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Effects of Imatinib Combined With Everolimus on Mouse Pituitary Tumor Cell AtT-20
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.
Insights
Imatinib and everolimus combination therapy significantly reduced pituitary tumor cell viability and ACTH production. This treatment also induced apoptosis and altered cell cycle progression in AtT-20 cells, suggesting potential as a therapeutic strategy.
Area of Science:
- Endocrinology and Molecular Oncology
- Pituitary Tumor Research
- Pharmacological Interventions
Background:
- Pituitary adenomas involve excessive adrenocorticotropic hormone (ACTH) production.
- The influence of cell-cycle regulation on ACTH production and tumor cell proliferation remains unclear.
- Imatinib has demonstrated potential in inducing tumor cell apoptosis.
Purpose of the Study:
- To investigate the effects of combining imatinib and everolimus on AtT-20 mouse pituitary tumor cells.
- To elucidate the molecular mechanisms underlying the combined drug action on cell proliferation, apoptosis, and ACTH production.
Main Methods:
- Cultured murine corticotropin tumor AtT-20 cells.
- Treated cells with imatinib and everolimus.
- Assessed cell viability (MTT), mitochondrial membrane potential, LDH leakage, apoptosis (TUNEL), protein expression (Western blot), mRNA expression (RT-PCR), ACTH concentration (ELISA), and cell cycle distribution (flow cytometry).
Main Results:
- The combination therapy significantly decreased cell viability and ACTH concentration.
- Increased markers of apoptosis and mitochondrial dysfunction were observed.
- Downregulation of p-Akt, p-CREB, and cyclin E, alongside upregulation of p27 and p53, indicated cell cycle arrest.
Conclusions:
- Imatinib and everolimus combination impacts the AtT-20 cell cycle via the PI3K/Akt/PKA pathway.
- The combined treatment inhibits cell proliferation and induces apoptosis in pituitary tumor cells.
- This drug combination shows promise as a potential therapeutic strategy for mouse pituitary tumors.
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