Hemin Enhances Radiosensitivity Of Lung Cancer Cells Through Ferroptosis

Since its discovery more than a century ago,radiation has been a critical component of clinic cancer treatment.The treatment is based on the concept that malignant tissues are more susceptible to radiation than normal tissues.To destroy cancer cells and protect non-cancerous cells from irradiation has been regarded as a critical challenge to radiologists.As an important result of irradiation and mediator of radiotherapy,the production of reactive oxygen species(ROS)result in oxidative stress in cells,causing damage of biological molecules,initiating various cellular signal pathways and even cell death.To some sense,the therapeutic efficacy of radiation treatment depends on the amount of ROS generation.Resistance of cancer cells to irradiation is a major problem in the curative treatment of cancer patients,as it results from high-capacity DNA repair,efficient antioxidants,avoidance of apoptosis mechanisms and a high chance of normal tissue complications,etc.Recently,it was discovered that radiation can damage the lipid bilayer of cell membranes via the interaction of ROS and polyunsaturated fatty acids(PUFA).Recent research has proved ferroptosis as a novel mechanism of radiation-induced cell death.Due to the fact that some kinds of cancer are characterized by resistance to apoptosis,ROS and iron-overload have been postulated as prospective anti-cancer therapeutic approaches.In other words,ferroptosis may potentially open up new treatment avenues for drug-resistant cancers.Hemin is a porphyrin that contains ferric chloride and is capable of forming heme groups.The enzyme heme oxygenase(HO)metabolizes Hemin,generating biliverdin,carbon monoxide,and ferrous iron as byproducts of the breakdown process.It is believed to be a pro-oxidant molecule because it releases iron,which results in the creation of oxidative radicals and so contributes to oxidative damage and,possibly,ferroptosis.The main objective of this study is to investigate the possible role of Hemin as a radiosensitizer overloading iron,possibly initiating the Fenton reaction to amplify ROS produced by irradiation,resulting in an increase of lipid peroxidation and eventually leading to ferroptosis death in lung cancer cells.Additionally,the possible mechanism of Hemin radiosensitizing malignant lung cells,as well as that of Hemin acting as an antioxidant in normal lung cells,will be discussed.The enzymatic reaction of HO-1 was increased in this study as a result of Hemin administration.Thus,Hemin may be a source of intracellular iron required for ferroptosis.As a result,we discovered that combining Hemin with fractionated irradiation increased the radiosensitivity of lung cancer cells via amplifying ROS generation up to 70 and 8 folds in A549 and H460 cells,respectively.This increase in ROS caused excessive lipid peroxidation in lung cancer cells following Hemin treatments.We suggested that increased ROS and lipid peroxidation might be due to Fenton and Haber-Weiss reactions.Thus,manipulating intracellular ROS and iron overload via Hemin degradation has the potential to boost radiosensitivity,making it a possibly useful approach and unique therapeutic strategy in lung cancer therapy.Additionally,as GPx4 is a critical antioxidant that plays a role in preventing cell death induced by ferroptosis,we found that the presence of Hemin in lung cells boosted GPx4 ubiquitination activity and resulted in considerable degradation after 24 to 48 h in both cancer and normal lung cells.Furthermore,GPx4 silencing or inhibition with RSL3 confirmed the role of GPx4 in radiosensitivity and suggested that it might be involved in lung cell death.Moreover,we found that lung cancer cells failed to store iron in stable FTH1.Therefore,we speculate that Hemin may contribute free iron as an additional factor in radiation-induced cell death.By contrast,Hemin was discovered to protect normal lung cells from radiationinduced cell death via inhibiting the production of ROS and lipid peroxidation after fractionated irradiation.We hypothesized that this protection in normal lung cells(Beas-2B)could be a result of increased kinetic synthesis of Bilirubin,which was previously identified as a lipophilic ROS antioxidant.Additionally,we demonstrated a significant increase in FTH1 expression(10-15 folds)in normal lung cells compared to lung cancer cells,indicating that iron does not contribute to IR-induced cell death in normal lung cells.It has been demonstrated clearly that FTH1 is necessary not only for acting as an iron buffer and storing excess iron,but also for reducing pro-oxidant activity.Our findings established that Hemin acted in a dual approach,increasing the radiosensitivity of ferroptosis in lung cancer cells while also protecting normal lung cells from irradiation.We believe that Hemin’s ability to discriminate between cancer and normal lung cells as radiosensitive sensitizers may provide a good opportunity to expand the therapeutic window of radiosensitivity.