Methylseleninic Acid Results in Rapid Changes in Intracellular Selenium Species and Sensitises Human Lymphoma Cells to Doxorubicin.

Abstract

Background: We have previously shown that selenium compounds such as methylseleninic acid (MSA) demonstrate cytostatic and cytotoxic effects in human lymphoma cell lines (Last et al., ASH 2004). We have now further investigated the activity of MSA in combination with standard cytotoxic agents and set up a novel method to measure total intracellular selenium concentration after incubation with MSA, and the appearance of other selenium species. Additionally, we have examined whether NF-κ B may be involved in any interaction seen.Procedures: The effects of MSA alone, and in combination with doxorubicin, on cell number and viability were investigated in a panel of MSA sensitive (CRL-2261 and SUD-4) and less sensitive (DHL-4 and DoHH2) diffuse large B-cell lymphoma cell lines using a Viacount assay on an automated cell analyzer system (GUAVA PCA-96). MSA activity in normal cells was investigated in PBMC's, separated from whole blood using a ficoll density gradient method. To investigate MSA uptake into cells the combination of flow injection analysis with quadrupole inductively coupled plasma mass spectrometry (ICP-MS) was used. An ion-pair reverse phase HPLC method, with ICP-MS, was developed for the determination of intracellular selenium species. NF-κ B activity was studied following TNF-α induction using a luciferase based reporter gene assay (pLUC-NFκ B).Results: MSA inhibited the growth of human lymphoma cells in a concentration and time-dependent manner, but had a reduced effect in normal PBMC's. MSA increased the potency of doxorubicin in all lymphoma cell lines when used in combination for 48h. Doxorubicin (1–3μ M) showed no effect in terms of cell kill, but the addition of sub-toxic concentrations of MSA resulted in a marked increase in cytotoxicity, with an 80–90% reduction in cell viability in MSA sensitive cells at 1μ M MSA, and a 60–70% reduction in less sensitive MSA cells at 20μ M MSA. It was notable that cell lines that were less sensitive to MSA also had lower basal intracellular selenium levels. Cell lines also varied in their ability to take up MSA after a 4h and 24h exposure. Preliminary data suggest that MSA was mainly converted to methylselenocysteine (70%) at 4h, with a smaller amount appearing as selenomethionine (20%). We also investigated whether MSA exposure altered NF-κ B activity, and observed a concentration-dependent inhibition of NF-κ B in the TNF-α induced system used, resulting in 35% and 75% reduction at 5μ M and 10μM MSA, respectively, in DoHH-2 and DHL-4 cells.Conclusions: Our continuing investigations in lymphoma cell lines confirmed the cytotoxic activity of selenium species as a single agent, but importantly, and for the first time in the lymphoma setting, showed a synergistic effect in terms of cell kill when used in combination with a standard anti-lymphoma agent. The novel ICP-MS technique used will prove useful in our ongoing studies of selenium in combination with chemotherapeutics in an animal model by characterising intracellular changes in selenium species. The effect of MSA on NF-κ B activity suggests a molecular mechanism that may be mediating the interaction observed. In conclusion these findings support the use of selenium compounds in the treatment of lymphomas.