Sigma 2-Agonist CB-64D Induces Apoptosis in Non Hodgkin's Lymphoma Cells: Association with Decreased Akt Expression.

Abstract

Background and aim: Sigma (σ) receptors are expressed at high density in a variety of tumor cell types, with an increased expression of σ2-subtype in rapidly proliferating tumors. CB-64D is a recently developed sigma agonist, showing a higher affinity for σ2- receptor subtype. We evaluated the cytotoxic activity of CB-64D in the extensively characterized Non-Hodgkin's Lymphoma (NHL) cell lines DB, RL, and Ramos.Materials and methods: Human DB, RL (both derived from patients with diffuse large cell lymphoma), and Ramos (Burkitt's lymphoma) cells were exposed to increasing concentrations of CB-64D for various periods of time. Cells were harvested and stained with Annexin-V and PI and apoptosis was evaluated by flow cytometry. Expression of caspases, Akt and Bcl-2 was determined by Western blotting.Results: CB-64D induced apoptosis in all NHL cells tested in both a time- and dose- dependent manner. Apoptosis was detected as early as 3 hours (7.5%) and reached its maximum at 12 hours (75%). As little as 5 μM CB-64D was able to induce apoptosis and maximum effects were noted with 40 μM CB-64D. To investigate the mechanisms responsible for the observed apoptosis, expression of caspases, Bcl-2, and Akt was evaluated. Western blot experiments revealed that CB-64D induced the expression of activated caspases 3, 8, and 9. Inhibitors of both caspase 3 and caspase 8 significantly decreased CB-64D-induced apoptosis. The induction of apoptosis was not associated with changes in Bcl-2 levels. In contrast, CB-64D induced a major decrease of Akt as determined by Western blot. Northern blot analysis demonstrated that Akt mRNA was not affected by CB-64D suggesting that the decreased expression of Akt was controlled at the post-transcriptional level. Treatment with the proteasome inhibitor PS-341 failed to prevent CB-64D-induced Akt down-modulation suggesting that the decreased expression of Akt is not due to proteasome degradation.Conclusions: Our data demonstrate that the σ2-agonist CB-64D has a potent anti-lymphoma activity. CB-64D directly induces apoptosis through caspase-dependent mechanisms. Our data also show that CB-64D-induced apoptosis is associated with a decreased Akt expression that is controlled at the post-transcriptional level. The present results warrant the exploration of CB-64D in a pre-clinical model.