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Here we report on organic arsenical darinaparsin (DAR, ZIO-101, S-dimethylarsino-glutathione) and its anti-myeloma activity compared with inorganic arsenic trioxide (ATO). 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 ATO. In contrast to ATO, intracellular GSH does not appear to be important for darinaparsin metabolism, as an inhibitor of GSH synthesis, buthionine sulfoximine (BSO), had little effect on drug activity. This discrepancy was resolved when we determined the effects of thiols on drug uptake. The addition of exogenous glutathione, L-cysteine, or D-cysteine prevented darinaparsin cellular uptake and cell death, but had no effect on the uptake or activity of ATO, 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 ATO. While both arsenicals induced a transient heat shock response, only ATO 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 ATO-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 ATO-resistant hematological cancers.
Multiple myeloma (MM) is a progressive hematological 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. 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 (ATO) 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 pre-clinical studies as well as clinical trials. Treatment of multiple myeloma cell lines and newly isolated patient samples with clinically achievable doses of ATO resulted in growth inhibition and apoptosis (2, 5-6). Similarly, in a phase II trial of refractory or relapsed MM patients, ATO exhibited modest activity and was well tolerated (7-8). Interestingly, the cytotoxic effects of ATO appear to be strongly related to the intracellular glutathione concentration of target cells (9). Indeed, combining ATO treatment with glutathione 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 ATO as a single agent and 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 ATO (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 pharmacological 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 (DAR) is a novel organic arsenical consisting of glutathione conjugated dimethylarsenic. Initial studies indicate this arsenical is active against xenograft tumors and has a maximum tolerated dose that is 35-fold higher than ATO in mice (13). Furthermore, phase I studies have indicated its safety in cancer patients and various phase II clinical studies with oral and IV administration are underway (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 ATO and darinaparsin in order to determine the likely usefulness of darinaparsin in ATO-resistant myeloma. The mechanism of cellular uptake, metabolism, and signaling of protective responses appears to differ from that of ATO, suggesting darinaparsin may be an effective treatment for ATO-resistant multiple myeloma.
Multiple myeloma cell lines U266 and 8226/S were purchased from the American Type Culture Collection (ATCC, Manassas, VA). MM.1s cell line was obtained from Dr. Steven Rosen (Northwestern University, Chicago, IL), and KMS11 cell line was provided by Dr. P. Leif Bergsagel (Mayo Clinic, Scottsdale, AZ). Cells were maintained on supplemented RPMI-1640 media, as previously described (15). 8226/S-ATOR05 was maintained on supplemented RPMI-1640 media containing 1 μM ATO.
Buthionine sulfoximine (BSO), Glutathione (GSH), L-cysteine, D-cysteine, propidium iodide (PI) and Igepal CA-360 were purchased from Sigma-Aldrich (St. Louis, MO). Nitric acid (HNO3), trace metal grade, and hydrogen peroxide (H2O2), ACS grade, were purchased from Fisher Scientific (Pittsburgh, PA). Standards (1000 mg/L) of arsenic (As) and yttrium (Y) in 2% HNO3, ICP-MS grade, were purchased from GFS Chemicals (Powell, OH). Annexin-V-Fluorescein Isothiocyanate (FITC) was purchased from Biovision (Palo Alto, CA). N-acetylcysteine (NAC) was purchased from Bedford Laboratories (Bedford, OH). Darinaparsin (ZIO-101), dimethylarsino-glutathione, was provided by Ziopharm Oncology, Inc. (New York, NY). Arsenic trioxide (ATO) was provided by Cephalon, Inc. (Frazer, PA).
Cells were cultured at a concentration of 2.5 × 105 cells/mL in supplemented RPMI-1640 media, as previously described (15), and incubated with the indicated concentrations of darinaparsin or ATO and/or 100 μM BSO, 10 mM NAC, 5 mM GSH, 5 mM L-cysteine, or 5 mM D-cysteine.
Cell viability was measured by Annexin-V-FITC and PI staining as previously described (6). Data were acquired on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using CellQuest software (Becton Dickinson).
