Metformin Induces Cell Death in a Human Neuroblastoma Cell Line through Affecting Multiple Survival Pathways.
Hong Chai, Priya Weerasinghe, Robert E Brown. University of Texas- Medical School, Houston
Background: Neuroblastoma is the most common extracranial solid cancer in childhood. Its tumorigenicity is enhanced by the expression of survival pathways such as Akt and signal transducer and activator of transcription (STAT)3 as well as an inappropriate level of mammalian target of rapamycin (mTOR) activity. Metformin is one of the most widely used diabetes drugs. It has shown inhibiting effects on mTOR, which may prevent the growth of cancer cells. In this study, we will examine the efficacy of metformin on a human neuroblastoma cell line and also investigate its possible mechanisms.
Design: A neuroblastoma cell line, SK-N-AS, was purchased from ATCC. After reaching 50-60% confluence, cells were treated with various concentrations of metformin (0, 1, 5, 10, 20, 40 mM) for different period of times (0, 30 min, 2, 6, 24, 48 and 72 hours). The cell viability was determined by MTT colorimetric assay. The apoptotic responses were measured with TUNEL assay. Phosphorylation of Akt1/2/3 (p-Akt1/2/3 [Thr308]), AMP-activated protein kinase alpha1 subunit (p-AMPKa1 [Thr172]), p42 MAP Kinase (p-ERK2), STAT3 (p-STAT3 [Ser727]), and mTOR (p-mTOR [S2448]) and total mTOR (mTOR [7C10]) protein expression levels were determined with Western blot assay.
Results: As early as 6 hours post-treatment, cell viability was significantly decreased at 10 mM concentration. In 24 and 48 hours treatment groups, metformin at 5 mM and above concentrations significant decreased cell viability. TUNEL assay showed similar results. Western blots showed a decrease of p-Akt2 and p-STAT3 expression levels in metformin treatment groups. An increase of p-ERK2, p-AMPKa1 was also observed. Total mTOR levels were slight increased while the phosphorylation at S2448 site was significantly decreased.
Conclusions: Our results indicate that metformin significantly decreased cell viability and increased cell death in a human neuroblastoma cell line in association with down-regulation of p-Akt, p-STAT3 and p-mTOR survival pathways. These data suggested a potential clinical application of metformin in the treatment of neuroblastoma cases with constitutive activation of Akt, STAT3 and mTOR.
Monday, February 28, 2011 11:30 AM
Platform Session: Section H 2, Monday Morning