparsin as well as compare it to arsenic trioxide, four multiple myeloma cell lines were treated with either drug for 24 or 48 h (Fig. 1). Darinaparsin was able to effectively kill three of the four cell lines in a dose-dependent manner, whereas the fourth cell line, U266, was somewhat resistant to the drug at the doses and time points investigated (Fig. 1A and C). In contrast, arsenic trioxide was effective at killing all four cell lines in a dose- and time-dependent manner (Fig. 1B and D). Interestingly, these four multiple myeloma cell lines were not equally responsive to the two drugs. For arsenic trioxide, U266 and 8226/S display similar sensitivities (IC50 1.9 μmol/L at 48 h), which are lower than those of KMS11 (IC50 1.2 μmol/L) and MM.1s (IC50 1.0 μmol/L), as indicated by apoptosis at 48 h. In contrast for darinaparsin, U266 (IC50 3.7 μmol/L) is the least sensitive, whereas 8226 (IC50 1.6 μmol/L) and KMS11 (IC50 1.2 μmol/L) are more sensitive than MM.1s (IC50 1.9 μmol/L) until one reaches the higher concentrations. However, if one normalizes these data to the number of arsenic atoms per molecule (1 for darinaparsin and 2 for arsenic trioxide), then 8226/S and KMS11 appear to be more sensitive to the organic form. These results suggest that these two arsenicals are either metabolized or function via distinct mechanisms.
To further investigate the mechanistic differences between these two arsenicals, we chose to focus on the two cell lines that were differentially affected by darinaparsin, KMS11 and 8226/S.
Buthionine Sulfoximine Increases the Activity of Arsenic Trioxide but Has No Effect on the Activity of Darinaparsin
The role of GSH in inorganic arsenic detoxification is well established; therefore, we tested its role in the metabolism of the organic arsenical darinaparsin. NAC increases intracellular cysteine for enhanced production of GSH, whereas buthionine sulfoximine inhibits the rate-limiting enzyme in GSH synthesis, γ-glutamate cysteine ligase, thereby blocking GSH production (18). To fully observe the protective effects of NAC, 8226/S and KMS11 cells were treated with 3 μmol/L darinaparsin or arsenic trioxide alone or in combination with 10 mmol/L NAC for 48 h. In contrast, to better observe the effects of buthionine sulfoximine, 8226/S and KMS11 cells were treated with a low concentration of either darinaparsin or arsenic trioxide. Based on dose curves from Fig. 1, 1.5 μmol/L darinaparsin or 1 μmol/L arsenic trioxide was used, and cells were treated with either arsenical alone or in combination with 100 μmol/L buthionine sulfoximine for 48 h.
Figure 2A shows NAC protected 8226/S and KMS11 cells from both darinaparsin-induced and arsenic trioxide-induced cell death, suggesting that increasing GSH production provides an advantage to cells treated with arsenicals regardless of the formulation. However, depletion of GSH with buthionine sulfoximine had distinct effects on darinaparsin and arsenic trioxide activity. Buthionine sulfoximine cotreatment resulted in increased arsenic trioxide activity in both cell lines. In contrast, cotreatment of cells with darinaparsin and buthionine sulfoximine did not result in any increased activity, yielding viabilities very similar to darinaparsin alone (Fig. 2B). These results suggest that GSH does not play the same role in darinaparsin metabolism as it does with arsenic trioxide.
Exogenous Cysteine and GSH Are Able to Block Darinaparsin Uptake and Cell Death
Darinaparsin is thought to be unstable in aqueous solutions, breaking down into its component parts, dimethylarsenic and GSH (19). Due to this, and the discrepancy in the effects of NAC and buthionine sulfoximine on darinaparsin activity, we wanted to examine the possibility that NAC could be acting outside the myeloma cells by binding to dimethylarsenic and thereby preventing drug entry and cell death. To test this, we first treated cells with 3 μmol/L darinaparsin or arsenic trioxide alone or in the presence of 5 mmol/L l-cysteine or d-cysteine. Although both forms of cysteine can be transported into the cell, they enter much less efficiently than NAC. Moreover, only l-cysteine is readily used for GSH production (18). If the free thiol group from cysteine is binding to dimethylarsenic outside the cell, we would expect to see protection from darinaparsin-induced cell death with both isomers. Indeed, both isomers of cysteine were able to protect cells from darinaparsin-induced cell death, whereas only l-cysteine provided any protection from arsenic trioxide-induced apoptosis (Fig. 2C).
