Endogenous TH metabolite 3-iodothyronamine (T1AM) and synthetic thyronamine-like analogues SG-1 and SG-2 induce autophagy in human glioblastoma cells (U-87MG)

Endogenous TH metabolite 3-iodothyronamine (T1AM) and recently developed thyronamine-like synthetic analogues SG1 and SG2 have emerged as neuroprotective agents. Autophagy has proved to be an effective therapeutic approach for neurodegenerative diseases. Therefore we investigated whether these compounds can induce autophagy in human glioblastoma cells (U-87MG). Cultured U-87MG cells were initially treated with 1 μM T1AM, SG-1, SG-2 or vehicle for 30’, 4, 8 and 24 h and autophagy was monitored morphologically by assessing the presence of autophagic vacuoles and LC3-II puncta formation with transmission electron microscopy (TEM) and immunofluorescence (IF) microscopy, respectively. In addition, cellular lysates were subjected to western blotting to assess the accumulation of microtubule-associated protein 1 light chain 3 (LC3). This protein is widely used as a marker for autophagy because it is a structural protein vital in autophagosome formation. TEM and IF microscopy showed a significant time dependent increase of autophagy-like vacuoles density and LC3 punta formation in U-87MG cells exposed to the treatment with test compounds, with T1AM and SG-1 being the most effective. Along with extensive cytoplasmic vacuolization, western blotting analysis revealed significant up regulation of LC3-II expression (P<0.01). Finally, we extended our analysis to examine the role played by our test compounds on the PI3K–AKT–mTOR pathway that has been suggested to play an important role in the regulation of autophagy in mammalian cells. We carried out WB for Akt, phosphorylated Akt (pAkt) and we calculated the ratio pAkt/Akt in vehicle-treated cells following test compounds administration. We found that 1 μM T1AM, SG-1 and SG-2 decreased such a ratio after 30’ and 4 h treatment, suggesting that they might induce autophagy through the modulation of pAkt level. In conclusion, T1AM and synthetic analogues induce autophagy in a glioblastoma cell line. The potential pathophysiological and/or therapeutic implications of this finding remain to be determined.