The Effect And Mechanism Of Artesunate On Inducing HCC Cell Death By Mobilizing Intracellular Labile Iron Accumulation In Endoplasmic Reticulum

BackgroundLiver cancer is a malignancy with high frequent lethality and morbidity,among 85%-90%is hepatocellular carcinoma(HCC),the incidence of which is steadily increasing in China.The common causes of HCC are virus infection,aflatoxin,and drug or alcohol abuse.In addition,the heavy metal intake including cadmium(Cd),mercury(Hg),lead(Pb),arsenic(As),zinc(Zn)and copper(Cu)can also induce HCC.Currently,the present situation of HCC clinical treatment still needs to be improved.With the rapid development of HCC treatments,great progress has been made in transcatheter arterial chemoembolization(TACE),nano-drugs,targeted therapy and immuno-therapy.However,Post-chemoembolization Syndrome,metabolic toxicity of nano-drugs,inflammatory response and drug resistance are major limiting factors.But these researches based on targeting biological characteristics of tumour cells are promising candidate treatments for HCC.Iron is important for proliferation,invasion,and metastasis of tumor cells,and high level of iron is one of the major biological characteristics of tumor cells.More importantly,recent studies showed that chelating agents of iron and inducers of ferroptosis induced growth inhibition and cell death(ferroptosis)of tumor cells,indicating that intervention of iron metabolism could be an effective strategy for anti-cancer.However,the lack of targeting and potential clinical toxicity limits the further application of the chelating agents of iron and inducers of ferroptosis.Thus,searching for new safe agents which effectively and controllably regulate iron metabolism is meaningful.Regulatory mechanisms of iron are complex but ordered with a dynamic balance,and the disruption of the balance could impact the normal physiological processes of tumour cells such as proliferation and cell death.LIP,indicated a pool of chelatable and redox-active iron,most of which are Fe²⁺and a few are Fe³⁺,is mainly derived from extracellular intake and ferritin(a storage protein of iron)Interestingly,although LIP only accounts only for 2%-5%of the total iron pool,a slight increase LIP could induce a high level of ROS which could cause severe damage the cell membrane structure if the antioxidants such as GSSH are exhausted.As we know,LIP is also regulated in a dynamic balance to meet the iron needs of the cell,which is mainly controlled by extracellular transportation,synthesis and degradation of ferritin,and dynamic distribution in organelles.Thus,LIP might play an important in iron metabolism,and targeting and regulating LIP could be a potential therapeutic target for HCC.Artemisinin and its derivatives are the most effective treatments for malaria,and their safety has been extensively verified.Recently,artemisinin and its derivatives have been reported to have anti-cancer effect.Interestingly,iron metabolism was reported to involve in the anti-cancer effect of artemisinin and its derivatives.It is reported that artemisinin promotes Fenton reaction by a inducing high level of iron ions,then causes lethal ROS,resulting in cell death.Although artemisinin has been reported to regulate iron metabolism,the detailed mechanisms are still unknown.Whether artemisinin promotes the intake of extracellular iron,the degradation of the ferritin,or the distribution in organelles is worth studying.In addition,whether LIP is related to the anti-cancer effect of artemisinin is also unclear.Thus,based on these issues,we aimed to study the anti-cancer effect of Artesunate(ART)and how the iron metabolism involves in mechanisms of ART in HCC cells,and then to identify the potential of ART in HCC treatment.Methods and Results1.Labile iron involves in the anti-cancer effect of ART in HCC cells1.1 Anti-cancer effect of ART in different HCC cellsFour HCC cell lines with different status of P53 including Hep3B(null,p53-/-),SMMC7721(low,p53WT),Hep G2(middle,p53WT)and huh7(high,p53MT)were selected.Cell viability(CCK-8)and colony formation assay were used to study the cell growth inhibitory effects of ART in HCC cells.Our results showed that ART inhibited the cell growth of four HCC cells at a dose and time-dependent manner,and SMMC7721 is the most sensitive cell line to ART in four HCC cell lines.In addition,FCM assay showed that ART induced cell cycle arrest and cell death of HCC cells.Interestingly,p53 seemed to have no effect on the reaction of HCC cells to ART.1.2 ART regulated iron metabolism in HCC cells.Then we detected the iron metabolism-related proteins through Western Blot.Our data showed that ART significantly increased the TFRC1,IRP1/2,DMT1 and ZIP14,which are contributed to increasing the intracellular iron level.Finally,tumor transplantation showed that ART inhibited the tumor growth in vivo and no obvious side effects were observed in the test period.1.3 ART increased LIP level in HCC cellsDeferoxamine Mesylate(DFO)was used to test the relationship of LIP and anti-cancer effect of ART,since DFO chelates the LIP.Our data showed that 4-hour pre-treatment of DFO rescued the induction of apoptosis of ART in HCC cells.In addition,ART increased the LIP level in HCC cells since the consistent increased signalling of Calcein AM(C-AM),an indicator of LIP,was observed in HCC cells after ART treatment,while DFO reversed the phenomenon.Then we detected the ferritin by WB.Our results showed that ART promoted the degradation of ferritin,and pre-treatment of DFO further promoted the degradation of ferritin and increased the signalling of C-AM,indicating ART might increase LIP by promoting the degradation of ferritin in HCC cells.Total iron and total ferrous were detected by ELISA assay,our data showed that ART had no impact on the total iron level or the total Fe²⁺level in HCC cells,and DFO chelated the labile iron.These results indicated that the labile iron pool is the key sentinel in