To determine if this protection was due to thiol binding or competition for uptake, a thiol compound was required that is not readily transported into the cell. GSH was chosen because many cells do not express the transporters required for its direct uptake (18). Additionally, data from our gene expression profiling studies show that neither GS-X nor GS-Y is expressed in myeloma cell lines (data not shown). 8226/S and KMS11 cells were treated with 3 μmol/L darinaparsin or arsenic trioxide in the presence or absence of 5 mmol/L GSH for 48 h. The addition of GSH to the culture medium protected 8226/S and KMS11 cells from darinaparsin-induced apoptosis. However, exogenous GSH was unable to effectively protect either cell line from arsenic trioxide-induced apoptosis (Fig. 2D). These results suggest protection from darinaparsin-induced death by thiol-containing compounds is primarily a result of binding outside the cell and not competition for an uptake pump or increase GSH synthesis inside the cell.
To more directly test this possibility, 8226/S cells were treated with 2 μmol/L darinaparsin or arsenic trioxide alone or in combination with 5 mmol/L GSH and total arsenic uptake was determined under each condition at 30 min, 3 h, and 6 h post-treatment. We found that GSH was able to block darinaparsin uptake significantly and by >90% at all time points, whereas the effect on arsenic trioxide uptake was minimal (Fig. 3A and B). Interestingly, the total amount of intracellular arsenic was much greater in darinaparsin-treated cells, than in arsenic trioxide-treated cells, at all time points evaluated. These results suggest that myeloma cells transport darinaparsin much more efficiently than arsenic trioxide. In addition, our data suggest that the uptake of darinaparsin occurs faster than that of arsenic trioxide.
Arsenic Treatment Induces Protective and Apoptotic Responses in Myeloma Cell Lines
To investigate potential differences in mechanism of action between these two forms of arsenic, we performed gene expression profiling. 8226/S and KMS11 cells were treated with either darinaparsin or arsenic trioxide for 0, 6, and 24 h. Analysis of the expression profiles indicated differential responses between the two drugs, including the up-regulation of genes involved in three protective responses, the metal response, the heat shock response, and the anti-oxidant/electrophile response.
Strong up-regulation of metallothionein transcription was seen following treatment of myeloma cells with arsenic trioxide but was completely absent in cells treated with darinaparsin (Supplementary Table S1).4
4Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
Increased transcription of heat shock genes was seen transiently following treatment of myeloma cells with darinaparsin and arsenic trioxide, indicating both drugs induce cellular stress (Supplementary Table S1).4 Heat shock proteins are involved in a variety of cellular processes, including housekeeping functions and stress protection (21, 22); therefore, it is difficult to determine exactly what the increased transcription seen here is related to. However, high expression of heat shock protein 70 family proteins has been shown to promote cancer cell growth and protect myeloma cells from drug-induced cell death (23, 24). Regardless, the increased transcription is present only transiently following arsenic treatment and is unlikely to protect either cell line from darinaparsin-induced or arsenic trioxide-induced cell death (Fig. 1).
The increase in transcription at 6 and 24 h of both heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1, and many enzymes involved in GSH synthesis, suggested that the Nrf2-Keap1 pathway was activated in response to arsenic trioxide treatment (Supplementary Table S1).4 These findings were confirmed at the protein level (Fig. 4A). Interestingly, stable up-regulation of these genes was not seen following treatment with darinaparsin, indicating that darinaparsin does not induce this protective response. The Nrf2-Keap1 pathway is often induced following treatment of cells with chemopreventive agents (25, 26), and it results in a signal transduction cascade that leads to the dissociation of Nrf2 from Keap1 and its translocation into the nucleus. Once in the nucleus, Nrf2 binds to promoters containing anti-oxidant response/electrophile response elements, thereby initiating the transcription of phase II detoxification enzymes, anti-oxidants, DNA repair enzymes, and chaperone proteins (25, 27). However, as with the heat shock response, activation of this protective pathway does not appear to be sufficient to protect cells from arsenic trioxide-induced cell death.5
5Morales AA, Gutman D, Cejas PJ, Lee KP, Boise LH. Reactive oxygen species are not required for an arsenic trioxide-induced anti-oxidant response or apoptosis. J Biol Chem. In press, 2009.
