Mechanism Of Programmed Cell Death Induced By Artesunate/Dihydroartemisinin In NSCLC Cells

Objective: The study elucidated that artesunate(ART)and dihydroartemisinin(DHA)could inhibit the non-small cell lung cancer(NSCLC)cells by inducing ROS dependent apoptosis and ferroptosis.Methods: Screening for natural products with therapeutic potential for NSCLC.A natural product library,which contained 173 natural products,was purchased from Selleck(Cat # L1400).We performed the screening assay on NCI-H1299,A549,LTEP-a-2,NCI-H23,and NCI-H358 cells according to the manufacturer’s recommendations.Then,the top 13 natural products that had significant inhibitory effect on NSCLC cells were screened out and divided into five categories according to their mechanisms.The effects of ART/DHA on cell proliferation and apoptosis in NSCLC cells.A549 cells were treated with ART or DHA for subsequent experiments.Cell proliferation of A549 and NCI-H1299 cells was detected by Alamar Blue assay.Apoptosis of A549 cells was detected by flow cytometry(Annexin V / PI double staining).The protein expression of VDAC was detected by western blot.The protein expression of cleaved caspase 3 was detected by immunofluorescence.The effects of ART/DHA on ferroptosis in NSCLC cells.After NSCLC cells were treated with ART or DHA alone or combined with NAC,cell proliferation was detected by Alamar Blue assay.A549 cells were treated with ART or DHA for subsequent experiments.The protein expression of cystine/glutamate transporter(x CT)and GPX4 was detected by western blot.The m RNA level of ferroptosis related gene(TFRC,x CT and SOD1)was detected by RT-q PCR.The effect of ROS on the inhibition of NSCLC cells by ART or DHA.A549 cells were treated with ART or DHA alone or combined with NAC for subsequent experiments.Apoptosis of A549 cells was detected by flow cytometry(Annexin V / PI double staining).The m RNA expression of transferrin receptor(TFRC)was detected by q PCR.The protein expression of VDAC and GPX4 was detected by western blot.Results: Screening for natural products with therapeutic potential for NSCLC.Through a natural product library screening assay,we found that a group of anti-insect drugs had significant inhibitory effect on the proliferation of NSCLC cells.Among the anti-insect drugs,two derivatives of artemisinin,i.e.,artesunate(ART)and dihydroartemisinin(DHA),a group of well-known anti-malarial drugs,have been shown to possess selective anti-cancer properties.Artemisinin derivatives induce apoptosis and ferroptosis in NSCLC cells.Compared with the control group,both ART and DHA induced apoptosis in A549 cells as revealed by flow cytometry.Western blot analysis showed that ART and DHA respectively downregulated the protein level of VDAC.Furthermore,ART and DHA noticeably increased the protein level of cleaved caspase 3 in A549 cells.Ultrastructural observation showed that ART/DHA induced mitochondria with disrupted cristae structure in A549 cells.The protein and m RNA level of x CT were markedly decreased by ART and DHA in A549 cells.ART and DHA decreased the protein level of GPX4 in A549 cells.In addition,the m RNA level of TFRC and SOD1 were robustly upregulated by ART and DHA.We found that the inhibitory effect of ART/DHA on the proliferation of A549 cells was partly reversed by Fer-1.ROS is a key regulator of ART/DHA-induced apoptosis and ferroptosis.Treatment of A549 cells with ART or DHA stimulated the production of ROS,which was reversed partly by NAC.Consistently,the inhibitory effect of artemisinin derivatives on the proliferation A549 and NCI-H1299 cells was partly reversed by the addition of NAC.ART and DHA decreased the protein level of VDAC,which was partly reversed by NAC.The addition of NAC decreased the m RNA level of TFRC induced by ART and DHA in A549 cells.Furthermore,the addition of NAC indeed significantly reversed the apoptosis induced by ART and DHA in A549 cells.These results established that artemisinin derivatives inhibited NSCLC cells through induction of ROS-dependent apoptosis/ferroptosis.The gene expression and clinical implication of TFRC and VDAC in lung cancer.We analyzed the expression of genes and proteins regulated by ART and DHA in lung cancer patients through the UALCAN database.We found that while TFRC was poorly expressed,VDAC was highly expressed in tumor tissues compared with normal tissues.Then,we explored the relationship between these molecules and the lung cancer patients’ survival by GEPIA 2 database.Our results revealed that the expression levels of TFRC and VDAC were positively and negatively correlated with the survival of lung cancer patients,respectively.All these data suggested that TFRC and VDAC were closely associated with the survival of lung cancer patients and can be used as potential therapeutic targets in lung cancer.Conclusion: 1.Artemisinin derivatives induce apoptosis and ferroptosis in NSCLC cells.2.ROS is a key regulator of ART/DHA-induced apoptosis and ferroptosis.