Therapy for patients with non-Hodgkin's Lymphomas (NHL) have significantly improved over the last decade, especially since the discovery of monoclonal antibodies and other biologic therapies. Although patients with B-cell NHL usually respond to conventional chemotherapy, they often relapse in spite of salvage therapy and stem cell transplantation. Early clinical studies of Bortezomib-based combinations, showed encouraging results both in Follicular Lymphoma (FL) as well as in Mantle Cell Lymphomas (MCL). In this study we hypothesize that combining Bortezomib with Enzastaurin or Lenalidomide would target separate signaling pathways increasing tumor-cell death.
Bortezomib, Lenalidomide and Enzastaurin alone and their combinations were tested in WSU-NHL, RL (FL cell lines) and Granta-519 and Jeko-1 (MCL cell lines) and primary cells from lymphoma patients. B-NHL cell lines were treated for 24–48 hours. The cell proliferation was determined by using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit and cell cytotoxicity with MTT-assay. The interaction between drugs was evaluated by isobologram analysis using the STACorp 8.2 software program based upon the Chou-Talalay method to determine if the combination were additive or synergistic. Apoptosis was evaluated by flow cytometry using Annexin V/Propidium Iodide (PI) staining. The effect on cell cycle was analyzed using PI by flow cytometry. Western blotting experiments were performed to determine whether the drugs combinations affected PI3K/Akt, PKC and MAPK/ERK pathways.
In the present study we have shown that Enzastaurin and Lenalidomide enhanced the cytotoxicity of Bortezomib in all B-NHL cell lines and primary cells from lymphoma patients. A clear synergistic interaction, confirmed by the Chou-Talalay method (combination index<1) was observed after 24 hours using low concentrations of all the drugs (Bortezomib 6 nM + Lenalidomide 6 μM; Bortezomib 6 nM + Enzastaurin 6 μM). The combination of Bortezomib with both Enzastaurin or Lenalidomide did not trigger relevant decrease in the viability of normal peripheral blood mononuclear cells (PBMNCs) and suppressed cell proliferation of B-NHL cell lines when co-cultured with bone marrow stromal cells (BMSCs) in a system that mimics the bone marrow microenvironment. In comparison with each single agents, the combination of Bortezomib with both Enzastaurin and Lenalidomide induced significant increase of apoptosis. This enhancement of apoptosis is mediated by an increased ratio of pro-apoptotic protein (Bim, Bad) to anti-apoptotic proteins (Bcl-2, Bcl-xL) which increased the threshold for caspases 3 and 9. The cycle analysis showed that the combination of Bortezomib with both Enzastaurin or Lenalidomide reduced the proportion of cells in the G0/G1, S and G2/M phase, increasing sub G0/G1. Western blot analysis showed that anti-proliferative events and pro-apoptotic effects were associated with dephosphorylation of PI3K/Akt and MAPK/ERK pathways.
In this study, we investigated the direct antitumor activity of Bortezomib combined with Enzastaurin or Lenalidomide in established B-NHL cells (Follicular Lymphoma and Mantle Cell Lymphoma) and freshly isolated patients cells in vitro. Our results demonstrated that the combination of Bortezomib with both Enzastaurin and Lenalidomide induces synergistic anti-proliferative and pro-apoptotic effects in all B-cell lymphoma cell lines and primary cells, even in the presence of the bone marrow microenvironment. This direct cytoxicity is mediated by signaling events involving PI3K/Akt, MAPK/ERK and Bcl-2 pathways leading to cell death. Hence, this in vitro studies to test combinations of these active agents in patients with Follicular Lymphoma and Mantle Cell Lymphoma.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.