Loss of pro-apoptotic Bax and Bak increases resistance to dihydroartemisinin-mediated cytotoxicity in normoxia but not in hypoxia in HCT116 colorectal cancer cells.

Tumor hypoxia is a major biological factor that drives resistance to chemotherapy and radiotherapy. We previously demonstrated that the pro-oxidative drug dihydroartemisinin (DHA) efficiently targeted normoxic and hypoxic cancer cells. Although well studied in normoxia, the mechanism behind DHA-mediated cytotoxicity in hypoxia is insufficiently explored. Here, we analyzed the effect of DHA in HCT116 wild type (wt) cells and in HCT116 Bax-/-Baksh cells with a defective intrinsic apoptosis pathway. Normoxic HCT116 wt cells underwent apoptosis shortly after treatment with DHA. Autophagy-associated cell death contributes to short-term cytotoxicity of DHA in normoxia. These cells switched to an apoptosis- and autophagy-independent cell death after treatment with DHA in hypoxia and displayed similar long-term survival in response to DHA in normoxia and hypoxia. In HCT116 Bax-/-Baksh cells, DHA induced cell cycle arrest shortly after treatment irrespective of oxygen levels. Later, HCT116 Bax-/-Baksh cells induced a delayed cell death after treatment with DHA in hypoxia followed by return to normoxia, while treatment with DHA in normoxia was hardly toxic. We identified lower glutathione levels in hypoxic HCT116 cells which correlated with higher lipid peroxidation after treatment with DHA. Moreover, insufficient expression of Bax/Bak counteracted hypoxia-mediated downregulation of mitochondrial function, thereby adding to DHA-induced ROS production and lipid peroxidation in hypoxia. In summary, DHA-mediated cytotoxicity in normoxia depended on Bax/Bak expression, while cytotoxicity after treatment with DHA in hypoxia was regulated independently of Bax/Bak in HCT116 colorectal cancer cells.

Activation of anti-oxidant Keap1/Nrf2 pathway modulates efficacy of dihydroartemisinin-based monotherapy and combinatory therapy with ionizing radiation.

The efficacy of radiotherapy depends not only on DNA damage but also on ROS production, both induced by ionizing radiation. Massive ROS production can induce cell death or activate protective pathways such as Keap1/Nrf2 pathway, which regulates intracellular cysteine availability through upregulation of SLC7A11, a subunit of xCT transporter, and subsequently glutathione synthesis, thus improving antioxidative defense. The anti-malaria drug dihydroartemisinin (DHA) shows anti-neoplastic potential. Previous publications suggested that DHA increased ROS production. We intended to enhance oxidative stress with DHA to improve the efficacy of radiotherapy. Therefore, we first analyzed the oxidative response to DHA in HCT116 colorectal and NCI-H460 lung adenocarcinoma cells. In response to DHA, we detected lipid peroxidation and protein oxidation, which resulted in mitochondrial damage and eventually in iron-dependent cell death. Concurrently, DHA activated Keap1/Nrf2 pathway in HCT116 cells, leading to increased SLC7A11 expression and glutathione level. In Keap1-mutant NCI-H460 cells, Nrf2 was constantly activated and responsible for high SLC7A11 and glutathione levels. Pancancer analysis revealed that lung cancer is the tumor entity with the most frequent Keap1 alterations. Although NCI-H460 cells reacted more refractory to DHA-induced cell death than HCT116 cells, eradication of clonogenic cells by DHA was more efficient in both cell lines when Keap1/Nrf2 pathway was inhibited. When applied simultaneously, radiotherapy and DHA more efficiently eradicated clonogenic cells than either therapy alone, but treatment schedule can mitigate the combinatory effect in HCT116 cells. In summary, DHA improved efficacy of radiotherapy, but treatment schedule must be considered with care especially in Keap1-wildtype cells.