The Tricyclic Coumarin GUT-70 Induces Apoptosis and Cell Cycle Arrest Preferentially in Mantle Cell Lymphomas with Mutant p53.

Abstract 1684

Poster Board I-710

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma resistant to standard chemotherapy. Since p53 inactivating mutations occur primarily in the aggressive and refractory MCL variants, development of novel compounds that target p53-independent signaling pathways is of considerable interest. We investigated the cytotoxic efficacy and molecular mechanisms of a newly discovered anticancer agent GUT-70 (synthesized at Nippon Shinyaku, Kyoto, Japan), a natural product derived from the stem bark of Calophyllum brasiliense, characterized as a tricyclic coumarin with the formula 5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b]dipyran-8-one (C₂₃H₂₆O₅). This agent has pronounced anti-tumor activity, but does not inhibit colony formation by normal hematopoietic progenitors or proliferation of normal human hepatocytes. (Kimura, Int J Cancer 2005;113:158) However, their mechanisms have not been fully investigated. In this study, cytotoxicity and mechanisms of action of GUT-70 were investigated in MCL cell lines with wild-type and mutant p53 (wt-p53: JVM-2, Granta-519, mt-p53: Jeko-1, MINO). Treatment with GUT-70 resulted in marked reduction in cell growth (trypan blue corrected cell numbers) and an increase in the apoptotic fraction (Annexin V), in a time- and concentration-dependent manner. Importantly, mt-p53 MCL were more sensitive than wt-p53 cells (IC₅₀ at 48 hrs: JVM-2, 4.5 μM; Granta 519, 6.3 μM; Jeko-1, 0.7 μM; MINO, 2.2 μM, % specific apoptosis of 5μM GUT-70 treated cell: JVM-2, 18.5%; Granta 519, 17.6%; Jeko-1, 38.1%; MINO, 30.9%; Annexin V). GUT-70 also impeded cell cycle progression, resulting in a decreased S-phase with increased G0/G1 cells independent of p53 status (S-phase was decreased by 8.2 % in JVM-2, 12.1% in Granta 519, 10.0 % in Jeko-1, 9.8 % in MINO). This was associated with a dramatic morphological change: bleb-like cytoplasmic enlargement without visible nuclear breakdown observed by phase-contrast time-lapse video microscopy. Next, the ability of GUT-70 to modulate cell cycle and apoptosis related proteins including p53 target genes was analyzed by western blotting. GUT-70 treatment significantly reduced cyclin D1, the hallmark of MCL, believed to be critical for lymphomagenesis, and increased p27 levels. Furthermore, GUT-70 inactivated and/or degraded Rb and repressed E2F1, effects similar to the action of the specific 26S proteasome inhibitors MG132 and bortezomib. GUT-70 induced mitochondrial apoptosis associated with caspase-9 and -3 activation, accompanied by transcriptional induction of the proapoptotic BH3-only protein Noxa. Notably, in highly sensitive Jeko-1 and MINO cells expressing mt-TP53, antiapoptotic Mcl-1 was not upregulated, whereas in less sensitive JVM-2 and Granta-519 cells with wt-TP53 GUT-70 caused Mcl-1 accumulation, which co-immunoprecipitated with Noxa. In addition, we observed higher levels of activated Bak in Jeko-1 and MINO cells compared to JVM-2 and Granta-519 cells. In summary, these data indicate that the novel anticancer agent GUT-70 depletes cyclin D1 and induces mitochondrial apoptotic cell death in MCL. Notably, these effects are more pronounced in MCL with mutant p53, a known negative prognostic factor for MCL. These findings suggest potential utility of GUT-70 for the treatment of MCL.