ON-TARGETplus SMART pool siRNAs were purchased from DHARMACON RNA Technologies Inc. (Chicago, IL). siCONTROL Non-targeting siRNA [si(-)], siBmf (BMF, L-004393), siNoxa (PMAIP1, L-005275), and siBim (BCL2L11, L-004383) were used. siRNAs were electoporated into 5 × 106 cells using Amaxa Program G-015 (Amaxa, Gaithersburg, MD) following manufacturer instructions, as previously described (15). Transfected cells were placed in 6-well plates with 3 mL of media for 16 h at 37°C and subsequently treated with 2 μM darinaparsin. Cells were collected at 6 and 24 h for protein expression analysis and at 24 and 48 h for apoptosis determination.
Total RNA was isolated from si(-) control and siBMF transfected samples at 6 and 24 hours following treatment, using the RNeasy® Mini Kit (Qiagen Valencia, CA). cDNA was synthesized from one μg of total RNA using the MuLV Reverse Transcriptase and random hexamer primers from the GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA). 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 manufacture protocol. TaqMan® human GAPDH (402869) was used as internal control and Bmf mRNA expression was calculated relative to GAPDH mRNA expression.
Western blot analysis was performed as previously described (16). Briefly, cells were washed once with PBS buffer and lysed in RIPA buffer containing protease inhibitors and protein concentrations were determined using the BCA Protein Assay (Pierce Biotechnology Inc., Rockford, IL). Twenty μg of total protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Protan® Nitrocellulose Transfer Membrane, Whatman GmbH, Dassel, Germany).
The following antibodies were used for Western blot analysis: Primary - mouse anti-Noxa (ab13654, Abcam, Cambridge, MA); rabbit anti-Bim (AB17003, CHEMICON International Inc., Temecula, CA); rabbit anti-Heme Oxygenase 1 (sc-10789, Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-NAD(P)H quinone oxidoreductase 1 (A180, Cell Signaling Technology, Danvers, MA) and rabbit anti-Actin (A 2066, Sigma-Aldrich, St. Louis, MO); Secondary - Anti-mouse IgG1-HRP Conjugate (03117766001, Roche Applied Science, Indianapolis, IN) and ECL Rabbit IgG, HRP-Linked Whole Ab (NA934V, GE HealthCare, Piscataway, NJ).
8226/S and KMS11 cell lines were treated for 0, 6, and 24 h with 2 μM ATO or 2 μM (8226/S) or 3 μM (KMS11) darinaparsin, and total RNA was subsequently isolated using the RNeasy® Mini Kit (Qiagen, Valencia, CA). Hybridization and initial data analysis were performed by Expression Analysis Inc (Durham, NC), probing Affymetrix Hu133 2.0 Plus Chips, as previously described (15). These data have been deposited in NCBI's Gene Expression Omnibus and are accessible by GEO Series accession number GSE14519.
Reporter constructs pGL3-Promoter, pGL3-ARE-Luc, and pGL3-HSE-Luc were kindly provided by Dr. Craig Logsdon (M.D. Anderson Cancer Center, Houston, TX) (17). Three μg of each construct was co-transfected with 0.3 μg of the pRL reporter plasmid (Promega, Madison, WI) by electroporation (Amaxa, Gaithersburg, MD) following manufacturer instructions. Briefly, 4 × 106 cells were electroporated in 100 μL of nucleofector solution (Amaxa Reagent V) with the appropriate volume of construct DNA, using the pre-selected Amaxa Program G-015. Electroporated cells were placed into 6-well plates with 3 mL of media for 16 h at 37°C. Cells were treated with 2 μM darinaparsin and samples were harvested at 6 hours for luciferase activity determination, using the Dual Luciferase Assay Kit from Promega (Madison, WI) following manufacturer protocol.
