Although recent reports suggest that selenium can modulate the activity of cytotoxic drugs, the mechanism underlying this activity remains unclear. This has been investigated using a panel of human B-cell lymphoma cell lines. The cytotoxic effects of chemotherapeutic agents (e.g., doxorubicin, etoposide, 4-hydroperoxycyclophosphamide, melphalan, and 1-β-d-arabinofuranosylcytosine) were increased by up to 2.5-fold when combined with minimally toxic concentrations (EC5-10) of the organic selenium compound, methylseleninic acid (MSA). DNA strand breaks were identified using comet assays, but the measured genotoxic activity of the combinations did not explain the observed synergistic effects in cell death. However, minimally toxic (EC10) concentrations of MSA induced a 50% decrease in nuclear factor-κB (NF-κB) activity after an exposure of 5 h, similar to that obtained with the specific NF-κB inhibitor, BAY 11-7082. Combinations of BAY 11-7082 with these cytotoxic drugs also resulted in synergism, suggesting that the chemosensitizing activity of MSA is mediated, at least in part, by its effects on NF-κB. Basal intracellular selenium concentration was higher in a MSA-sensitive cell line. After exposure to MSA, methylselenocysteine and selenomethionine were identified as the main intracellular species generated. Volatile selenium species, trapped using solid-phase microextraction fibers, were identified as dimethylselenide and dimethyldiselenide. These volatile species are thought to be the most biologically active forms of selenium. Taken together, these results show that the NF-κB pathway is one target for MSA underlying the interaction between MSA and chemotherapy. These data encourage the further clinical development of selenium as a potential modulator of cytotoxic drug activity in B-cell lymphomas. [Cancer Res 2007;67(22):10984–92]

Selenium is an essential trace element that has immunomodulatory activity (1, 2), and also has several functions relevant to cancer medicine. At low concentration, selenium compounds act as antioxidants, thereby protecting DNA, most likely via selenoproteins such as glutathione peroxidase and thioredoxin reductase (3). Selenium supplementation studies have reported a decreased incidence of specific cancers, most notably prostate cancer (4, 5). Large randomized chemoprevention studies, such as the Selenium and Vitamin E Cancer Prevention Trial, are in progress.

Several studies have reported that selenium, at supranutritional concentrations, may modulate the activity of cytotoxic chemotherapy. In a study from our own research group, serum selenium concentration at diagnosis was predictive of dose delivery, treatment response, and long-term survival in a group of 99 patients with aggressive non–Hodgkin's lymphoma. Response to first treatment was 54% in the lowest serum quartile compared with 88% in the highest quartile, with shorter overall survival in patients with lower serum selenium (6). Serum selenium remained predictive of outcome in a multivariate analysis that included clinical variables as cofactors, as also reported by Deffuant et al. (7) in cutaneous T-cell lymphoma patients. More recently, a dramatic increase in the maximum tolerated dose and antitumor efficacy of several cytotoxic agents has been reported when administered with selenium as either selenomethionine or methylselenocysteine, in both head and neck and lung cancer xenograft models (8, 9). At higher concentrations, selenium compounds are cytotoxic to tumor cells in vitro (10) and at high doses (15 mg/kg) are lethally toxic in nude mice (11).3

3

Our unpublished observations.

The precise mechanism whereby selenium modulates cytotoxic drug activity remains unclear, although modification of stress response pathways is emerging as a possible explanation (12). One of these stress response mediators, GADD153, is reported to suppress nuclear factor-κB (NF-κB) activity, known to be elevated in many cancer types. Furthermore, a number of reports have associated increased NF-κB activity with decreased cytotoxic drug activity and shown that NF-κB inhibition can sensitize cells to cytotoxic agents (1317). These data therefore provide a possible mechanism for the chemomodulatory activity of selenium compounds.

Our earlier report (6) on the influence of selenium status on treatment outcome in patients with non–Hodgkin's lymphoma suggests that selenium supplementation at the time of chemotherapy may be a useful treatment strategy in this disease. Although we have recently described the single-agent activity of methylseleninic acid (MSA) in a panel of B-cell lymphoma cell lines (12), there are no published data describing the activity of selenium in combination with cytotoxic drugs using a lymphoma model. We have therefore investigated the effects of selenium, as methylseleninic acid, in combination with standard antilymphoma agents in B-cell lymphoma cell lines. As the drugs used were DNA-damaging agents, the effects of MSA on the induction and repair of DNA strand breaks were studied using the comet assay, and changes in NF-κB activity after MSA exposure have been determined using a NF-κB reporter construct.

Identifying intracellular selenium species that might be mediating the effects reported is important to both maximize any interaction observed and to provide a potential molecular end point for subsequent clinical trials with selenium compounds. We have therefore also determined the intracellular selenium species generated from MSA using novel mass spectrometry (MS)–based methodologies, and identified the volatile and highly reactive selenium species dimethylselenide and dimethyldiselenide in the cell headspace after MSA exposure.

