Molecular Mechanism Of β-lapachone Induced Ferroptosis In Colorectal Cancer Cells

Background: Colorectal cancer(CRC)is one of the most common malignant tumors in the digestive system.In recent years,the incidence of CRC has increased due to the improvement of people’s living standards and changes in their eating habits.The treatment of colorectal cancer mainly includes surgical resection,chemotherapy,radiotherapy,immunotherapy and so on.Among them,chemotherapy is one of the most common adjuvant treatments.However,the clinical efficacy of chemotherapeutic agents remains limited in terms of side effects and resistance to oncologic agents.Therefore,it is urgent to find safe and effective anticancer drugs with less toxic and side effects.Ferroptosis is a new form of programmed cell death,Different from apoptosis,autophagic death and necrosis.Ferroptosis mainly occurs under the action of intracellular ferrous ion or ester oxygenase to catalyze unsaturated fatty acids on the cell membrane,resulting in lipid peroxidation and ultimately cell death.Studies have shown that ferroptosis is an effective strategy for tumor treatment.Ferroptosis can prevent tumor cells from developing acquired drug resistance to multiple drugs.Some drug-resistant tumor cells show more sensitivity to ferroptosis.In addition,metabolic reprogramming of tumor cells makes tumor cells more sensitive to ferroptosis.Therefore,ferroptosis is considered as a promising therapeutic target for cancer therapy.β-Lapachone is a natural naphthoquinone compound,which was first isolated and extracted from the lapacho tree(Tabebuia avellanedae).β-Lapachone has anti-fungal,anti-viral and anti-inflammatory activities.In addition,β-Lapachone shows a satisfied anti-tumor effect on various type of cancers.Studies have shown that β-Lapachone induces ROS accumulation and ultimately promotes tumor cells death by inducing DNA damage.In previous studies,β-Lapachone was predicted to induce ferroptosis in CRC cells using RNA-seq.However,the relationship betweenβ-Lapachone and ferroptosis has not been reported.Therefore,this study aims to investigate the role of β-Lapachone in the proliferation,apoptosis and ferroptosis of CRC cells,and explore the molecular mechanism underlying β-Lapachone induced ferroptosis.Objective: To investigate the role of β-Lapachone in the proliferation,apoptosis and ferroptosis of CRC cells.To explore the molecular mechanism underlyingβ-Lapachone induced ferroptosis in CRC cells.Materials and methods: CCK-8 assay was used to detect cell viability of normal human colorectal epithelial cells(HCo Epi C)and colorectal cancer cells(SW620,SW480,DLD-1)after different concentration of β-Lapachone(0,1,2,4,6,8 μmol/L)treatment.The effects of β-Lapachone on the proliferation of colorectal cancer cells were detected by Colony formation assay and Ed U assay.Flow cytometry and western blotting were used to detect the effects of β-Lapachone on apoptosis of colorectal cancer cells.RNA-seq was used to screen the differentially expressed genes(DEGs)afterβ-Lapachone treatment,and KEGG enrichment analysis was performed to predict the biological process of β-Lapachone.The effect of β-Lapachone on ROS production was detected to DCFH-DA staining.CCK-8 assay was used to determine whether ferrostatin-1 partially restored the inhibitory effect of β-Lapachone on the cell viability of CRC cells.MDA detection kit was used to determine the levels of lipid peroxidation products after β-Lapachone treatment.GSH detection kit was used to detect the glutathione content after β-Lapachone treatment in CRC cells.The expression of SLC7A11 and GPX4 were detected after β-Lapachone treatment by western blotting.The expression of SLC7A11 and GPX4 was detected in CRC cells after β-Lapachone treatment in the presence or absence of ROS inhibitor,NAC.The expression of differentially expressed genes was verified by real-time PCR and western blotting.The regulation of β-Lapachone on intracellular ferrous iron was determined by iron assay kit.The expression of iron metabolism-related and ferritinophagy-related proteins were detected by immunofluorescence(IF)and western blotting.The expression of autophagy-related protein in CRC cells after β-Lapachone treatment was detected via western blotting and IF assay.The expression of ferritinophagy-related protein,levels of intra-cellular iron and lipid peroxidation were detected in CRC cell after β-Lapachone treatment in the presence or absence of autophagy inhibitor,3-MA.Results: 1.