Stearoyl-Coa Desaturase 1 Potentiates Hypoxic Plus Nutrient-deprived Pancreatic Cancer Cells Ferroptosis Resistance

Objective:Hypoxia and nutrient starvation(H/NS)microenvironment,a notable characteristic of pancreatic carcinoma,plays a critical role in cell death resistance and tumor recurrence.Inhibition of stearoyl-Co A desaturase 1(SCD1)expression can inhibit tumor proliferation,invasion,metastasis and induce cell death.High expression of SCD1 is associated with poor prognosis of tumors.Recent studies have revealed the role of SCD1 in regulating tumor cell ferroptosis and inducing pancreatic cancer cells to a ferroptosis-resistant state.Taken together,our findings reveal a new role of H/NS microenvironment against ferroptosis by activation of SCD1 and the molecular mechanism of its regulation of ferroptosis,and suggest a potential therapeutic strategy for overcoming ferroptosis resistance in pancreatic cancer cells.Methods:(1)CCK-8 assay was used to screen the ferroptosis of pancreatic cancer cells induced by Erastin,RSL3,and sulfasalazine.Microscope observation of the number and morphology of pancreatic cancer cells after treatment.CCK8 kit detected the viability of PANC1 and Patu8988 cells after 24 h of ferroptosis inducers(Erastin,RSL3,and sulfasalazine treatment under H/NS condition.MDA Assay Kit detected the MDA level of PANC1 and Patu8988 cells.(2)Western blot and q RT-PCR were used to detect the efficiency of interference with GPX4.Cell viability was measured in Ctrl sh RNA and GPX4 sh RNA PANC1 cells cultured in normal or H/NS condition for 24 h.(3)The expression of SCD1 and its clinical significance in pancreatic cancer was analyzed by using bioinformatics methods.Real-time PCR and western blot was used to detect the expression of SCD1 in different pancreatic cancer cells(PANC1,Pa Tu8988,BXPC3,SW1990)under H/NS condition.(4)The Human MNSFA ELISA KIT was used to detect the content of MUFAs in human pancreatic cancer cell lines PANC1 and Patu8988 under Nor and H/NS condition.(5)TCGA database analyzed the correlation between SCD1 and ferroptosis-related genes in pancreatic cancer.The efficiency of interference with SCD1 in human pancreatic cancer cell lines PANC1 and Patu8988 was detected by fluorescence microscope,western blot and q RT-PCR.SYTOX Green staining and CCK-8 assay were used to access the effect of inhibition of SCD1 expression on ferroptosis of PANC1 and Patu8988 cells under H/NS condition.The Human MNSFA ELISA KIT was used to access the effect of inhibition of SCD1 expression on MUFAs in human pancreatic cancer cell lines PANC1 and Patu8988.(6)MDA Assay Kit was used to detect the level of MDA in PANC1 and Patu8988 cells that have been knocked down by SCD1 sh RNA after erastin treatment under H/NS condition.PI staining and CCK-8 assay kit was used to access the cell viability of pancreatic cancer cells in the combination group of SCD1 inhibitor A939572 and erastin.BODIPYC11581/591 staining detects the lipid peroxidation degree of PANC1 cells in different treatment groups: DMSO group;Erastin group;A939572 group;Erastin+A939572 group and takes pictures.(7)The Panc02 subcutaneous tumor model were treated with the following agents: DMSO(control),Erastin,A939572,Erastin+A939572.The tumor volume of each group was measured.Lysis the subcutaneous tumor tissue,MDA Kit detects the MDA level of the tumor tissue of the mice in each group.Results:(1)The results of CCK-8 assay showed that Erastin,RSL3,and sulfasalazine can induce ferroptosis in pancreatic cancer cells(PANC1 and Patu8988).Pancreatic cancer cells resist ferroptosis induced by ferroptosis inducers under H/NS condition and the level of MDA was decreased.(2)GPX4 protein levels were significantly down-regulated in PANC1 cells transfected with Ctrl sh RNA and GPX4 sh RNA,Cell viability was measured in Ctrl sh RNA and GPX4 sh RNA PANC1 cells cultured in normal or H/NS condition for 24 h.(3)The analysis of TCGA database showed that the expression of SCD1 in PAAD tissue was significantly higher than that in normal pancreatic tissue.Compared with patients with low SCD1 expression,the survival time and disease-free survival time of PAAD patients with high SCD1 expression was significantly shorter.The results of q RT-PCR and western blot showed that SCD1 levels were significantly increased in PDAC cell lines PANC1 and Patu8988 under H/NS condition,while weakly expressed in BXPC3 and SW1990 cells.(4)The Human MNSFA ELISA results showed that under H/NS condition,the content of MUFAs in PANC1 and Patu8988 cells was significantly higher than that under Nor condition.(5)Fluorescence microscopy,western blot and q RT-PCR showed that the interference plasmid of SCD1 sh RNA was successfully transferred into PANC1 and Patu8988 with high interference efficiency.SYTOX Green staining and CCK-8 results indicated that inhibition of SCD1 expression significantly enhanced erastin-induced ferroptosis of PANC1 and Patu8988 cells under H/NS condition and the levels of MUFAs were decreased in PANC1 and Patu8988 cells.(6)The levels of MDA in PANC1 and Patu8988 cells with SCD1 knockdown after erastin treatment increased significantly under H/NS condition.The results of PI staining and CCK-8 showed that the activity of pancreatic cancer cells in the combination group of SCD1 inhibitor A939572 and erastin was significantly reduced.The results of BODIPYC11581/591 staining showed that under H/NS conditions,PANC1 cells treated with erastin and A939572 had an increase in lipid peroxidation and lipid ROS levels.(7)The tumor volume and size were reduced in erastin group,A939572 group and erastin +A939572 group.Compared with single-drug group,tumors of erastin+A939572 group were significantly reduced in Panc02 subcutaneous tumors.After the tumor tissues in each group were fully lysed,the MDA results showed that the combined treatment of A939572 and erastin significantly increased the MDA level in pancreatic cancer tumor tissues.Conclusion:(1)Hypoxic and nutrient-deprived condition protects pancreatic cancer cells from ferroptosis.(2)SCD1 expression is positively related to pancreatic cancer H/NS condition and progression.The analysis of TCGA database showed that the expression of SCD1 in PAAD tissue was significantly higher than that in normal pancreatic tissue.Compared with patients with low SCD1 expression,the survival time and disease-free survival time of PAAD patients with high SCD1 expression was significantly shorter.(3)SCD1 is a suppressor of ferroptotic pancreatic cancer cell death under H/NS condition.(4)Inhibition of SCD1 activity sensitizes pancreatic cancer ferroptosis in vitro and in vivo.