anti-cancer effect of ART in HCC cells.2.The distribution of labile iron in HCC cells2.1 Lysosome involve in the increased labile iron level in HCC cells after ART treatmentCCK-8 showed,baf A1,a lysosomal inhibitor,reversed the growth inhibitory effect of ART in HCC cells.The fluorescence confocal C-AM living cells showed that baf A1 also inhibited the increased labile iron after ART treatment.These results indicated that lysosome plays an important role in ART increasing labile iron of HCC cells.2.2 ART promoted the degradation of ferritin through autophagy-independent pathwayWestern Blot showed that ART promoted the degradation of ferritin in a dose-dependent manner.Results of Western Blot showed that although baf A1 rescued the ART-induced degradation of ferritin,the ratio of LC3I/II indicated that ART only induced autophagy in SMMC7721 and Hep G2 cells.In addition,baf A1 inhibited the degradation of ferritin but had no effect on autophagy flux.These results showed that ART promoted the degradation of ferritin through autophagy-independent pathway.2.3 ART acidified lysosome in HCC cellsLysosome p H was detected,the results showed that ART increased lysosome acidity in a time-dependent manner,which was assessed by lysosomal p H probe and lysotracker Red.In addition,pre-treatment of baf A1 also reversed the increased lysosomal acid after ART treatment in HCC cells.The results showed that the ART decreased HCC lysosomal acidification,promoted Ferritin degradation and increased the content of LIP.2.4 ART induced the distribution of labile iron in HCC cellsMultiple organelle dyes and labile iron indicator ferro Orange and Mito-ferro Green were used to identify the distribution of the ART-induced labile iron in HCC cells through confocal microscopy.Our data showed that ART mainly accumulated the ROS level in endoplasmic reticulum and mitochondria.3.The mechanisms of ART induced lethal effect on HCC cells by regulating labile iron3.1 Lethal ROS induced by ART was related to LIPThe results of confocal showed that ART significantly increased lipid peroxidation(LPO),DFO reversed this effect.CCK-8 and apoptosis assays showed that lipid oxidation inhibitors(Ferrostatin-1 and Liproxstatin-1)failed to effectively inhibit ART growth inhibition and death in HCC cells,only DFO significantly reversed ART induced growth inhibition and death.Western blot showed that ART increased GPX4 and SLC7A11,of which are key proteins against ferroptosis.CCK-8,apoptosis assays and Western blot showed apoptosis-related proteins PARP and Caspase-3 increased in varying degrees in different HCC cells.However,the Caspase kinase inhibitor Z-VAD-FMK only slightly reversed effect of ART on SMMC7721,which was not consistent with the change of apoptosis protein.The results suggest that ART induced HCC cell death does not have typical ferroptosis and Caspase dependent apoptosis characteristics.FCM results showed that ART significantly increased the ROS level in HCC cells including total ROS and mitochondrial ROS,but pre-treatment of DFO reversed the phenomenon,indicating LIP is necessary for ATR to induce the increased ROS level.FCM showed that NAC,a ROS scavenger,inhibited the induction of apoptosis of ART in HCC cells,but the Mito Q,a mitochondrial ROS scavenger,had no effect on the induction of apoptosis of ART.3.2 The lethal ROS was distributed on ER in HCC cells.Interestingly,ART decreased the signalling of TMRM in Hep3B?SMMC7721 and Huh7cells,while increased the signal of TMRM in Hep G2,but pre-treatment of DFO increased the signalling of TMRM in four HCC cells.Then the mitochondrial structure was observed through confocal microscopy,our data showed that although ART changed the mitochondrial morphology and decreased the numbers of mitochondria,but did not activate the mitochondrial apoptosis pathway since no cleaved caspase-9 was observed after ART treatment.These results showed that the lethal ROS induced by ART is not derived from mitochondria.Our results showed that ART significantly increased labile iron mainly distribute in the ER,indicating that ART induced lethal ROS by increased the distribution of labile iron in ER.Multiple organelle dyes and ROS indicator were used to identify the distribution of the ART-induced ROS in HCC cells through confocal microscopy.Our data showed that ART mainly increased the ROS level in the ER,and pre-treatment of DFO decreased the ROS level in ER.3.3 ART induced continuous ER stress in HCC cellsOur results showed that ART increased lipid peroxidation in ER via colocalization of lipid peroxidation probe and ER-tracker.In addition,ART induced obvious ER stress since the related proteins increased in HCC cells after ART treatment through Western blot.However,4-PBA,a classical ER stress inhibitor,could not reverse the inhibitory effect of ART on HCC cells.Disrupted ER was observed and the signalling of ER-tracker decreased in HCC cells after 24-hour treatment of ART,while pre-treatment of DFO reversed the disruption.These results showed that disruption of ER could be the cause of cell death induced by ART.Conclusions1.Anti-cancer effect of ART is p53-independent and ART has low cytotoxicity in normal tissues.2.ART increases LIP level by promoting the degradation of ferritin via increasing lysosomal acidity.3.ART induced lethal ROS in ER by redistributing labile iron on the ER,the accumulation of lipid oxidation destroys the integrity of the ER membrane.4.ART-induced cell death is related to LIP but significantly different from ferroptosis or mitochondrial apoptosis,and the lipid peroxidation induced oxidative damage is labile iron-dependent.Our study indicated that ART can effectively inhibit HCC cells but has low tissue toxicity,and targets iron metabolism by promoting redistribution of labile iron in ER and lipid peroxidation,resulting in structural destruction of ER and cell death.These findings raised our awareness of ART and provide an important basis for ART in HCC therapy.