Cell culture. A panel of human B-cell lymphoma cell lines, a normal B-cell line and human peripheral mononuclear cells (PBMC) separated from whole-blood samples, were used for this study. The DHL-4 cell line was obtained from the Dana-Farber Cancer Institute (kind gift from Dr. Margaret Shipp). DoHH-2, CRL-2261, and SUD-4 cell lines were obtained from The Centre for Haematology, Barts and the London School of Medicine (kind gift from Professor Finbarr Cotter). DHL-5 cells and an EBV-transformed normal B-cell line (NC-NC) were obtained from DSMZ GmbH. With the exception of DHL-5 cells (wild-type p53), all lymphoma cells had heterozygous (DoHH2; ref. 18) or homozygous p53 mutations.

All cells were cultured in a standard cell humidifier in RPMI 1640 (Sigma-Aldrich Company, Ltd.) supplemented with 10% fetal bovine serum (heat-inactivated) and penicillin/streptomycin (100 units/mL). For NF-κB studies, cells were incubated with tumor necrosis factor-α (TNF-α; R&D Systems Europe, Ltd.) by direct addition to the culture medium at a final concentration of 10 ng/mL. BAY 11-7082, used as a specific NF-κB inhibitor, was purchased from Merck Biosciences, Ltd.

Selenium compounds. MSA was obtained from PharmaSe, Inc., and dissolved in sterile double-distilled water (ddH2O) as stock solutions of 10 mmol/L, which were stored at −80°C. Further dilutions were made in RPMI 1640 cell culture medium before adding to cell suspensions.

Cell viability and EC50 values. Cell viability assays to determine the activity of MSA and chemotherapeutic agents alone, and in combination, were carried out by plating 5 × 103 cells/well into 96-well microtiter plates. After drug incubation for 4 to 72 h, ViaCount Flex reagent (Guava Technologies, Inc.) was added and cell viability was then determined using a PCA-96 automated cell analyzer with Cytosoft analysis software (both from Guava Technologies). EC50 values were calculated in GraphPad Prism (GraphPad Software) using a sigmoidal dose-response model with variable slope.

Alkaline single-cell gel electrophoresis assay. To investigate potential DNA damage caused by increased selenium exposure alone, and in combination with the established chemotherapy agents doxorubicin and etoposide, comet assays were carried out according to Singh et al. (19), with slight modifications. In brief, cells were plated in 24-well plates at a density of 5 × 103/mL and treated with the drugs of interest for 24 to 48 h. The cell pellets were then washed twice and resuspended in 50 μL PBS. Precooled microscope glass slides were coated with 1% regular agarose. A low-melting-point agarose (0.5%) suspension at 37°C was then added to the cell suspension at a ratio of 16:1 and immediately transferred to a slide precoated with regular agarose. The cells on the slides were lysed with ice-cold high-salt lysis buffer [2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris (pH 10.0), 1% Triton X-100] for 1 h in the dark. Cells were then covered in alkali solution [1 mmol/L EDTA, 300 mmol/L sodium hydroxide (pH >13.0), 10 μg/mL proteinase K] for 45 min followed by electrophoresis [300 mmol/L NaCl, 100 mmol/L Tris (pH 9.0)] under low light at 20 V, 140 mA (1 V/cm) for 15 min at 4°C. The slides were neutralized using 1 mol/L ammonium acetate in ethanol and washed with ddH2O twice before being dried overnight.

The slides were rehydrated with ddH2O and stained with propidium iodide solution (2.5 μg/mL in ddH2O) for 15 min before rinsing with ddH2O and drying in the dark. Comet images were visualized by using an inverted fluorescent microscope (DIASHOT model TMD, Nikon) at 510 to 560 nm excitation and 590 nm barrier filter at ×320 magnification. Images were analyzed from duplicate slides, 25 images per slide, using Komet assay software (Kinetic Imaging).

Where not apparent from plotted data, statistical comparisons were carried out using the Wilcoxon signed rank test for matched pairs (Minitab v14). A P value of <0.05 was considered statistically significant.

Transient transfection and measurement of NF-κB activity. Cells were plated in 24-well plates at a density of 5 × 104/mL/well and were transiently transfected with the reporter plasmid pNF-κB-Luc containing binding sites for NF-κB: (TGGGGACTTTCCGC)5 upstream of the firefly luciferase gene in a pLuc-MCS construct (Stratagene). The pRL-TK Renilla luciferase plasmid (Promega) was cotransfected with the NF-κB plasmid as an internal standard for equal transfection efficiency using Effectene Transfection Reagent (Qiagen, Ltd.). At 24 h posttransfection, recombinant TNF-α was added for 1 h followed by MSA for another 4 h. Cellular extracts were then incubated with the Dual-Glo Luciferase Assay System substrate (Promega) and luciferase activity was measured using a luminometer (Polarstar Optima, BMG Labtech, Ltd.). NF-κB activity was expressed as the percentage change of the activity of firefly luciferase normalized to the Renilla luciferase signal obtained from untreated control cells.

