ABSTRACT: We synthesized novel fluorescent thalidomide analogs (2,6-dialkylphenyl-4/5-amino-substituted-5,6,7-trifluorophthalimides) that localize specifically to lipid droplets. By using affinity chromatography interacting proteins were identified: Rab7, Rab10, Rab11, Sec22b, sorting nexin 2, calnexin, protein disulfide isomerase, GRP78, GRP94 among others that are involved in vesicular transport and ER stress response. Amino-trifluoro-phthalimides exerted potent anticancer activities in vitro on different cell lines including melanoma, leukemia, hepatocellular carcinoma, glioblastoma. They are non-toxic to adult animals up-to 1 g/kg but are highly teratogenic to zebrafish embryos at micromolar concentrations resulting defects in developing muscle. The analogs resulted in calcium release from the endoplasmic reticulum (ER), induction of reactive oxygen species (ROS), ER stress and finally apoptosis. Gene expression analysis confirmed the induction of ER stress-mediated apoptosis pathway components, such as growth arrest- and DNA damage-inducible gene 153 (GADD153), ATF3, Luman/CREB3 recruitment factor (LRF) and the ER-associated degradation-related gene HERPUD1. Tumor suppressors, P53, LATS2 and ING3 were also up-regulated in various cell lines after drug treatment. Exogenous docosahexaenoic acid or eicosapentaenoic acid induced calcium release and ROS and synergized with the analogs in vitro, while oleic acid had no such an effect. Different antioxidants partially, intracellular calcium chelator almost completely diminished ROS production. Amino-phthalimides down-regulated the expression of CCL2, which is implicated in tumor metastasis and angiogenesis. Because of the anticancer, anti-angiogenic action and the wide range of applicability of the immunomodulatory drugs, lipid droplet-binding members of this family could represent a new class of agents by affecting ER-membrane integrity and perturbations of ER homeostasis.