β-Lapachone inhibited proliferation of colorectal cancer cells:CCK-8 assay showed that β-Lapachone inhibited the cell viability of normal human colorectal epithelial cells and three types of colorectal cancer cells in a concentration-dependent manner(p<0.05).The results of Colony formation and Ed U assay showed that β-Lapachone significantly inhibited the proliferation of colorectal cancer cells compared with the control group(p<0.01).2.β-Lapachone induced apoptosis of colorectal cancer cells: Compared with the control group,the apoptotic rates of colorectal cancer cells with β-Lapachone treatment were significantly increased(p<0.01).The results of western blotting showed that Cleaved-caspase3/Pro-caspase3 and Bax/Bcl-2 ratios were significantly increased after β-Lapachone treatment in colorectal cancer cells(p<0.01).3.β-Lapachone promoted intracellular ROS production and induced ferroptosis in colorectal cancer cells: RNA-seq and KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in pathways,including P53,ferroptosis,colorectal cancer,etc.DCFH-DA staining showed that β-Lapachone promoted ROS production in colorectal cancer cells compared with the control group(p<0.01).CCK-8 results showed that ferrostatin-1,an ferroptosis inhibitor,partially reversed the inhibitory effect of β-Lapachone on colorectal cancer cell viability(p<0.01).The results of MDA assay showed that β-Lapachone significantly increased the production of malondialdehyde(MDA),a lipid peroxidation products,in colorectal cancer cells(p<0.01).The results of intracellular glutathione detection showed that the GSH/GSSG ratio was significantly decreased after β-Lapachone treatment(p<0.05).The results of western blotting showed that β-Lapachone significantly down-regulated the expression levels of SLC7A11 and GPX4 in colorectal cancer cells(p<0.01).Moreover,pre-treatment of ROS inhibitors,NAC partially reversed this results(p<0.01).4.β-Lapachone induced ferroptosis in colorectal cancer cells by promoting intracellular iron accumulation: The differentially expressed genes enriched in ferroptosis,which were obtained from RNA-seq,were verified by real-time PCR.The results showed that the m RNA expression of TFRC and LC3 B were up-regulated,while the m RNA expressions of SLC3A2,HMOX1 and NCOA4 were down-regulated(p<0.01),The expression of FTH1 has no significantly.The results of western blotting showed that the expression of TFRC and NCOA4 were up-regulated after β-Lapachone treatment(p<0.05),while the expression of SLC3A2,HMOX1 and FTH1 were down-regulated(p<0.05).The results of Immunofluorescence assay showed that β-Lapachone increased the expression of NCOA4 and decreased the expression of FTH1 in colorectal cancer cells.Iron assay kit showed that β-Lapachone promoted the production of ferrous ion in colorectal cancer cells(p<0.01).5.β-Lapachone induced ferroptosis in colorectal cancer cells by promoting ferritinophagy: The results of western blotting showed that treatment with β-Lapachone up-regulated the ATG7 and LC3 B expression in colorectal cancer cells(p<0.05),while the expression of P62 was down-regulated(p<0.05).Immunofluorescence assay showed that β-Lapachone increased the expression of LC3 B in colorectal cancer cells.Pre-treatment with autophagy inhibitor,3-MA could partially reverse the effect of β-Lapachone on regulating the expression of NCOA4 and FTH1(p<0.05).In addition,pre-treatment with 3-MA also reversed the effect of β-Lapachone on regulating intra-cellular ferrous iron and MDA levels.Conclusions: 1.β-Lapachone inhibits the proliferation,and induces apoptosis and ferroptosis in colorectal cancer cells.2.β-Lapachone induces the intracellular ferrous ion accumulation through mediating ferritinophagy and increases intracellular ferrous iron levels,leads to lipid peroxidation,and eventually induces ferroptosis in colorectal cancer cells.