Induction of Cell Cycle Arrest and Apoptosis by Dephosphorylation of ERK, Akt, and Bcl-2 and Phosphorylation of JNK by LY293111 in Human Anaplastic Large Cell Lymphoma (ALCL).

Abstract

The prognosis of patients with anaplastic large cell lymphoma (ALCL) treated with standard chemotherapy remains poor. Leukotriene B4 (LTB4) receptor has been reported to exhibit a growth-promoting activity in lymphoma, since it augments DNA synthesis and increases cell proliferation of lymphocytes. LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a Leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. LY293111 inhibits pancreatic cancer cell growth, induces apoptosis, and reduces growth of colon cancer in vivo. In the present study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in an ALCL cell line. We utilized the Sup/M2 cell line derived from human anaplastic large cell lymphoma as a model. Dose-response studies demonstrated that the IC50 of LY293111 was 4μM after 5 days of treatment. LY293111 caused retardation of Sup/M2 cell entry into S phase in a dose-dependent fashion as measured by BrdU/propidium iodide flow cytometry. LY293111 at 2.5μM induced complete G1-S cell cycle arrest in cells synchronized by serum starvation. LY293111 upregulated p21 and p27 protein expression and reduced cyclin E mRNA and protein. Pre-treatment with LY293111 for 4 hours resulted in profound inhibition of serum-induced phosphorylation of ERK1/2, Akt, and Bcl-2. Concomitantly, phosphorylation of stress-activated kinase JNK dramatically increased following LY293111 treatment. LY293111 induced pronounced apoptosis of Sup/M2 cells and caspase inhibition by Benzyloxycarbonyl -Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) abrogated this effect. LY293111 induced cleavage of the caspase-9, -3, PARP and XIAP, but had no effect on caspase-8, suggesting activation of the intrinsic apoptotic pathway. Accordingly, early loss of mitochondrial membrane potential was observed in a dose-dependent fashion. At 72 hrs, LY293111 decreased Bcl-2 expression levels. These findings provide the first evidence that LY293111 inhibits ALCL proliferation by arresting cells in G1 phase of the cell cycle, as a result of cyclin E downregulation and induction of cell-cycle inhibitory proteins p21 and p27. LY293111 induces apoptosis in Sup/M2 lymphoma cells, with activation of the mitochondrial apoptosis pathway, including cleavage of caspase 9, caspase 3, PARP, XIAP, and Bcl-2. Furthermore, LY293111 treatment markedly shifts signaling toward the JNK stress-related pathway and away from cytoprotective MEK/ERK and AKT signaling. These results suggest that LY293111 may have a utility in the management of aggressive non-Hodgkin's lymphomas.