| OBJECTIVE:Hepatocellular carcinoma(HCC),which represents 90% of primary liver malignancy,is a deadly cancer with high mortality.There are two first-line systemic medications available,namely sorafenib and lenvatinib,for advanced HCC.However,studies have shown that sorafenib treatment could only benefit HCC patients with extended survival of 2-3 months.Many HCC patients developed acquired resistance to sorafenib,severely weakening the anticancer effect of subsequent sorafenib treatment.Therefore,it is of great clinical significance to increase sorafenib sensitivity by combining with clinically well-validated drugs,in order to overcome the cancer resistance to sorafenib and to improve HCC treatment efficacy.Artesunate,as a magic anti-malaria drug,was found to be effective in killing multiple cancer cells and capable of synergizing with many medications in cancer treatment.The aim of this study is to investigate whether artesunate could synergize with sorafenib in inducing HCC cell death and unravel the involved molecular mechanisms.METHODS: Firstly,sorafenib and artesunate were used separately or in combination in HCC cells to clarify the death forms of HCC cells.Secondly,the molecular mechanisms of the combined treatment against HCC were elucidated by detecting the changes of mitochondrial and lysosomal functions,and of ferroptosis pathways.Lastly,by using a nude mice HCC xenograft experimental model,the therapeutic effect of combined drugs against HCC tumor in vivo was verified.The inhibition of tumor growth and the changes of ferroptosis related pathway were examined after combined treatment.1.Cell growth and death study: MTT,flow cytometry(sub G1 and PI exclusion assay)and colony formation assay were used to detect cell viability,cell death and colony forming capacity respectively.2.Ferroptosis study: By pretreatment with relevant ferroptosis inhibitors(including lipid peroxidation inhibitors and iron chelators)and other cell death inhibitors,the main form of cell death involved in the combined treatment of sorafenib and artesunate was determined using PI exclusion assay.The lipid peroxidation level was determined by Bodipy 581/591 C11;and the production of lipid peroxidation metabolite MDA was determined by the corresponding detection kit.3.Mitochondrial function study: After indicated cell treatment,the levels of general reactive oxygen species(ROS)and mitochondrial derived ROS were measured by DCF or Mito SOX red respectively.Changes of mitochondrial membrane potential were detected by TMRM.The cellular ATP levels were determined by biochemical assays.Then mitochondrial respiratory oxygen consumption rate(OCR)was measured by Seahorse metabolic assay.4.Lysosomal function study: Western blotting analysis was conducted to examine the effects of combined drug treatment on ferritin expression with/without bafilomycin A1(lysosomal inhibitor)pretreatment;lyso Tracker dye was utilized to determine the changes of lysosomal acidification;cathepsin L and cathepsin B Magic Red assay to quantify lysosomal enzyme activities;and flow cytometry to access cell survival.5.In vivo nude mice xenograft tumor study: HCC xenograft in nude mice were established and treated with various drug therapy as indicated,in order to observe the changes of tumor growth.In the end of the experiment,the xenograft nodules were collected.Then H&E staining of the tumor section was used to show the histopathological changes and necrosis in tissues;the immunohistochemical detection of ki-67 changes to evaluate the proliferation status of tumor cells;and MDA assay to determine the production of lipid peroxidation.RESULTS: 1.The cell-killing effects of either artesunate alone or sorafenib alone on HCC cells were not obvious,but artesunate synergized with sorafenib to kill HCC cells significantly.The apoptosis inhibitor z-VAD could not effectively protect the cell death caused by the combined treatment,but the oxidative stress inhibitor NAC showed significant protection,suggesting that the cell death caused by combined treatment was dependent on oxidative stress.Colony growth experiments further demonstrated that the combined drug treatment had a significant inhibitory effect on cell colony proliferation.2.After cell pretreatment with lipid peroxidation inhibitors(Liprox)or iron chelators(DFO),the combined drug treatment-induced cell death was abolished significantly,suggesting that the form of cell death was ferroptosis.The degree of lipid peroxidation is more pronounced after the combined treatment.The assay results revealed that lipid peroxidation product MDA was increased but scavenger GSH was decreased,further supporting the occurrence of ferroptosis.3.Further studies of the source of lipid metabolism using DCF and Mito Sox found that both intracellular ROS and mitochondria-derived ROS production were increased significantly after combined treatments or sorafenib alone,indicating that sorafenib may be important for the production of mitochondria-involved oxidative stress.The mitochondrial membrane potential was also decreased.More importantly,the ability of mitochondria to produce ATP was significantly inhibited,but it can be reversed by NAC.Seahorse metabolic assay revealed a corresponding decrease in ATP-associated respiratory OCR,while an increase in proton pump leakage associated respiratory OCR.4.Lastly,the origin of free iron was also investigated,and it was found that ferritin light chain FTL and heavy chain FTH were significantly down-regulated after the combined treatment;and the pretreatment of lysosomal inhibitor(bafilomycin A1)could reverse the above changes.More importantly,bafilomycin A1 attenuated cell death by the combined medication,suggesting that lysosome may play a key role in cell death.Detection of lysosomal activity by Lyso Tracker further found that lysosomal activity increased after the combined treatment.Note that this action also occurred after treatment with artesunate alone,although at a lesser extent.Consistently,the detection of cathepsin B/L activities by Magic Red kits also showed enhanced lysosomal enzyme activities after the combined treatment.5.In nude mice xenograft experiment,there was no significant difference in mice body weight after the combined treatment,but the growth of xenograft tumor reduced significantly after the combined treatment.The H&E staining of the xenograft tissues revealed larger area of cell death in the combined treatment group.The immunohistochemical staining of ki-67 results showed that tumor cell proliferation status was also significantly inhibited in the combined treatment group.Importantly,the combined drug treatment also significantly down-regulated ferroptosis-related FTL protein and increased the content of lipid peroxidation metabolite MDA in vivo.CONCLUSIONS:1.The combination of sorafenib with artesunate had a synergistic effect on killing HCC cells.Ferroptosis,rather than apoptosis,was the major form of the induced cell death.2.Mechanically,sorafenib acted mainly through inhibition of mitochondrial functions and GSH synthesis,whereas artesunate primarily enhanced lysosomal function and the degradation of Ferritin.The combined treatment synergistically led to lipid peroxidation,free iron release and ferroptosis.3.The in vivo nude mice xenograft experiment further demonstrated that the combination of sorafenib with artesunate inhibited xenograft tumor growth and promoted ferroptotic cell death in xenograft cancer tissues. |
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