Cold Atmospheric Plasma and Silymarin Nanoemulsion Activate Autophagy in Human Melanoma Cells

Background: Autophagy is reported as a survival or death-promoting pathway that is highly debatable in different kinds of cancer. Here, we examined the co-effect of cold atmospheric plasma (CAP) and silymarin nanoemulsion (SN) treatment on G-361 human melanoma cells via autophagy induction. Methods: The temperature and pH of the media, along with the cell number, were evaluated. The intracellular glucose level and PI3K/mTOR and EGFR downstream pathways were assessed. Autophagy-related genes, related transcriptional factors, and autophagy induction were estimated using confocal microscopy, flow cytometry, and ELISA. Results: CAP treatment increased the temperature and pH of the media, while its combination with SN resulted in a decrease in intracellular ATP with the downregulation of PI3K/AKT/mTOR survival and RAS/MEK transcriptional pathways. Co-treatment blocked downstream paths of survival pathways and reduced PI3K (2 times), mTOR (10 times), EGFR (5 times), HRAS (5 times), and MEK (10 times). CAP and SN co-treated treatment modulates transcriptional factor expressions (ZKSCAN3, TFEB, FOXO1, CRTC2, and CREBBP) and specific genes (BECN-1, AMBRA-1, MAP1LC3A, and SQSTM) related to autophagy induction. Conclusion: CAP and SN together activate autophagy in G-361 cells by activating PI3K/mTOR and EGFR pathways, expressing autophagy-related transcription factors and genes.