Elucidation of OSW-1-Induced Stress Responses in Neuro2a Cells

Abstract

OSW-1, a steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, is a promising compound for an anticancer drug; however, its cytotoxic mechanisms have not been fully elucidated. Therefore, we analyzed the stress responses triggered by OSW-1 in the mouse neuroblastoma cell line Neuro2a by comparing it with brefeldin A (BFA), a Golgi apparatus-disrupting reagent. Among the Golgi stress sensors TFE3/TFEB and CREB3, OSW-1 induced dephosphorylation of TFE3/TFEB but not cleavage of CREB3, and induction of the ER stress-inducible genes GADD153 and GADD34 was slight. On the other hand, the induction of LC3-II, an autophagy marker, was more pronounced than the BFA stimulation. To elucidate OSW-1-induced gene expression, we performed a comprehensive gene analysis using a microarray method and observed changes in numerous genes involved in lipid metabolism, such as cholesterol, and in the regulation of the ER–Golgi apparatus. Abnormalities in ER–Golgi transport were also evident in the examination of secretory activity using NanoLuc-tag genes. Finally, we established Neuro2a cells lacking oxysterol-binding protein (OSBP), which were severely reduced by OSW-1, but found OSBP deficiency had little effect on OSW-1-induced cell death and the LC3-II/LC3-I ratio in Neuro2a cells. Future work to elucidate the relationship between OSW-1-induced atypical Golgi stress responses and autophagy induction may lead to the development of new anticancer agents.

Keywords

• CREB3
• Golgi stress
• OSW-1
• TFE3
• TFEB