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Elucidation of OSW-1-Induced Stress Responses in Neuro2a Cells
Kentaro Oh-Hashi1,2,3, Hibiki Nakamura1, Hirotaka Ogawa2
1Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
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.
Insights
OSW-1, a potential anticancer drug, triggers unique Golgi stress responses and autophagy in neuroblastoma cells. Further research into these mechanisms could lead to novel cancer therapies.
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Cancer Research
Background:
- OSW-1, a steroidal saponin from Ornithogalum saundersiae, shows anticancer potential but its cytotoxic mechanisms are unclear.
- Understanding cellular stress responses is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To investigate the cytotoxic mechanisms of OSW-1 in Neuro2a cells by analyzing Golgi and ER stress responses.
- To compare OSW-1's effects with brefeldin A (BFA), a known Golgi disruptor.
- To elucidate OSW-1-induced gene expression changes and their relation to lipid metabolism and ER-Golgi transport.
Main Methods:
- Comparative analysis of OSW-1 and BFA in Neuro2a cells.
- Assessment of Golgi stress sensors (TFE3/TFEB, CREB3) and ER stress markers (GADD153, GADD34).
- Microarray analysis for gene expression profiling.
- Evaluation of secretory activity using NanoLuc-tag genes.
- Generation and analysis of oxysterol-binding protein (OSBP) knockout Neuro2a cells.
Main Results:
- OSW-1 induced TFE3/TFEB dephosphorylation but not CREB3 cleavage, with minimal ER stress.
- OSW-1 significantly increased autophagy marker LC3-II, more so than BFA.
- Microarray analysis revealed OSW-1 affects genes in lipid metabolism and ER-Golgi regulation.
- Abnormalities in ER-Golgi transport were observed.
- OSBP deficiency did not significantly alter OSW-1-induced cell death or autophagy.
Conclusions:
- OSW-1 elicits atypical Golgi stress responses and potent autophagy induction in Neuro2a cells.
- The compound impacts lipid metabolism and ER-Golgi transport.
- Further investigation into the interplay between OSW-1-induced Golgi stress and autophagy may yield new anticancer strategies.