Instrumentation for total intracellular selenium and generated selenium species. Analysis using high-performance liquid chromatography (HPLC) linked to an inductively coupled plasma MS (ICP-MS) were performed with an Agilent Technologies 1100 HPLC system for chromatographic separations and an Agilent 7500i ICP-MS for element-specific detection. Reversed-phase HPLC was performed on an Agilent Zorbax Rx-C8 connected to the microflow concentric nebulizer of the ICP-MS. Chromatographic data were analyzed using Agilent Technologies ICP-MS chromatographic software (G1824C Version C.01.00). For the HPLC-electrospray ionization (ESI) QTrapMS experiments, a 4000 QTRAP mass spectrometer (ABI/MDS Sciex) and an Agilent Technologies 1100 HPLC system were used. Data acquisition and processing were performed using the ABI Analyst software version 1.4.1. The effluent from the reversed-phase HPLC column was fed directly into the electrospray source. For optimum chromatographic separation conditions and instrumental parameters for online measurements with ICP-MS and electrospray–tandem MS (MS/MS), see Supplementary Data.

Selenium standards, including sodium selenite, Se-dl-methionine (SeMet), Se-methylselenocysteine (MSC), dimethylselenide, and dimethyldiselenide were purchased from Sigma-Aldrich unless stated otherwise. The standards of l-γ-glutamyl-Se-methylseleno-l-cysteine and MSA were purchased from PharmaSe. For the preparation of SeMet stock solutions, 0.1 mol/L hydrochloric acid was used; all other standards were diluted in ultrapure water and kept at 4°C in the dark.

A standard solution of 10 μg/kg of Se in methanol-water-0.1% formic acid was prepared for the daily optimization of the ICP-MS parameters.

Total Se determination by online flow injection analysis with ICP-MS. Quantification of total Se in cell extracts was performed by flow injection analysis combined online with ICP-MS detection. Cellular extracts were injected in a stream of ultrapure water as the sample carrier. For calibration, standard addition at three concentration levels, monitoring the isotope 82Se, was used. Rhodium was used as the internal standard. An integration time per mass of 300 ms was selected for obtaining the best signal-to-noise ratio.

Quantification of intracellular Se species by reversed-phase HPLC-ICP-MS. Cell extracts were analyzed by reversed-phase HPLC-ICP-MS using a water-methanol (98+2, v/v) mixture containing 0.1% (v/v) trifluoroacetic acid (TFA) as the mobile phase. For chromatographic and ICP-MS conditions, see Supplementary Data. Calibration was carried out by the standard addition technique at three concentration levels, using peak area measurements of the chromatographic signals by monitoring the 82Se signal. The standard solution used for calibration was characterized for its total Se content by ICP-MS.

Verification of the presence of MSC by HPLC-ESI MS/MS. For identification of the Se peaks detected by HPLC-ICP-MS, cell extracts were injected onto the reversed-phase column. The separation and elution of the different Se species were achieved using the same chromatographic column as for the ICP-MS analysis (see Supplementary Data) but with a mobile phase containing formic acid, which is, unlike TFA, compatible with ESI. Mass spectra were recorded over a m/z range of 50 to 1,000. For Se standards, product ion spectra were acquired in the collision-induced dissociation mode. MS/MS spectra for Se standards and Se species in the cell extracts were acquired in the selected reaction monitoring mode. The most abundant transitions m/z 184 > m/z 167 (loss of OH) and m/z 184 > m/z 95 (formation of the ion fragment SeCH3+) for MSC and m/z 198 > m/z 181 (loss of OH), and m/z 198 > m/z 109 (formation of the ion fragment CH3SeCH2+) for SeMet were monitored.

Solid-phase microextraction capture of volatile Se compounds. To capture volatile selenium species, a 75 μm Carboxen polydimethylsiloxane solid-phase microextraction (SPME) fiber (Supelco) was placed in the headspace above DHL-4 cells in culture before and after MSA treatment. The fiber was then held in the inlet liner of the gas chromatogram for the duration of the complete chromatographic run. Sampling of Se volatile compounds from the headspace of a standard mixture solution containing dimethylselenide and dimethyldiselenide was performed using the same procedure.

Identification of volatile Se species by gas chromatography time-of-flight MS. Gas chromatography-time-of flight MS (GC-TOFMS) measurements were performed using an Agilent 6890 GC coupled to the Leco Pegasus III TOFMS (Leco). A HP Ultra2 column was used for separation of the volatile species. TOF mass calibration was performed using perfluorotributylamine. A splitless injection mode was used with helium as the carrier gas. Mass spectra were recorded over a m/z range of 50 to 400. Data acquisition and processing were performed using the LECO ChromaTOF software version 2.25 (optimized for Pegasus).

