Orexin A induces autophagy in HCT-116 human colon cancer cells through the ERK signaling pathway

Orexins are a class of peptides which have a potent influence on a broad variety of cancer cells. Autophagy is closely associated with tumors; however, its function is not yet completely understood. In this study, we aimed to determine whether orexin A induces autophagy in HCT-116 human colon cancer cells and to elucidate the molecular mechanisms involved. For this purpose, HCT-116 cells were treated with orexin A, and cell viability was then measured by MTT assay, and apoptosis was determined by flow cytometry. The expression levels of autophagy-related proteins were measured by western blot analysis. Quantitative analysis of autophagy following acridine orange (AO) staining was performed using fluorescence microscopy, and cellular morphology was observed under a transmission electron microscope. In addition, the HCT-116 cells were treated with the extracellular signal-regulated kinase (ERK) inhibitor, U0126, or the autophagy inhibitor, chloroquine, in combination with orexin A in order to examine the activation of ERK. We found that orexin A significantly inhibited the viability of the HCT-116 cells. Both autophagy and apoptosis were activated during the orexin A-induced death of HCT-116 cells. When the HCT-116 cells were treated with orexin A for 24 h, an accumulation of punctate microtubule-associated protein-1 light chain 3 (LC3) and an increase in LC3-II protein levels were also detected, indicating the activation of autophagy. Moreover, orexin A upregulated ERK phosphorylation; however, U0126 or chloroquine abrogated ERK phosphorylation and decreased autophagy, compared to treatment with orexin A alone. Therefore, our findings demonstratedm that orexin A induced autophagy through the ERK pathway in HCT-116 human colon cancer cells. The inhibition of autophagy may thus prove to be an effective strategy for enhancing the antitumor potential of orexin A as a treatment for colon cancer.