Five hundred μL of 0.5% Igepal (V/V) was added to the previously washed cell pellets. With the use of an ultrasonic probe (Fisher Scientific, Pittsburgh, PA) the cells were lysed and then homogenized. Five μL of the solution was removed for protein analysis (BCA Assay, Pierce Biotechnology Inc., Rockford, IL) and 500 μL of concentrated HNO3 was added to the remaining sample. The samples were incubated for 1 hour, and then 250 μL of 30% H2O2 was added.
One-40 μg/L of As standards were prepared from a 1 mg/L stock, previously prepared from the 1000 mg/L standard in 2% HNO3. Y was used as an internal standard (IS) at a concentration of 20 μg/L. For sample analysis, 250 μL of the cell digest was added to the IS and diluted to 5 mL with DDI water.
An Elan DRC-e (Perkin Elmer, Waltham, MA) 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 As, 89 for Y, 77 for ArCl and 82 to monitor, and correct for, any polyatomic interferences resulting from the presence of chloride (Cl) in the samples. Data was treated with Elan software version 3.4 (Perkin Elmer Sciex, Waltham, MA).
All data presented as the mean, plus or minus the standard error, of at least three independent experiments. GraphPad QuickCalcs online t test calculator was used for determination of statistical significance.
In order to determine the sensitivity of multiple myeloma (MM) cell lines to darinaparsin, as well as compare it to ATO, four MM cell lines were treated with either drug for 24 or 48 hours (Figure 1). Darinaparsin was able to effectively kill 3 of the 4 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 (Figure 1A and C). In contrast, ATO was effective at killing all four cell lines in a dose- and time-dependent manner (Figure 1B and D). Interestingly, these four MM cells lines were not equally responsive to the two drugs. For ATO, U266 and 8226/S display similar sensitivities (IC50 1.9 μM at 48 hours), which are lower than those of KMS11 (IC50 1.2 μM) and MM.1s (IC50 1.0 μM), as indicated by apoptosis at 48 hours. In contrast for darinaparsin, U266 (IC50 3.7 μM) is the least sensitive where as 8226 (IC50 1.6 μM) and KMS11 (IC50 1.2 μM) are more sensitive than MM.1s (IC50 1.9 μM) 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 ATO) 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.
In order 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.
The role of glutathione (GSH) in inorganic arsenic detoxification is well established; therefore we tested its role in the metabolism of the organic arsenical darinaparsin. N-acetylcysteine (NAC) increases intracellular cysteine for enhanced production of glutathione, whereas buthionine sulfoximine (BSO) inhibits the rate-limiting enzyme in GSH synthesis, γ-glutamate cysteine ligase, thereby blocking GSH production (18). In order to fully observe the protective effects of NAC, 8226/S and KMS11 cells were treated with 3 μM darinaparsin or ATO alone or in combination with 10 mM NAC for 48 hours. In contrast, to better observe the effects of BSO, 8226/S and KMS11 cells were treated with a low concentration of either darinaparsin or ATO. Based on dose curves from Figure 1, 1.5 μM darinaparsin or 1 μM ATO was used, and cells were treated with either arsenical alone, or in combination with 100 μM BSO for 48 hours.
Figure 2A shows NAC protected 8226/S and KMS11 cells from both darinaparsin and ATO-induced cell death, suggesting increasing GSH production provides an advantage to cells treated with arsenicals regardless of the formulation. However, depletion of GSH with BSO had distinct effects on darinaparsin and ATO activity. BSO co-treatment resulted in increased ATO activity in both cell lines. In contrast, co-treatment of cells with darinaparsin and BSO did not result in any increased activity, yielding viabilities very similar to darinaparsin alone (Figure 2B). These results suggest that GSH does not play the same role in darinaparsin metabolism as it does with ATO.
Darinaparsin is thought to be unstable in aqueous solutions, breaking down into its component parts, dimethylarsenic [DMA(III)] and GSH (19). Due to this, and the discrepancy in the effects of NAC and BSO on darinaparsin activity, we wanted to examine the possibility that NAC could be acting outside the myeloma cells, by binding to DMA(III) and thereby preventing drug entry and cell death. In order to test this, we first treated cells with 3 μM darinaparsin or ATO alone or in the presence of 5 mM L-cysteine or D-cysteine. While 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 glutathione production (18). If the free thiol group from cysteine is binding to DMA(III) 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 ATO-induced apoptosis (Figure 2C).