Sensitivity of malignant and normal B-cell lines to MSA. The effect of MSA was studied in lymphoma and normal B-cell lines and in human PBMCs. The lymphoma cell lines differed in their sensitivity to MSA as shown in Fig. 1A. CRL-2261, SUD-4, and DHL-5 cell lines were the most sensitive to MSA with EC50 values of 4.7 μmol/L [95% confidence interval (95% CI), 3.3–6.8], 6.5 μmol/L (95% CI, 5.3–7.9), and 2.3 μmol/L (95% CI, 1.8–2.9), respectively, after a 72-h exposure. DHL-4 and DoHH-2 cells were considerably less sensitive, with EC50 values of 373 μmol/L (95% CI, 198.1–703.1) and 168.6 μmol/L (95% CI, 110.7–256.6), respectively. The cell line panel was therefore grouped into MSA-sensitive and MSA-resistant cell lines for further experiments. The normal B-cell line (NC-NC) and PBMCs were also less sensitive to MSA with EC50 values of 27 μmol/L (95% CI, 22.4–32.5) and 84.2 μmol/L (95% CI, 11.0–641.0), respectively (Fig. 1B).

Figure 1.

Cytotoxic effects of MSA (mean ± SD) on five different malignant B-cell lines after an exposure time of 72 h (A), and normal B-cells for 24 and 72 h (B). Cell viability was measured using the automated cell system GUAVA PCA-96 with the ViaCount Flex assay (GUAVA Technologies). The obtained results were analyzed using a sigmoidal dose-response model with variable slope to generate EC50 values (Graphpad Prism 3.03).

Figure 1.

Cytotoxic effects of MSA (mean ± SD) on five different malignant B-cell lines after an exposure time of 72 h (A), and normal B-cells for 24 and 72 h (B). Cell viability was measured using the automated cell system GUAVA PCA-96 with the ViaCount Flex assay (GUAVA Technologies). The obtained results were analyzed using a sigmoidal dose-response model with variable slope to generate EC50 values (Graphpad Prism 3.03).

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Two malignant B-cell lines (one sensitive and one resistant) were further studied using a methylcellulose colony formation assay system. After a 14-day incubation period, a reduction in colonies was observed in MSA-treated cells at 5 and 10 μmol/L MSA for the SUD-4 cell line and at 100 and 200 μmol/L MSA for DHL-4 cells, whereas untreated control cells showed clear colony formation (data not shown).

Enhanced efficacy of standard chemotherapy when combined with MSA. Cell lines were incubated simultaneously for 48 h with nontoxic or minimally toxic concentrations of MSA and specific cytotoxic drugs used to treat B-cell lymphomas. In SUD-4 cells, representing a MSA-sensitive cell line, the cytotoxicity of doxorubicin alone could be increased from 21% (±3.2) cell kill at 75 nmol/L to 49% (±4.9) after 48 h when combined with low (nontoxic) concentrations of MSA (0.5 μmol/L), resulting in a clear synergistic effect (Fig. 2A). Synergy was also observed when etoposide was combined with 0.5 μmol/L MSA, with an increase in cell kill from 32% (±1.3) with 10 μmol/L etoposide alone to 60% (±3.6) when combined with MSA. The activity of 1 μmol/L 4-hydroxycyclophosphamide (the active form of cyclophosphamide) was increased from 19% (±0.9) when used alone to 50% (±1.6) in the presence of 0.5 μmol/L MSA (Fig. 2A). Similar effects were observed in the selenium-resistant DHL-4 cell line. For doxorubicin (225 nmol/L), the cytotoxic activity was increased from 8% (±3.4) alone to 44% (±17.3) when combined with MSA (10 μmol/L; Fig. 2B), for etoposide from 4% (±1.4) to 22% (±1.0), and for 4-hydroxycyclophosphamide from 7% (±1.5) to 22% (±3.1). Only additive effects were observed when MSA was added to melphalan or 1-β-d-arabinofuranosylcytosine in these cell lines. The best enhancement of cytotoxic drug activity was obtained with simultaneous exposures with MSA rather than sequential treatments (data not shown).

Figure 2.

Effects of combination studies (mean ± SD) using two different B-cell lines exposed to MSA or the chemotherapeutic agents doxorubicin (Dox), etoposide (Etop), or cyclophosphamide (4-HC) alone, and simultaneously in combination with MSA in SUD-4 (A) and DHL-4 (B) cell lines for 48 h. Different combinations are also shown for the common NF-κB inhibitor BAY 11-7082 (BAY) in two representative cell lines: SUD-4 (C) and DHL-4 (D). Conc., concentration.

Figure 2.

Effects of combination studies (mean ± SD) using two different B-cell lines exposed to MSA or the chemotherapeutic agents doxorubicin (Dox), etoposide (Etop), or cyclophosphamide (4-HC) alone, and simultaneously in combination with MSA in SUD-4 (A) and DHL-4 (B) cell lines for 48 h. Different combinations are also shown for the common NF-κB inhibitor BAY 11-7082 (BAY) in two representative cell lines: SUD-4 (C) and DHL-4 (D). Conc., concentration.

