Induction of ferroptosis of glioblastoma cells using combined treatment with chloramphenicol and 2-deoxy-d-glucose

Little is known about the relationship between glioblastoma and mitochondria. We focused on mitochondria as they are key organelle targets in the treatment of glioblastoma²¹. In addition, hypoglycemic and hypoxic conditions exist in the tumor environment^14,15. In our previous study, we focused on the development of treatment for glioblastoma in hypoglycemic conditions⁹. The results of this earlier study were as follows: mitochondria were activated under glucose starvation conditions and antimicrobial agents were effective under glucose starvation conditions. Because the tumor environment is hypoglycemic and deficient in various nutrients, previous findings represented promising treatments. However, under glucose deficiency conditions, the glucose level (100 mg/L) was much lower than under normal glucose conditions (1000 mg/L)⁹. Given tumor heterogeneity, some nutrient-rich lesions may have higher glucose levels than nutrient-poor lesions. It is important to develop an effective treatment with different concentrations of oxygen under normal and low glucose conditions to overcome these malignant tumors. Therefore, in this study, we focused on normal blood glucose and hypoxic conditions and developed a combination treatment using CAP and 2-DG. This treatment drastically inhibited cell growth under normal glucose conditions under normal and hypoxic conditions. Thus, the combination treatment may hold promise for patients with glioblastoma.

According to our results, the combination treatment was ineffective under high blood glucose conditions (4500 mg/L) compared to normal blood glucose conditions, highlighting the importance of maintaining glucose levels within the normal range. According to a previous study, blood glucose levels are associated with prognosis in patients with glioblastoma, with higher glucose levels leading to poorer prognoses.²². Moreover, OCR was higher under normal glucose conditions than under high glucose conditions, suggesting that mitochondria-targeted therapy is more effective under normal glucose conditions than under high glucose conditions.⁹a glucose concentration of 1000 mg/L being considered normal in humans.

The blood-brain barrier (BBB) ​​prevents certain agents from invading the brain²³. Thus, one must ask whether an agent can cross the BBB. Recently, nanotherapeutic techniques have been developed to overcome the BBB to improve the treatment of glioblastoma²³. Among the different agents available, we have selected two agents, CAP and 2-DG, which can easily cross the BBB and directly affect the mitochondria^24,25.

CAP is an antimicrobial agent that induces mitochondrial dysfunction²⁶. Additionally, this agent has been used to treat meningitis²⁷. Dunkel et al.²⁷ reported that the effective blood concentration for preterm infants with central nervous system infection was 46–154 μM. Moreover, a concentration of 100 μM is reasonable and less harmful. In addition, when using this agent, repositioning of the drug is expected. Metformin has recently been used to treat cancers, including glioblastoma²⁸. Kim et al.²⁸ reported that 2-DG combined with metformin inhibits proliferation of glioblastoma cells. We studied the effect of metformin under conditions of glucose deprivation, and our study reported that CAP was effective under conditions of glucose deprivation.⁹while metformin was not very effective (Fig. S8). This difference is probably due to differences in the mechanisms of action of these two drugs. CAP inhibits mitochondrial ribosomes and causes mitochondrial dysfunction²⁶. Metformin would inhibit mitochondrial complex 1; however, another theory has been proposed, and the exact mechanism has not been fully elucidated²⁹. PAC is effective under both glucose-starved and normal conditions. Therefore, we believe that CAP is a key drug for the treatment of glioblastoma.

2-DG is used to treat cancers targeting glycolysis¹². Stein and Raez conducted a clinical trial to treat cancers or solid tumors using 2-DG and presented its safety at appropriate concentrations. Stein recommended a dose of 45 mg/kg, while Raez recommended a safe dose of 63 mg/kg/day^12,13. Moreover, the C_maximum of a dose of 45 mg/kg is 73.7 μg/ml (449 μM), whereas that of a dose of 63 mg/kg/day is 116 μg/ml (almost 700 μM). However, Sasaki et al.³⁰ reported that although 2-DG successfully treats cancer, effective doses induce serious adverse effects. They therefore recommended a new device that delivers 2-DG in poly-lactic-co-glycolic acid nanoparticles. In our study, the concentration of 2-DG was 300 μM, which is within the safe range.

2-DG inhibits glycolysis and provides conditions similar to glucose starvation. Under conditions of glucose starvation, as noted earlier, mitochondria become dominant. 2-DG also increases the expression of COX1 and OXPHOS, implying mitochondrial dominance. Furthermore, we consider that this nutrient deficiency leads to an increase in fatty acid oxidation activity and assume that the increase in OXPHOS is due to fatty acid activations. Etomoxir is used to detect fatty acid oxidation activity, and high-dose etomoxir is reported to inhibit mitochondrial complex 1³¹. OXPHOS decreased after etomoxir injection, although the decrease was less than expected. Thus, there may be other pathways, including the amino acid pathway. Further investigation is needed to elucidate this.

Based on the study results, ferroptosis may have occurred. Several markers of ferroptosis, including KEAP1, NRF2, HO-1, GpX4, TFRC, FTH1 and xCT, have recently been reported¹⁷. A previous study showed that PAC induces ferroptosis via the p-p62-KEAP1-NRF2-HO-1 pathway⁹. In this study, we used two different agents; it was therefore difficult to confirm the existence of a single ferroptosis flux. However, both agents significantly affected iron dynamics and showed a combined effect on FTH1, GpX4 and KEAP1. In addition, to confirm whether these agents induce ferroptosis, it is important to use an inhibitor of this pathway; we used DFO accordingly. DFO inhibited both agents, and we believe that the mechanism underlying cell death in this case is ferroptosis. In this study, we evaluated other cell death pathways, including apoptosis and necroptosis. Expression of caspase 3, which involves apoptosis, was not altered in each agent. Moreover, an increase in the level of RIP3K mRNA, which is linked to necroptosis, was not detected. Additionally, one of the reasons 2-DG causes ferroptosis may be that glucose deprivation induces blockade of the serine synthesis pathway, a pathway derived from glycolysis.³². This leads to the depletion of glutathione, a GpX4 activator protecting against ferroptosis³³.

A limitation of this study is that the data was collected in vitro rather than in vivo; thus, further in vivo studies in mice are needed. However, the development of an effective treatment is worth mentioning. In conclusion, we have developed an effective treatment combining CAP with 2-DG to treat glioblastoma cell lines under normal glucose conditions. Therefore, the results of our study appear beneficial for developing treatments for glioblastoma.