In order 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. Glutathione was chosen because many cells do not express the transporters required for its direct uptake (18). Additionally, data from our gene expression profiling (GEP) studies show that neither GS-X nor GS-Y is expressed in myeloma cell lines (not shown). 8226/S and KMS11 cells were treated with 3 μM darinaparsin or ATO in the presence or absence of 5 mM GSH for 48 hours. The addition of GSH to the culture media protected 8226/S and KMS11 cells from darinaparsin-induced apoptosis. However, exogenous GSH was unable to effectively protect either cell line from ATO-induced apoptosis (Figure 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 glutathione synthesis inside the cell.
To more directly test this possibility, 8226/S cells were treated with 2 μM darinaparsin or ATO alone or in combination with 5 mM GSH and total arsenic uptake was determined under each condition at 30 min, 3 hr, and 6 hr post treatment. We found that GSH was able to block darinaparsin uptake significantly, and by greater than 90% at all time points; whereas the effect on ATO uptake was minimal (Figure 3A and B). Interestingly, the total amount of intracellular arsenic was much greater in darinaparsin treated cells, than in ATO treated cells, at all time points evaluated. These results suggest myeloma cells transport darinaparsin much more efficiently than ATO. In addition, our data suggest that the uptake of darinaparsin occurs faster than that of ATO.
In order to investigate potential differences in mechanism of action between these two forms of arsenic, we performed gene expression profiling (GEP). 8226/S and KMS11 cells were treated with either 2 μM darinaparsin or ATO for 0, 6 and 24 hours. 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 antioxidant/electrophile response.
Strong up-regulation of metallothionein (MT) transcription was seen following treatment of myeloma cells with ATO, but was completely absent in cells treated with darinaparsin (Table 1, Supplement). Metallothioneins are stress-inducible proteins required for the regulation of intracellular zinc concentration, heavy metal detoxification, and cellular response to oxidative stress (20).
Increased transcription of heat shock genes was seen transiently following treatment of myeloma cells with darinaparsin and ATO, indicating both drugs induce cellular stress (Table 1, Supplement). Heat shock proteins (HSP) 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 HSP70 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 or ATO-induced cell death (Figure 1).
The increase in transcription at 6 and 24 hours of both heme oxygenase -1 (HMOX-1) and NAD(P)H dehydrogenase quinone 1 (NQO1), and many enzymes involved in glutathione synthesis, suggested that the Nrf2-Keap1 pathway was activated in response to ATO treatment (Table 1, Supplement). These findings were confirmed at the protein level (Figure 4A). Interestingly, stable up regulation of these genes was not seen following treatment with darinaparsin, indicating 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 antioxidant response/electrophile response elements (ARE/EpREs), thereby initiating the transcription of phase II detoxification enzymes, antioxidants, 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 ATO-induced cell death (manuscript, submitted).
To further investigate the activation of HSE and ARE related genes, we transiently transfected KMS11 cells with pGL3-ARE-Luciferase, pGL3-HSE-Luciferase, or pGL3-promoter reporter constructs, and 16 hours later, treated the cells with 2 μM ATO or darinaparsin for 6 hours. As expected, treatment of cells with darinaparsin or ATO resulted in activation of the HSE reporter while only ATO activated the ARE-driven reporter (Figure 4B).
In order to determine the possible mechanism of darinaparsin-induced cell death, the GEP was examined for changes in the transcription of BH3-only proteins, as these have been shown by our lab to be involved in ATO-induced cell death (15). Table 1 (Supplement) shows changes in transcription of Noxa, Bim and Bmf, three BH3-only proteins important for ATO-induced apoptosis (15, 28). Following treatment of 8226/S cells with darinaparsin, transcription of Noxa and Bim was up regulated, while 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 (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 μM of drug, and apoptosis was assessed. Real-time PCR was used to confirm Bmf silencing, comparing samples transfected with si(-) and siBmf (Figure 5A). Western blot analysis verified silencing of Noxa and Bim (Figure 5B). Silencing of Noxa and Bim partially protected 8226/S from darinaparsin-induced apoptosis, while Bmf silencing had no effect (Figure 5C). These data are consistent with the GEP 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).