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The effect of MSA on the generation of DNA damage. To investigate the genotoxic activity of selenium alone and its effects on DNA single-strand breaks generated by doxorubicin, we used an alkaline version of the comet assay. This is a sensitive method for the quantitative detection of low levels of DNA damage and, after a drug-free recovery period, DNA repair. A MSA-sensitive (SUD-4) and a more MSA-resistant (DHL-4) B-cell line were used for these studies. MSA alone induced a concentration-dependent increase in DNA damage in both cell lines, with an increase in the DNA content in the comet tail of 2.6-fold with 1 μmol/L MSA and 3.5-fold increase with 5 μmol/L MSA in the sensitive cell line SUD-4 (Fig. 3A). Similar concentration-related DNA damage was seen in DHL-4 cells, with a 1.2-fold increase in comet tail DNA with 10 μmol/L MSA and a 2.2-fold increase with 200 μmol/L MSA (Fig. 3B). Both cell lines showed the expected increase in DNA damage with doxorubicin over the 24-h exposure period. Doxorubicin-induced DNA damage was concentration dependent between 37.5 and 750 nmol/L, and showed a clear relationship with cell viability, in SUD-4 cells. In DHL-4 cells, this relationship was more complex, with a concentration-dependent increase in DNA damage occurring at lower concentrations than required for cell kill (data not shown).

Figure 3.

Summary of DNA damage in two different B-cell lines after exposure to increasing MSA concentrations, doxorubicin, or the combination of both, for 24 h. Columns, DNA damage shown as percentage DNA in the comet tail in SUD-4 cells (A) representing a MSA-sensitive B-cell line, and DHL-4 cells (B) as a more MSA-resistant cell line; bars, SE. Data were analyzed using a Wilcoxon signed rank test comparing doxorubicin alone versus doxorubicin + MSA (#) resulting in P < 0.0001 for both cell lines. The predicted versus the observed effect of the combination treatment (*) were not significant for either cell line [P = 0.101 for SUD-4 cells (A) and P = 0.213 for DHL-4 cells (B)].

Figure 3.

Summary of DNA damage in two different B-cell lines after exposure to increasing MSA concentrations, doxorubicin, or the combination of both, for 24 h. Columns, DNA damage shown as percentage DNA in the comet tail in SUD-4 cells (A) representing a MSA-sensitive B-cell line, and DHL-4 cells (B) as a more MSA-resistant cell line; bars, SE. Data were analyzed using a Wilcoxon signed rank test comparing doxorubicin alone versus doxorubicin + MSA (#) resulting in P < 0.0001 for both cell lines. The predicted versus the observed effect of the combination treatment (*) were not significant for either cell line [P = 0.101 for SUD-4 cells (A) and P = 0.213 for DHL-4 cells (B)].

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The combination of doxorubicin with MSA resulted in an increase in DNA damage, but this was not more than additive, as shown in Fig. 3, and was below the level at which maximal DNA damage was achieved with doxorubicin alone in both cell lines. Statistical analysis using the Wilcoxon signed rank test showed that the amount of DNA damage from the combination was greater than that obtained with doxorubicin alone (Fig. 3A and B; P < 0.0001 for both cell lines). However, for the combinations, the difference between the measured and expected (the sum of each agent used singly) DNA damage was not statistically significant (SUD-4 cells, P = 0.101; DHL-4 cells, P = 0.213).

The addition of MSA to doxorubicin resulted in no change in the amount of DNA repair after a 4-h recovery period compared with doxorubicin alone.

Inhibition of NF-κB activity in MSA-treated B-cell lymphoma cells. As changes in DNA damage did not fully explain the synergistic effects in cell death obtained in the combination experiments, we studied the NF-κB pathway, known to be involved in apoptosis and chemosensitivity, by measuring TNF-α–induced NF-κB activity in DHL-4 cells. The transcriptional activity of NF-κB after 5-h exposure to MSA was decreased in a concentration-dependent manner, down to 52% (±11) activity at 5 μmol/L MSA and 33% (±11) at 10 μmol/L MSA (Fig. 4). The ability of MSA to reduce NF-κB activity was also seen in other B-cell lines (e.g., DoHH-2; data not shown). This reduction was similar to that seen with the specific NF-κB inhibitor, BAY 11-7082, where activity was decreased to 67% (±6) at 1 μmol/L and 40% (±1) at 3 μmol/L BAY 11.

Figure 4.

Alterations in NF-κB activity in transiently transfected DHL-4 cells using a pLucNF-κB reporter construct encoding an NF-κB binding site (TGGGGACTTTCCGC)5 and a pRL-TK Renilla luciferase construct as internal control for 24 h. Cells were then incubated with either MSA or BAY 11-7082 for 1 h followed by another 4 h of TNF-α stimulation (10 ng/mL) before lysis and analyzed for NF-κB activity. The changes are shown as percentage of control cells (mean ± SD) using firefly luciferase activity normalized to the pRL-TK Renilla luciferase signal of TNF-α–induced cells.