To further investigate the activation of HSE- and ARE-related genes, we transiently transfected KMS11 cells with pGL3-ARE-Luc, pGL3-HSE-Luc, or pGL3-promoter reporter constructs and, 16 h later, treated the cells with 2 μmol/L arsenic trioxide or darinaparsin for 6 h. As expected, treatment of cells with darinaparsin or arsenic trioxide resulted in activation of the HSE reporter, whereas only arsenic trioxide activated the ARE-driven reporter (Fig. 4B).
To determine the possible mechanism of darinaparsin-induced cell death, the gene expression profiling was examined for changes in the transcription of BH3-only proteins, as these have been shown by our laboratory to be involved in arsenic trioxide-induced cell death (15). Supplementary Table S14 shows changes in transcription of Noxa, Bim, and Bmf, three BH3-only proteins important for arsenic trioxide-induced apoptosis (15, 28). Following treatment of 8226/S cells with darinaparsin, transcription of Noxa and Bim was up-regulated, whereas Bmf transcription remained unchanged. Bmf data were verified by real-time RCR; however, Western blot analysis only revealed an up-regulation of Noxa and not Bim, which is constitutively expressed at the protein level (data not shown). In KMS11 cells, however, Noxa and Bmf transcription was up-regulated, whereas Bim transcription was not.
To further investigate the roles of these proteins in darinaparsin-induced apoptosis, 8226/S cells were transiently transfected with siRNA for the above-mentioned BH3-only proteins, treated with 2 μmol/L of drug, and apoptosis was assessed. Real-time PCR was used to confirm Bmf silencing, comparing samples transfected with si(-) and siBmf (Fig. 5A). Western blot analysis verified silencing of Noxa and Bim (Fig. 5B). Silencing of Noxa and Bim partially protected 8226/S from darinaparsin-induced apoptosis, whereas Bmf silencing had no effect (Fig. 5C). These data are consistent with the gene expression profiling data that show up-regulation of Bim and Noxa and no transcription of Bmf in 8226/S cells. In KMS11 cells, silencing of all three BH3-only proteins partially protected against darinaparsin-induced cell death; however, the degree of silencing, and therefore protection, was not as great as that seen in 8226/S (data not shown).
Darinaparsin Induces Cell Death in an Arsenic Trioxide-Resistant Myeloma Cell Line
We have created an arsenic trioxide-resistant cell line, 8226/S-ATOR05.6
6In preparation.
Discussion
The results presented in this article suggest that the method of metabolism may play an important role in the overall potency of arsenic-containing drugs. Intracellular GSH is an important factor in numerous cellular processes, including detoxification of xenobiotics and anti-oxidant responses (18, 27, 29). Although the role of GSH in inorganic arsenic metabolism is well established, little is known regarding its role in the metabolism of organic arsenic. Similar to arsenic trioxide, multiple myeloma cells were protected from darinaparsin-induced cell death when cocultured with NAC, suggesting a role for GSH in darinaparsin metabolism. Alternatively, cotreatment of multiple myeloma cells with buthionine sulfoximine and darinaparsin had no additional activity unlike the increased activity seen with cotreatment of arsenic trioxide and buthionine sulfoximine. These results are consistent with a recent study of darinaparsin in leukemia cell lines; however, no explanation for the findings was provided (30).
The paradoxical nature of the NAC and buthionine sulfoximine results with darinaparsin led us to consider the possibility that NAC is exerting its effect on drug activity outside the cell rather than through the increased production of GSH. GS-conjugates can be in equilibrium with GSH outside the cell (19). Therefore, it is reasonable to propose that darinaparsin, which consists of GS-dimethylarsenic, could be in equilibrium with GSH and free dimethylarsenic. Assuming this is the situation, the addition of an exogenous thiol-containing compound should drive the equilibrium toward complex formation, thereby preventing the release of dimethylarsenic and its subsequent entry into the cell. The effects of exogenous GSH on total arsenic uptake suggest that this is a likely explanation for this paradox. Consistent with this possibility, the addition of exogenous cysteine or GSH prevented darinaparsin-induced apoptosis. Furthermore, preventing the breakdown of GSH into its constituent amino acids and the subsequent cysteine transport with acivicin did not inhibit darinaparsin-induced apoptosis.7
7Unpublished data.
Together, these data suggest that the active form of darinaparsin is actually free dimethylarsenic and that its disassociation from GSH is necessary for cellular uptake. However, this raises questions as to why inorganic arsenic is not affected by exogenous thiols. A likely possibility is that the requirement to bind three free thiols to neutralize arsenic trioxide is too great in the absence of conjugating enzymes, whereas dimethylarsenic can be inactivated by a single event. Perhaps this also explains why, in vivo, inorganic arsenic is converted to the more toxic dimethylarsenic for inactivation.