We have created an ATO-resistant cell line, 8226/S-ATOR05 (manuscript in preparation). As the myeloma cell response to ATO and darinaparsin appears to differ, we wanted to determine if the ATO-resistant cell line was sensitive to darinaparsin. 8226/S parental cells and 8226/S cells grown along side the resistant cell line (8226/S-CR) were used as controls. These three cell lines, 8226/S, 8226/S-CR and 8226/S-ATOR05, were treated with varying concentrations darinaparsin (Figure 6A) for 24 hours. As expected, ATO was unable to induce cell death in the resistant cell line (Figure 6B). In contrast, darinaparsin was able to induce apoptosis in the ATO-resistant cell line in a dose-dependent manner.
The results presented in this paper suggest that the method of metabolism may play an important role in the overall potency of arsenic-containing drugs. Intracellular glutathione (GSH) is an important factor in numerous cellular processes, including detoxification of xenobiotics and antioxidant responses (18, 27, 29). While 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 ATO, MM cells were protected from darinaparsin-induced cell death when co-cultured with NAC, suggesting a role for GSH in darinaparsin metabolism. Alternatively, co-treatment of MM cells with BSO and darinaparsin had no additional activity, unlike the increased activity seen with co-treatment of ATO and BSO. 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 BSO 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 glutathione outside the cell (19). Therefore it is reasonable to propose that darinaparsin, which consists of GS-DMA(III), could be in equilibrium with GSH and free DMA(III). 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 DMA(III) 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 glutathione 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 (unpublished data).
Together, these data suggest the active form of darinaparsin is actually free DMA(III), and that its disassociation from glutathione 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 ATO is too great in the absence of conjugating enzymes, while DMA(III) can be inactivated by a single event. Perhaps this also explains why, in vivo, inorganic arsenic is converted to the more toxic DMA(III) for inactivation.
Similar to glutathione, 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 ATO to activate the Nrf2-Keap1 pathway and regulate gene transcription from the ARE/EpRE. Treatment of MM cells with ATO resulted in transcription from AREs, whereas darinaparsin treatment resulted in transcription just above background. Production of ROS following ATO treatment is well documented, and in at least one study, darinaparsin was shown to produce a higher amount of reactive oxygen than ATO (30). However, we have shown here that in MM cells the AREs are 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 cell's response. We have previously reported that this is the case in some MM cell lines, where ROS production is caspase-dependent (16). Additionally, it is possible that the activation of the ARE by ATO 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 MM cells with ATO is not due to the production of ROS (manuscript, submitted). This activation of Nrf2 by ATO is probably due to direct binding and crosslinking of vicinal thiols on Keap1 (25-26). DMA(III) 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 (MT) gene expression. In addition to induction by metals such as zinc and cadmium, MT genes are often up-regulated during oxidative stress signaling (20). While MT genes are the most up-regulated genes in response to ATO, 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 MM cells with either ATO 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 demonstrating activation of heat shock factor 1 (HSF-1) by sodium arsenite (31). Together these results demonstrate that ATO and darinaparsin not only enter myeloma cells through distinct mechanisms, but once they are in the cell, they are sensed as different types of cellular stress.
In contrast to the protective responses, GEP analysis also revealed the up-regulation of apoptotic responses. The three BH3-only proteins Noxa, Bim, and Bmf which are important for ATO-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, while ATO has only had modest effects in the treatment of myeloma, it is capable of killing MM 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 MM cells and maintains activity under conditions of ATO resistance. Thus, further investigation of this novel agent in myeloma and other ATO-resistant diseases is warranted.
Grant Support: NIH Grants: R0-1 CA97243 and R0-1 CA127910