Figure 4.

Alterations in NF-κB activity in transiently transfected DHL-4 cells using a pLucNF-κB reporter construct encoding an NF-κB binding site (TGGGGACTTTCCGC)5 and a pRL-TK Renilla luciferase construct as internal control for 24 h. Cells were then incubated with either MSA or BAY 11-7082 for 1 h followed by another 4 h of TNF-α stimulation (10 ng/mL) before lysis and analyzed for NF-κB activity. The changes are shown as percentage of control cells (mean ± SD) using firefly luciferase activity normalized to the pRL-TK Renilla luciferase signal of TNF-α–induced cells.

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Do synergistic effects of chemotherapeutic agents with MSA occur due to NF-κB inhibition? We next investigated whether the synergistic activity observed when MSA was combined with chemotherapy was attributable to the MSA-induced decrease in NF-κB activity. The down-regulation of NF-κB activity with the inhibitor BAY 11-7082 significantly increased the cytotoxic activity of etoposide and doxorubicin in both cell lines studied, as shown for the MSA-sensitive cell line SUD-4 (Fig. 2C) and a MSA-resistant cell line DHL-4 (Fig. 2D). In SUD-4 cells, the cell kill with doxorubicin and BAY 11-7082 in combination was 51% (±8.5) compared with 23% (±3.3) with doxorubicin alone, whereas for etoposide BAY 11-7082 increased cell kill from 34% (±1.3) to 54% (±6.4). Again, similar effects were seen in the more resistant cell line, DHL-4, in which 1 μmol/L BAY 11-7082 enhanced the cell death from 12% (±3.4) with doxorubicin alone to 51% (±3.7) with BAY 11-7082, and from 7% (±1.4) with etoposide alone to 39% (±3.0) with BAY 11-7082 (Fig. 2D).

Cytotoxic drugs alone induced NF-κB activity, markedly so in the case of etoposide, which resulted in a 2.7-fold increase in activity. However, in the presence of 10 μmol/L MSA, NF-κB continued to show reduced activity even in co-incubations with 10 μmol/L etoposide (72.9% of control) or 375 nmol/L doxorubicin (66.2% of control).

Intracellular MSA uptake and the resulting selenium levels in B-cell lymphoma cells. A novel LC-ICP-MS method showed that B-cell lymphoma cell lines differed in their basal selenium levels. In CRL-2261 cells, a MSA-sensitive cell line, the baseline selenium content was 55 ng/mL, considerably higher than that found in the two MSA-resistant cell lines DHL-4 (16 ng/mL) and DoHH-2 (18 ng/mL).

After 4-h exposure to MSA, selenium concentrations increased in the DHL-4 cell line, notably by 25-fold at 400 μmol/L MSA exposure. In DoHH-2 cells, there was a 4-fold change in selenium content after a longer exposure time of 24 h (data not shown). In contrast, the MSA-sensitive cell line CRL-2261, with high baseline selenium content, showed only a small change in intracellular selenium after 4 h at MSA concentrations up to 50 μmol/L, but returned to pretreatment levels during a 24-h exposure.

Intracellular selenium metabolites generated after MSA exposure in B-cell lymphoma cells. To investigate further the cellular metabolites generated by MSA, a method capable of detecting selenium species in cell extracts was developed using reversed-phase HPLC-ICP-MS analysis. We first determined selenium species in cells exposed to 200 and 400 μmol/L MSA for 4 h, the time point showing the highest intracellular uptake, in the DHL-4 cell line. Two main peaks were detected, with retention times of 4.6 and 9.9 min, identified as MSC and SeMet, with relative Se distribution (as total Se areas of eluted peaks) of 70% (±5) and 20% (±2), respectively. The time course of selenium species generation was then investigated further, as shown in Fig. 5A. None of the parent compound MSA was detected intracellularly in any sample during the 4-h incubation period, even after 10 min. MSC concentration was maximal after 30 min at 4,666 ppm (data not shown) and decreased thereafter to 1,232 ppm after 2 h (Fig. 5A). The second detected selenium metabolite, SeMet, was formed much more slowly than MSC. A slight increase from pretreatment levels was detected after 10-min exposure to MSA with a further increase at 2 h from 290 to 620 ppm (Fig. 5A).

Figure 5.

A, HPLC-ICP-MS elution profile of a selenium standard containing different species (top) showing the retention time of MSA (3.3 min), MSC (4.6 min), and SeMet (9.9 min). Appearance of selenium species in B cells before and after 10 and 120 min of MSA exposure. B, GC-ICP-MS chromatograms of the volatile selenium standards dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) and the generated selenium species collected with SPME fibers in the headspace of DHL-4 cell cultures before and after they were exposed to MSA (200 μmol/L) for 10 and 20 min.

