XCT Inhibition Reverses The Cisplatin Resistance Of Lung Adenocarcinoma Through Inducing Ferroptosis

Objective: Lung cancer is one of the most common type of cancer worldwide,which is always the leading cancer-related lethality.Non-small cell lung cancer(NSCLC)almost consists of 85% of lung cancers.NSCLC is firstly treated with surgery,but it will relapse in five years.Most of the advanced NSCLC have lost the chance of operation.Multi-course chemotherapy is an important treatment for NSCLC.However,the 5-year survival rate of NSCLC patients is very low.Cisplatin(CDDP)resistance is an important reason for poor prognosis.Thus,exploring effective adjuvant therapies has become a key issue in prolonging patient survival and improving patient outcomes.CDDP is the first-line chemotherapy of NSCLC.CDDP activates DNA damage by inducing DNA damage and induces apoptosis of tumor cells.Another important mechanism of CDDP-induced apoptosis in malignant tumor cells is up-regulation of intracellular reactive oxygen species(ROS)levels,imbalance of the homeostasis,oxidative stress,oxidative/antioxidant imbalance.A variety of tumor cells represented by lung adenocarcinoma have apoptotic pathway signal transduction obstacles,anti-apoptotic proteins are activated and are affected by the high expression of multidrug resistance genes,and the resistance to apoptosis mechanism is very obvious.In the past,most of the tumor cells were aimed at signal Transduction disorders target new drug development or the combination of a variety of chemotherapy drugs to address the primary and secondary apoptosis resistance,in order to improve the chemotherapy drug-mediated tumor cell apoptosis levels.Unfortunately,due to the existence of an extremely complete complementarity compensation mechanism of apoptotic pathways that is not easily inhibited or activated by small molecule drugs for a long time,combined with unpleasant side effects caused by the combination of multiple chemotherapeutic drugs,etc.This leads to making poor progress in this area.Induction of non-apoptotic death in lung adenocarcinoma CDDP-resistant cells is an important strategy for overcoming CDDP resistance.Nuclear factor erythroid 2-related factor 2(Nrf2)is a major regulator of antioxidant system,expresses a large number of anti-oxidative genes and plays an important role in anti-oxidative defense.Nrf2 is activated by Keap1 mutation and takes part in chemotherpy resistance.The Nrf2/ARE downstream gene such as xCT has not yet been tested in lung adenocarcinoma.Ferroptosis is an iron-dependent form of regulated cell death that is distinct from apoptosis,necroptosis,and autophagic cell death.Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides(L-ROS)to lethal levels.Ferroptotic cells exhibit changed mitochondrial morphology and cristae structure.Smaller than normal mitochondria with increased mitochondrial membrane density and reduction/vanishing of mitochondria crista have been observed in ferroptosis.Inhibition of necrosis,apoptosis,and necroptosis by small molecule inhibitors(e.g.,necrostatin-1,Z-VAD-FMK,and BOC-D-FMK)cannot reverse ferroptosis.In contrast,iron chelators(e.g.,deferoxamine mesylate,DFO),ferrostatin-1(Fer-1)and antioxidants(e.g.,vitamin E)block cell death.In addition,the inhibition of cystine/glutamate antiporter(xCT),a key molecule related to ferroptosis,may induce the eradication of cancer cell resistance to conventional chemotherapy by inhibiting the import of cystine,leading to glutathione depletion.It can be induced by the small molecules such as Erastin and Sorafenib.Unfortunately,there isn't any studies about ferroptosis in lung adenocarcinoma.Taken together,we need to confirm:(1)whether the activation of Nrf2/xCT pathway causes CDDP resistance to lung adenocarcinoma cell lines.(2)whether the inhibition of xCT could enhance the sensitivity of CDDP-resistant lung adenocarcinoma to CDDP.