Similar to GSH, phase II detoxification enzymes are important cellular defenses against the effects of reactive oxygen species (ROS). Transcription of these enzymes is regulated by the Nrf2-Keap1 pathway (25, 26). Similar to the differences in metabolism observed, we also found differences in the ability of darinaparsin and arsenic trioxide to activate the Nrf2-Keap1 pathway and regulate gene transcription from the antioxidant response/electrophile response elements. Treatment of multiple myeloma cells with arsenic trioxide resulted in transcription from ARE, whereas darinaparsin treatment resulted in transcription just above background. Production of ROS following arsenic trioxide treatment is well documented, and in at least one study, darinaparsin was shown to produce a higher amount of reactive oxygen than arsenic trioxide (30). However, we have shown here that, in multiple myeloma cells, the ARE is not activated following treatment with darinaparsin. It is possible that the generation of ROS with darinaparsin occurs late in metabolism and as a result has a minimal affect on the response of the cell. We have previously reported that this is the case in some multiple myeloma cell lines, where ROS production is caspase dependent (16). Additionally, it is possible that the activation of the ARE by arsenic trioxide is not related to ROS production. Consistent with this possibility, we have recently observed that the activation of the Nrf2-Keap1 pathway following treatment of multiple myeloma cells with arsenic trioxide is not due to the production of ROS.4 This activation of Nrf2 by arsenic trioxide is probably due to direct binding and crosslinking of vicinal thiols on Keap1 (25, 26). Dimethylarsenic can only bind to a single cysteine and would be unable to crosslink the thiols in Keap1. Also consistent with differences in cellular stress signaling are the patterns of metallothionein gene expression. In addition to induction by metals such as zinc and cadmium, metallothionein genes are often up-regulated during oxidative stress signaling (20). Although metallothionein genes are the most up-regulated genes in response to arsenic trioxide, they remain essentially unexpressed in darinaparsin-treated cells.
In addition to the up-regulation of phase II detoxification enzymes, increased transcription of chaperone proteins is also a documented result of Nrf2-Keap1 pathway activation (25, 26). However, activation of the stress-induced HSE only occurred transiently after treatment of multiple myeloma cells with either arsenic trioxide or darinaparsin, suggesting an alternative mechanism of up-regulation. Based on the activation of the HSE reporter constructs, we predict that HSE-driven, not ARE-driven, transcription is the most likely explanation. This is consistent with a previous report showing activation of heat shock factor-1 by sodium arsenite (31). Together, these results show that arsenic trioxide and darinaparsin not only enter myeloma cells through distinct mechanisms but also, once they are in the cell, are sensed as different types of cellular stress.
In contrast to the protective responses, gene expression profiling analysis also revealed the up-regulation of apoptotic responses. The three BH3-only proteins Noxa, Bim, and Bmf, which are important for arsenic trioxide-induced apoptosis (15), also appear to play a role in darinaparsin-induced cell death. However, the responses were somewhat different. Noxa transcription increased following treatment with darinaparsin in 8226/S and KMS11 cells. Bim transcription was up-regulated in 8226/S cells following darinaparsin treatment; however, no change in protein level was observed. Bmf transcription increased in KMS11 cells but remained unchanged in 8226/S cells. Transient silencing of Noxa and Bim partially protected 8226/S cells from darinaparsin-induced apoptosis, suggesting a role for these two BH3-only proteins in the mechanism of darinaparsin-induced death. The inability of Bmf silencing to protect 8226/S cells from darinaparsin-induced apoptosis suggests that inhibition of anti-apoptotic protein Mcl-1 could be sufficient for induction of cell death in this cell line. However, it is also possible that an alternative BH3-only sensitizer capable of inhibiting anti-apoptotic Bcl-xL, such as Bad, is involved in the mechanism of darinaparsin-induced cell death in 8226/S cells (32, 33).
Finally, although arsenic trioxide has only had modest effects in the treatment of myeloma, it is capable of killing multiple myeloma cell lines in vitro at clinically achievable concentrations. This disconnect between in vitro and in vivo responses is probably due to drug uptake and effects of the microenvironment. Therefore, from a clinical perspective, it is encouraging that darinaparsin is much more readily taken up by multiple myeloma cells and maintains activity under conditions of arsenic trioxide resistance. Thus, further investigation of this novel agent in myeloma and other arsenic trioxide-resistant diseases is warranted.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
References
Competing Interests
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