Figure 5.

A, HPLC-ICP-MS elution profile of a selenium standard containing different species (top) showing the retention time of MSA (3.3 min), MSC (4.6 min), and SeMet (9.9 min). Appearance of selenium species in B cells before and after 10 and 120 min of MSA exposure. B, GC-ICP-MS chromatograms of the volatile selenium standards dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) and the generated selenium species collected with SPME fibers in the headspace of DHL-4 cell cultures before and after they were exposed to MSA (200 μmol/L) for 10 and 20 min.

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Volatile selenium species formed by lymphoma cells when exposed to MSA. Analysis of rapidly generated volatile selenium species collected by SPME fibers before and after MSA exposure from DHL-4 cells followed by GC-ICP-MS analysis showed dimethyldiselenide formed after 10-min MSA exposure with a retention time of 1.4 min (Fig. 5B). After 20-min exposure, a massive increase in dimethyldiselenide was observed, with the appearance of a second metabolite identified as dimethylselenide with a retention time of 0.9 min. Neither MSA nor cells alone in cell culture medium showed any peaks at these elution times, indicating that the formation of the volatile selenium species occurred intracellularly and was time dependently formed in the first few minutes of MSA exposure. The presence of these two main metabolites was confirmed by oven cooling-GC-TOFMS and showed exactly the same masses compared with library data.

Ion-pair reversed-phase HPLC-ESI-MS/MS analysis to confirm the intracellular species detected. To verify the presence of MSC as the main intracellular selenium species after MSA exposure in DHL-4 cells, ion-pair reversed-phase HPLC with ESI-MS/MS in selected reaction monitoring mode was used. Specific transitions from the precursor ion (m/z 184) to product ions with m/z of 95 (SeCH3+) and 167 (loss of OH), also observed in the standard, were monitored (Fig. 6). In cells exposed to MSA (200 μmol/L) for 30 min, both transitions were detected at the two retention times of MSC (Fig. 6C), confirming the presence of MSC as the major intracellular selenium species after exposure to MSA (Fig. 6D).

Figure 6.

HPLC-ESI-MS/MS chromatograms for intracellular selenium species. A, a chromatogram showing a mixture of selenium standards, with the m/z spectrum for the product ions generated from MSC (m/z 184) shown in B. A representative chromatogram is shown in the right panel for a MSC standard (C) and cell extracts of human B cells (D), with m/z transitions of 184/95 and 184/167, to verify the presence of MSC as the main intracellular species detected after 30-min exposure to MSA.

Figure 6.

HPLC-ESI-MS/MS chromatograms for intracellular selenium species. A, a chromatogram showing a mixture of selenium standards, with the m/z spectrum for the product ions generated from MSC (m/z 184) shown in B. A representative chromatogram is shown in the right panel for a MSC standard (C) and cell extracts of human B cells (D), with m/z transitions of 184/95 and 184/167, to verify the presence of MSC as the main intracellular species detected after 30-min exposure to MSA.

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The emerging role of selenium as a potential chemomodulatory agent led us to explore the supplementation effect of MSA when combined with established cytotoxic drugs, with the aim of improving the treatment of lymphoid malignancies. First attempts have been made to increase the therapeutic efficacy of standard cytotoxic drugs using selenium compounds (20, 21). The present study results confirmed that MSA reduces cell viability in a panel of B-cell lymphoma cell lines (10), was less cytotoxic to normal B-cells, and importantly at low concentration showed synergistic activity when combined with cytotoxic drugs used in the treatment of lymphomas. This makes MSA, or similar compounds that rapidly generate reactive selenium species such as MSC, good candidates for further studies in cancer therapy.

The synergistic effects on cell kill observed with three of the five chemotherapeutic agents when combined with nontoxic MSA concentrations showed that the chemosensitization was drug specific but not cell line restricted. Even chemotherapy-resistant cell lines, such as DHL-4, were sensitized to cytotoxic drugs when combined with MSA, suggesting that selenium supplementation could modify drug sensitivity in resistant B-cell lymphomas. Similar results have been reported with other selenium compounds such as MSC and SeMet as selective modulators of anticancer agents in in vivo models (11). We are currently evaluating whether this approach translates to the clinic in a phase 1/2 trial in which selenium is given with chemotherapy.

To explore the mechanisms by which the synergistic effects seen might be mediated, we first addressed whether the increased apoptosis observed was due to MSA-induced changes in DNA damage, or DNA damage repair, resulting from cytotoxic drug exposure. MSA and the DNA-damaging agent doxorubicin, either alone or in combination, induced permanent DNA damage (no DNA repair was seen after a 4 h drug-free period). There was a linear relationship between doxorubicin-induced DNA strand breaks and cell viability in SUD-4 cells, whereas in the MSA-resistant cell line, DHL-4, a concentration-dependent increase in DNA damage was observed at lower doxorubicin concentrations than those required to induce cell kill, suggesting that the resistance to cytotoxic drugs observed in this cell line is in the apoptotic response to DNA damage. In both cell lines, the combination of MSA with doxorubicin resulted only in additive DNA damage, indicating that the increased cell kill observed was not due to enhanced DNA damage alone.

