The Induction of Apoptosis in Lymphoma Cells by Methylseleninic Acid and Selenodiglutathione.

Abstract

Introduction. We have previously demonstrated that presentation serum selenium predicted not only for response to treatment and duration of first remission, but also importantly for overall survival in aggressive non-Hodgkin's lymphoma (Journal of Clinical Oncology 2003, 21:2335–2341). To investigate a possible causal association as the explanation for these findings we have studied the activity of 2 selenium species, methylseleninic acid (MSA) and selenodiglutathione (SDG). We provide the first report of the activity of these two compounds in lymphoma, using a panel of human cell-lines (CRL, DHL4, SUD4 and DoHH2) and a primary human lymphoma culture system.Methods. The effects of 3-day incubations with MSA or SDG on cell proliferation and cell viability (n = 3–6) were determined by Trypan blue exclusion assay. Cell cycle distribution (including a sub-G1 apoptotic fraction), generation of reactive oxygen species (ROS) and PI/annexin staining were analysed by flow cytometry. Apoptosis was further investigated by western blotting for poly-ADP ribose polymerase (PARP) and specific caspase (8 and 9) cleavage. The activity of MSA and SDG was also investigated in a primary lymphoma culture system utilising a feeder layer of CD40 ligand expressing CHO cells irradiated prior to plating primary lymphoma cells.Results. Both MSA and SDG demonstrated cytostatic and cytotoxic effects in all 4 cell-lines studied. The EC50 values ranged from 1.0 – 10.2 μM, with the exception of the % viability EC50 for MSA in DHL4 cells (166 μM). Flow cytometry revealed cell death was associated with an increase in the sub-G1 (apoptotic) fraction, without an obvious preceding G1, S or G2/M cell cycle arrest. Apoptosis was confirmed by flow cytometric analysis of PI/annexin dual labelled cells, and was associated with PARP cleavage and involvement of both caspase 8 and 9. SDG exposure resulted in marked increases in ROS, particularly in CRL and SUD4 cells (3-5-fold increase after 30 minutes exposure to the day-3 viability EC50 concentration). The addition of n-acetyl-cysteine reduced the cytotoxic activity of SDG, but not MSA, confirming that ROS contribute to the effects seen with SDG. In 5 different 2-day primary lymphoma cultures (n = 4–6) MSA and SDG showed a concentration dependent reduction in % viability, with day-2 EC50 values of approximately 30 μM for MSA and 10 μM for SDG.Conclusions. We have demonstrated for the first time the cytostatic and cytotoxic activity of two selenium species, MSA and SDG, in human lymphoma cell-lines and primary lymphoma cells and their induction of caspase mediated apoptosis. These data suggest that selenium species may play a causal role in the success of conventional chemotherapy in aggressive lymphoma. Selenium compounds merit further investigation in the clinical management of this disease.