RESUMO
Context: Pituitary adenoma is a clinical syndrome in which excessive production of pituitary corticotropin (ACTH). For ACTH tumor cells, researchers know little about the influence of the cell-cycle process on ACTH production and cell proliferation. Some research has shown that imatinib can induce apoptosis of tumor cells. Objective: The study intended to explore the effects and molecular mechanisms of imatinib combined with everolimus on AtT-20 cells in AtT-20 mouse pituitary tumors. Design: The research team performed a laboratory study using murine corticotropin tumor AtT-20 cells. Setting: The study took place at the Department of Neurosurgery at Renmin Hospital of the Hubei University of Medicine in Shiyan, Hubei, China. Intervention: The research team cultured the cells in AtT-20-cell-specific medium containing 100 µg/mL of streptomycin, 100 U/mL of penicillin, and 10% fetal bovine serum at 37°C and 5% CO2. The team divided the cells into a control group, a normal culture without the drug, and an intervention group, incubated for 24 hours with 1 µM of imatinib and 3 µM of everolimus when the cells grew to 40% confluence. Outcome Measures: The research team: (1) determined the effects of the combined drugs on cell viability using a methyl thiazolyl tetrazolium (MTT) assay; (2) detected the cell's mitochondrial membrane potential and LDH leakage using "sytox blue, 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide," CBIC2(3) or JC-1, and lactate dehydrogenase (LDH) assay kits, respectively; (3) detected AtT-20 cell apoptosis using a "terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling" (TUNEL) kit; (4) analyzed the expression of protein kinase B (p-Akt), cAMP-response element binding protein (p-CREB), p27, p53, and cyclin E using a Western blot test; (5) detected the mRNA expression of opioid melanin procorticotropin (POMC)), caspase-3, and pituitary tumor transforming gene 1 (PTTG1) using reverse transcription-polymerase chain reaction (RT-PCR); (6) measure the concentration of adreno-cortico-tropic-hormone (ACTH) in the supernatant using an enzyme-linked immunoassay (ELISA) kit; and (7) assessed the cell cycle distribution using flow cytometry. Results: No differences existed in cell viability between the groups at the baseline (0 h) of the culture period (P > .05). Compared to the control group, the intervention group's: (1) cell viability was significantly lower at 4, 8, and 12 hours and postintervention at 16 hours (P < .001); (2) LDH concentration was significantly higher (P < .001); (3) mitochondrial membrane potential was significantly lower (P < .001); (4) apoptosis rate of TUNEL was significantly higher (P < .001 ); (5) expression of p-Akt, p-CREB phosphorylation, and cyclin E was significantly lower (P < .001), (6) expression of p27 and p53 protein was significantly higher (P < .001); (7) mRNA expression of POMC and PTTG1 were significantly lower (P < .001); (8) mRNA expression of caspase-3 was significantly higher (P < .001); (9) concentration of ACTH was lower (P < .001); and (10) percentage of cells in the G0/G1 phase was significantly higher, while the percentage of cells in the S phase was significantly lower (P < .05). Conclusions: Imatinib combined with everolimus can affect the AtT-20 cell cycle through the signaling pathway of the phosphatidylin-ositol-3-kinase (PI3K)/Akt/ protein kinase A (PKA) system and can inhibit cell proliferation and induce cell apoptosis. Therefore, Imatinib and everolimus may be an effective combination of candidates for drugs for mouse pituitary tumor.