We next investigated whether the NF-κB pathway might be targeted by MSA. NF-κB is constitutively up-regulated in many cancers, including lymphomas, and targeting NF-κB using the proteasome inhibitor velcade has shown clinical activity in this setting (22). NF-κB is also involved in the cellular response to oxidative stress and plays a role in chemosensitivity (23, 24). MSA had a marked effect on this pathway, down-regulating NF-κB activity by 50% to 70% after a short exposure time of 5 h. We confirmed that inhibiting NF-κB increased sensitivity to chemotherapeutic drugs by conducting combination experiments with the same drugs and the specific NF-κB inhibitor BAY 11-7082, known to block the release and translocation of NF-κB into the nucleus followed by further activation of antiapoptotic genes, including cIAP-2, Bcl-2, Bcl-XL (25, 26), and also Cox-2 and cyclin D1 (2729).

A decrease in NF-κB activity is reported to overcome chemoresistance in many malignancies (30). As reduced NF-κB activity was observed in both MSA-sensitive and MSA-resistant cells, it is likely that the synergistic effects observed with drug combinations can be explained, at least partly, by changes in this pathway after MSA exposure, resulting in the down-regulation of cell survival pathways. Similar chemosensitization has been shown with other agents that target NF-κB (15), including the use of specific IκK inhibitors to overcome resistance to imatinib (31).

As MSA was shown to generate concentration-dependent DNA damage and a reduction in NF-κB activity, we next sought to identify the selenium species that might be mediating this cellular stress, using novel LC-ICP-MS and GC-TOFMS approaches. Our first observation was that the basal levels of intracellular selenium differed from cell line to cell line, despite maintaining these cells in the same culture conditions. Cell lines with low basal selenium levels showed a marked increase in total selenium after MSA exposure (by 5- to 25-fold), suggesting that differences in intracellular saturation levels for selenium differ between cell lines. These findings are likely to have clinical relevance in that even in the same disease, basal tumor selenium levels, and the ability to take up selenium, may vary considerably.

The two main selenium species identified, MSC and SeMet, were detected after only 10-min exposure, making the rapid intracellular conversion of MSA to other species very clear. The main compound generated, MSC, was verified by a separate organic MS method involving HPLC-ESI MS/MS, although SeMet was not detectable using this method due either to its reduced sensitivity or to a matrix-induced interference.

Some reports hypothesize that MSC would be generated by MSA via methylselenol, before further reduction to selenite and selenol (32). As these selenium species are very volatile, they would be expected to have a very short half-life. During incubation experiments with MSA, we did detect the characteristic “bad smell,” which is described by many others when cells are forming methylselenol. We were able to trap such volatile selenium metabolites, and/or their methylation products, in the headspace of malignant B cells during MSA exposure using SPME fibers with subsequent compound determination by GC-ICP-MS (33). The two main detected species, namely dimethylselenide and dimethyldiselenide, were almost undetectable in untreated cells, but showed a massive increase after just 10 to 20 min of exposure to MSA. These two active metabolites of selenol are the most potent biologically active forms of selenium (5).

Taken together, the present study suggests a cascade in the rapid generation of active forms of selenium in human B cells after exposure to MSA: MSA→MSC→SeMet→γ-glutamyl and to dimethylselenide and dimethyldiselenide as volatile components. The appearance of these reactive species is associated with the generation of DNA damage, also reported by others using methylated selenium species (34), and a decrease in the transcriptional activity of NF-κB. This series of events, triggered by low concentrations of selenium, results in the observed synergistic effects in cell kill. Importantly, this synergistic interaction was achieved at MSA concentrations that had little, if any, effect on the viability of normal B cells.

MSA, or related compounds such as MSC, seem to be excellent candidates for use as novel chemosensitizing agents in the clinic. Additionally, the availability of the methods described for measuring selenium species in intracellular and volatile forms will be an important tool for monitoring changes in selenium status in a planned clinical trial.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: Leukemia Research Fund, London, United Kingdom (S.P. Joel and J. Fitzgibbon).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Prof. J.A. Hartley and J.M. Hartley (Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College Medical School, University College London, London, United Kingdom) for providing the microscope and software to analyze the comet assay experiments; Dr. Julian Spallholz (PharmaSe) for providing the selenium compounds as well as for helpful discussions throughout this study; Emma Warburton for the valuable help to analyze volatile selenium species and Dr. Gavin O'Connor for the ESI MS/MS measurements (both LGC limited); and The United Kingdom Department of Trade and Industry funding through Valid Analytical Measurements program.

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Supplementary data