All Trans Retinoic Acid Nanodisks Enhance Retinoic Acid Receptor-Mediated Apoptosis and Cell Cycle Arrest in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is characterized by the translocation t (11; 14)(q13; q32), aggressive clinical behavior, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel pathways. All-trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. We hypothesized that since MCL cells express retinoid receptors, ATRA will exert anti-proliferative effects and thus may have a role in treatment. In the present study, we pursued a novel approach to deliver an ATRA payload to MCL cells in culture. This water insoluble bioactive lipid was stably incorporated into nanoscale lipid particles, termed nanodisks (ND). ND are comprised of a disk-shaped phospholipid bilayer whose periphery is stabilized by amphipathic apolipoproteins. It was hypothesized that following cellular uptake and/or liberation of ATRA from ND, this retinoid will interact with intracellular binding proteins and, ultimately, nuclear hormone receptors, leading to target gene transactivation, cell growth arrest and/or apoptosis.

We studied the mantle cell lymphoma cell lines Granta, NCEB and JEKO. ATRA-ND were prepared using recombinant human apolipoprotein A-I as the scaffold protein. Empty ND, lacking ATRA, were prepared in the same manner except that ATRA was omitted from the formulation contents. In various experiments ATRA was presented to cells using dimethylsulfoxide (DMSO) as vehicle (naked ATRA). Reactive oxygen species (ROS) were measured by oxidation of 2'7'dichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF) and quantified by fluorescence intensity using FACS. Apoptosis was quantified by Annexin V-FITC and PI using flow cytometry and FACS. Cell cycle analysis was measured by flow cytometry using FACS. PARP cleavage, caspase activation and analysis of cell cycle regulator proteins was measured by Western blotting, and retinoic acid receptor activity was measured by RT-PCR.

We found that ATRA-ND induced significantly more cell death than naked ATRA (in dimethylsulfoxide) or empty ND. In all three cell lines, ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. Incubation of cells with the antioxidant, N-acetylcysteine, inhibited ATRA-ND-induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 cell cycle arrest. Cyclin dependent kinases (CDK) regulate checkpoints that integrate mitogenic and growth inhibitory signals during cell cycle transitions. CDK inhibitors bind to CDK and inhibit kinase activity, leading to cell cycle arrest. P21 and p27 negatively regulate CDKs, and since p21 is induced by the tumor suppressor p53 in response to DNA damage, the expression level of these proteins was examined in Granta cells as a function of ATRA exposure. Compared to untreated control cells, no changes in the level of these proteins were seen following incubation with empty-ND. Whereas naked ATRA induced a modest increase in p21, p27 and p53, much larger increases were induced by ATRA-ND. Further ATRA-ND resulted in striking decrease in cyclin-D1. At ATRA concentrations that induce apoptosis, expression levels of the retinoic acid receptor- α (RAR α) and retinoid X receptor- γ (RXR γ) were increased. Furthermore, the RAR antagonist, Ro41-5253, inhibited ATRA-ND-induced ROS generation and abrogated ATRA-ND-induced cell growth arrest and apoptosis. Taken together, we found that ATRA-ND-induced apoptosis is dependent on ROS generation and RAR activation. We do not know if ATRA in ND is more stable or resists degradation during the course of cell incubations or whether ATRA-ND are taken up by the cells via receptor mediated endocytosis. Further work is required to elucidate the molecular basis of the enhanced biological activity of ATRA when presented to cells as a component of ND.

ATRA-ND greatly enhanced apoptosis and cell cycle arrest in MCL cell lines, and resulted in increase in p21, p27 and p53 with a decrease in cyclin D1. It is conceivable that solubilization of ATRA in the ND hydrophobic milieu effectively concentrates this bioactive lipid and provides a means for more efficient delivery to target cells. Nonetheless, results obtained in this study suggest ATRA-ND represent a potentially effective approach to the treatment of MCL and should be explored further.

No relevant conflicts of interest to declare.