Dihydroartemisinin efficiently induces cytotoxicity in normoxic and hypoxic cancer cells and improves radiotherapy

The efficacy of radiotherapy depends on DNA damage induced either directly by IR or, most of all, indirectly in response to oxidative stress via ROS production. Massive ROS stress can either induce cell death or activate protective mechanisms such as the Keap1/Nrf2 pathway, improving the antioxidative defense and limiting IR-induced lethal damage. Moreover, hypoxic areas in tumor tissues restrict the efficacy of radiotherapy. Reduced ROS production and a lowered induction of irreparable, oxidative damage in an oxygen-deficient environment is a reason for this limitation. The present study aimed to improve the efficacy of radiotherapy in normoxia and hypoxia by inducing an additional and cytotoxic ROS boost with the prooxidative anti-malaria drug DHA. Due to its anti-neoplastic effects, low toxicity and known safety, DHA has received increasing attention in cancer research.
Initially, the oxidative response was analyzed in the colorectal cancer cell line HCT116 and the lung cancer cell line NCI-H460 treated with DHA in normoxia. DHA efficiently induced iron-dependent oxidative damage to lipids, proteins and mitochondria. At the same time, DHA activated the protective Keap1/Nrf2 pathway in HCT116 cells, while, in NCI-H460 cells, Nrf2 was already constitutively activated due to a Keap1 mutation. An activated Keap1/Nrf2 pathway is related to resistance towards chemo- and radiotherapy. However, the results of the present thesis revealed that a concurrent combined treatment of IR and DHA markedly improved radiotherapy, independent of Keap1 mutations. In HCT116 cells responding to DHA with an activation of the protective Keap1/Nrf2 pathway, the treatment regimen can limit the combinatory effect when DHA is applied prior to IR. An application of the prooxidative drug after IR was more toxic than either treatment alone in the colon cancer cell line. The cytotoxic impact evoked by the combinatory therapy was even more efficient when cells were treated under hypoxic conditions. The results revealed, that hypoxia-mediated radioresistance was overcome when combining IR with DHA. Thus, the present study demonstrates that combining ROS-based therapies is an effective anti-cancer strategy.
In order to improve our understanding on the mechanisms behind DHA-induced cytotoxicity in normoxia and hypoxia, short-term cell death and long-term survival were analyzed in HCT116 wild type (wt) cells and in HCT116 Bax-/-Baksh cells with a defective intrinsic apoptosis pathway. The results revealed apoptosis and autophagy-associated cell death as the prevalent mechanisms contributing to DHA-induced cell death of HCT116 colon cancer cells treated in normoxia. Our data identified the pro-apoptotic proteins Bax and Bak as factors regulating sensitivity to DHA in normoxic conditions. In addition, in HCT116 Bax-/-Baksh cells, treatment with DHA in normoxia initially induced cell cycle arrest, which most likely allows repair of DHA-induced oxidative damage, resulting in an improved long-term survival. In hypoxia, sensitivity to DHA was independent of Bax and Bak expression. While DHA induced cell death shortly after treatment with DHA in hypoxic HCT116 wt cells, treatment with DHA in hypoxia initially induced cell cycle arrest in HCT116 Bax-/-Baksh cells, followed by a delayed ferroptosis-like cell death. Lower glutathione levels correlated with a higher lipid peroxidation in hypoxic HCT116 Bax-/-Baksh cells. Moreover, incubation in hypoxia resulted in reduced oxidative phosphorylation after returning to normoxia, providing the cells with reduced energy production. This effect was less pronounced in HCT116 Bax-/-Baksh than in HCT116 wt cells, providing HCT116 Bax-/-Baksh cells with a better energy supply via oxidative phosphorylation than HCT116 wt cells after return to normoxia.
In summary, we identified a) DHA-induced activation of Keap1/Nrf2 pathway leading to improved antioxidant defense and b) the pro-apoptotic proteins Bax and Bak as critical factors regulating sensitivity to DHA in HCT116 colon cancer cells in normoxia, while impaired Nrf2 activation and reduced glutathione levels improved the cytotoxic response to DHA in hypoxia despite less ROS production and independent of Bax and Bak expression.



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