In vitro effects of ascorbic acid on viability and metabolism of patients’ osteosarcoma stem cells

Stagnation in novelties of osteosarcoma (OS) treatment indicates the need for new therapeutic methods. OS cancer stem cells (OS-CSC) are taught to have the ability to self-renew and develop mechanisms of anticancer drug resistance, and this is why it is difficult to eradicate them. Their metabolism has been recognized as a potential target of therapeutic action. Ascorbic acid (AA) is considered to act pro-oxidative against OS-CSC in vitro by oxidative effect and by inhibition of glycolysis. This study examined an in vitro impact of AA on OS-CSC metabolism isolated from patients' biopsies, with the aim of better understanding of OS-CSC metabolism and the action of AA on OS-CSC. OS-CSC were isolated using a sphere culture system and identified as stem cells using Hoechst 33342 exclusion assay. Determination of the dominant type of metabolism of OS-CSC, parental OS cells, human mesenchymal stem cells (hMSC) and U2OS OS lineage before and after AA treatment was done by Seahorse XF (Agilent). Cytotoxicity of high-dose AA was confirmed by the MTT test and was proven for all the examined cell types as well as HEK293. Seahorse technology showed that OS-CSC can potentially use both glycolysis and oxidative phosphorylation (OXPHOS), and can turn to glycolysis and slow metabolic potential in unfavorable conditions such as incubation in AA.