Signaling Pathways of All-Trans Retinoic Acid (ATRA)-Induced Apoptosis in Mantle Cell Lymphoma (MCL).

MCL, characterized by the t(11;14), results in the overexpression of cyclin D1, and typically has an aggressive clinical course. The disease is not curable by conventional therapy, and new modalities are needed. A MCL cell line, Granta, has been studied and has a complex karyotype and genetic alterations of cell cycle regulatory genes. ATRA is a metabolic derivative of retinoic acid (RA). Upon binding with ATRA, its receptors become transcriptionally activated and transactivate their target genes leading to cell growth arrest or apoptosis. We hypothesized that ATRA would lead to apoptosis or cell growth arrest in Granta MCL cells. We used ATRA nanodisks for increased solubilization and in vivo delivery. Reactive oxygen species (ROS) were measured with fluorescence-activated cell sorting (FACS) following incubation at 6 hours (h) and 24 h with ATRA (12.5–50nM). At 6 h, ATRA induced dose-dependent generation of ROS, which returned to baseline at 24 h. The levels of ROS in untreated controls were unchanged over all the incubation time points. To determine the effects of ATRA on apoptosis, Granta cells were treated with various concentrations for 6 h, 24 h, and 48 h; apoptosis was measured with annexinV/propidium iodide (annexin/PI) binding and FACS analysis. We found increasing apoptosis in Granta at 24 h, reaching significant levels at 48 h. At 48 h, the lowest doses of ATRA (12.5nM) resulted in >70% increase (as a percentage of control) in annexin/PI. To determine the biologic consequences of ATRA exposure in Granta, RARα protein levels were measured in control and ATRA treated cells at 2 h, 6 h, and 24 h. RARα protein was marginally increased at 6 h and significantly downregulated following 24 h ATRA exposure (50nM). Furthermore, ATRA significantly increased p21 protein expression at 24 h as assessed by immunoblotting. RT-PCR data showed that SESN1(T2) (an oxidant gene) transcript was virtually absent in Granta, and that SESN2 and CDKN1A transcripts were not affected by ATRA exposure. In summary, these data demonstrate that ATRA, complexed with nanodisk delivery particles, stimulates ROS generation as early as 6 h in Granta cells. In addition, ATRA induced significant apoptosis at 48 h, downregulated RARα protein, and increased p21 protein. Further studies investigating the role of ATRA in MCL are warranted.