ABSTRACT
PURPOSE: Cushing's disease is associated with significant morbidity; thus, additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway, which plays regulatory role in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, on cell viability, apoptosis, cell cycle phase distribution, and ACTH production in mouse corticotroph tumor cells. METHODS: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using an MTS-based assay. Apoptosis was evaluated by Annexin V staining. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Ccnd1, Ccne1, Cdk2, Cdk4, Myc, and Rb1) was assessed by qPCR. Protein expression of p27, total and phosphorylated Akt was assessed by Western blot. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. RESULTS: Treatment with BKM120 decreased AtT-20/D16v-F2 cell viability, induced a G0/G1 cell cycle arrest, reduced the phosphorylation of Akt at Serine 473, and increased p27 expression. Furthermore, BKM120 treatment diminished ACTH levels in the cell culture supernatants. CONCLUSION: In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in anti-proliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing's disease.
Subject(s)
Adenoma , Pituitary ACTH Hypersecretion , Pituitary Neoplasms , Adenoma/pathology , Adrenocorticotropic Hormone/metabolism , Aminopyridines , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , Corticotrophs/metabolism , Corticotrophs/pathology , Humans , Mice , Morpholines , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinase/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Pituitary ACTH Hypersecretion/metabolism , Pituitary Neoplasms/pathology , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.