(3)whether the inhibition of xCT could induce the ferroptosis of CDDP resistant lung adenocarcinoma cells.Methods: 1.NCI-H1299,A549,N2 and N5 cells were treated with a series of dose of CDDP(from 10 to 80 ?g/mL)for 48 h,and then cell survival rate was analyzed using CDK-8 kit.2.A549 and N5 cells were exposed to 20 ?g/mL CDDP for 3 to 12 h.And the expressions of Nrf2,xCT,HO-1 and NQO1 mRNA were evaluated by RT-qPCR and shown as normalized to GAPDH.3.A549 cell was suffered from 20 to 40 ?g/mL CDDP treatment for 12 h.Nrf2 and xCT protein expressions were detected by Western blotting analysis.Lamin B was an internal control.4.A549 and N5 cells were transfected with ARE luciferase reporter vector and Renilla luciferase vector.After 24 h of transfection,the cells were incubated in the presence of 20 ?g/mL CDDP for additional 12 h,and then subjected to a luciferase assay.Luciferase activities were normalized with Renilla luciferase activities.5.A549 cell was transfected with Nrf2 or xCT siRNA.Twenty-four hours later,the cell was treated with 20 ?g/mL CDDP for 48 h,and cell survival rate was determined.Nrf2 or xCT expression of A549 cell was inhibited by specific siRNA using the same methods as above.At 24 h posttransfection,whole cell lysate was extracted and Western blotting analysis was performed for Nrf2 and xCT expression detection.Lamin B was used as loading control.6.N5 cell was transfected with Nrf2 or xCT expression vector.And pcDNA3 vector was an internal control.After 24 h of incubation,the cell was suffered from 40 ?g/mL CDDP for 48 h,and the subjected to cell survival rate analysis.7.A549 and N5 cells were exposed to 20 ?g/mL CDDP or DMSO for 6 h.DCFDA fluorescence indicative for intracellular ROS level was analyzed by flow cytometry.8.N5 CDDP resistant(N5CP)variant cell line was established by continuous culturing with increasing concentrations of CDDP(1 to 10 ?g/mL)for six months.The culture medium of N5 CP cell was additionally contained 10 ?g/mL CDDP to maintain its drug resistance characteristic.9.N5 CP cell was treated with erastin(10 ?M),sorafenib(20 ?M),DFO(50 ?M)and Vit-E(100 ?M)in combined manner as indicated for 48 h,and then cell survival rate detection was perform.N5 CP cell was exposed to erastin(10 ?M),sorafenib(20 ?M)or DMSO for 48 h and subjected to PI staining analysis.10.Mice bearing N5 CP cell subcutaneous xenograft were randomly divided into 4 groups and intraperitoneal injected with CDDP,erastin,sorafenib or PBS(control).After 3 times injection,tumors were removed and weighted.Results: 1.NCI-H1299,A549,N2 and N5 cells were treated with a series of dose of CDDP(from 10 to 80 ?g/mL).A549 cells were the most resistant to CDDP.N5 cells were the most sensitive to CDDP.2.A549 and N5 cells were exposed to 20 ?g/mL CDDP for 3 to 12 h.And the expressions of Nrf2,xCT mRNA were higher than control,especially in A549.3.A549 cells were suffered from 20 to 40 ?g/mL CDDP treatment for 12 h.Nrf2 and xCT protein expressions were higher than control accordance with the result from PCR.4.A549 and N5 cells were transfected with ARE luciferase reporter vector,the cells were incubated in the presence of 20 ?g/mL CDDP for additional 12 h,then subjected to a luciferase assay.Luciferase activities of A549 were more than those of N5.5.A549 and N5 cells were exposed to 20 ?g/mL CDDP or DMSO for 6 h.DCFDA fluorescence indicative for intracellular ROS level was analyzed.CDDP induced the accumulation of ROS significantly in N5 cells.6.A549 cell was transfected with Nrf2 or xCT siRNA.Twenty-four hours later,the cell was treated with 20 ?g/mL CDDP for 48 h,and cell survival rate obviously decreased.7.N5 cell was transfected with Nrf2 or xCT expression vector.After 24 h of incubation,the cell was suffered from 40 ?g/mL CDDP for 48 h,and the cell survival rate increased.8.N5 CP cell was treated with erastin(10 ?M),sorafenib(20 ?M)for 48 h,and then cell survival rate decreased,the same as PI staining.N5 CP cell was treated with erastin(10 ?M),sorafenib(20 ?M)DFO(50 ?M)and Vit-E(100 ?M)combined manner.The survival rate increased.9.Mice bearing N5 CP cell subcutaneous xenograft were ntraperitoneal injected with CDDP,erastin,sorafenib.The tumor weight of erastin/sorafenib group was lighter than CDDP group.Conclusion: 1.The activation of Nrf2/xCT pathway is more important in CDDP resistance to lung adenocarcinoma.Nrf2/xCT pathway negatively regulates CDDP toxicity.Nrf2/xCT expression level determines CDDP sensitivity.2.The xCT inhibition induce CDDP resistant lung adenocarcinoma cells ferroptosis efficiently.