Study On The Mechanism Of Gambogic Acid Regulating Oxidative Stress Induced Ferroptosis Of Ovarian Cancer Cells A2780 Based On Nrf2/HO-1 Signaling Pathway

Ovarian cancer is one of the common malignant tumors of female genital organs.Because the mortality rate is the highest in gynecological cancer,it is the biggest disease that seriously threatens women’s lives.Iron death（Ferroptosis）is a kind of cell death caused by accumulation of reactive oxygen species（ROS）,which depends on intracellular iron.Previous studies have shown that Gambogenic acid（GNA）can play an anti-tumor role by regulating mitochondrial oxidative stress,but whether it is related to iron death has research value and significance.In this study,ovarian cancer cell line A2780 was used to study the inhibitory effect and mechanism of GNA on the proliferation of ovarian cancer cells,and to provide the corresponding biological basis for further elucidating the molecular mechanism of programmed cell death induced by GNA.Objective:To study the inhibitory effect of GNA on the proliferation of ovarian cancer cell line A2780,clarify the relationship between GNA-induced ovarian cancer cell death and Ferroptosis,clarify the role of Nrf2/HO-1 signaling pathway in GNA-induced ovarian cancer cell death,and further explain its mechanism.Method:Human ovarian cancer cell line A2780 was selected as the research object.CCK-8 and trypan blue exclusion method were used to detect the cell viability of GNA-treated cells and calculate IC50 value;LDH leakage rate of lactate dehydrogenase in A2780 cells treated with GNA was detected by colorimetry;Z-VAD-FMK（apoptosis inhibitor）,Necrostatin-1（necrosis inhibitor）,3-Methyladenine（autophagy inhibitor）,Ferrostatin-1 and iron chelator DFO were detected to observe the effect of GNA on cell proliferation.;The contents of iron and the changes of malondialdehyde（MDA）,superoxide dismutase（SOD）,glutathione（GSH）and glutathione peroxidase（GPX）were detected by colorimetry;the morphological changes of cells were observed by DAPI staining and fluorescence microscopy;Fluorescence staining of H2DCF-DA and BODIPY-C11 was used to detect the levels of cytoplasm and lipid reactive oxygen species（ROS）by flow cytometry;Western blot was used to detect the expression of Ptgs2 and TFR-related proteins in cells;Western blot was used to detect the expression of Nrf2,HO-1 and GPX4 proteins;q RT-PCR was used to detect the expression of Nrf2,HO-1 and GPX4 genes;fluorescence microscopy was used to observe the transfection efficiency after Nrf2 gene was knocked out by lentivirus;LDH leakage rate and MDA content were detected by colorimetry;Western blot was used to detect the expression of Nrf2,HO-1 and GPX4 proteins.The expression of HO-1 and GPX4 was detected by q RT-PCR.Results:1 GNA inhibits the proliferation of A2780 cells（1）CCK-8 and Trypan blue staining showed that GNA inhibited the proliferation of A2780 cells in a dose-and time-dependent manner（P<0.01）;（2）The results of colorimetric assay showed that the LDH leakage rate of A2780 cells induced by GNA was significantly increased（P<0.01）;（3）Morphological observation showed that the cells became smaller and rounder after GNA treated,the refractive index decreased and the adherence ability weakened.2 GNA Induces ferroptosis in A2780 Cells（1）Apoptosis inhibitor（Z-VAD-FMK）,autophagy inhibitor（3-Methyladenine,3-MA）,necrosis inhibitor（Necrostatin-1,Nec-1）,Ferroptosis inhibitor（Ferrostatin-1,Fer-1）and deferoxamine（DFO）were used to pretreat A2780 cells with 4μM GNA.The results of CCK-8 indicated that Z-VAD-FMK,Fer-1 and DFO could reduce the inhibition rate of the drug in a certain concentration range（P<0.01）,while Nec-1 and 3-MA did not produce this effect（P>0.05）.（2）DAPI staining showed that GNA could induce apoptosis in A2780 cells.Fer-1pretreatment could not avoid apoptosis.（3）The morphology of mitochondria was observed by transmission electron microscopy.It was found that the mitochondria of the cells decreased after the treatment of A2780cells with GNA,and the mitochondria morphology gradually increased to normal after Fer-1 pretreatment.（4）The content of Fe²⁺in the cells was detected by enzyme-labeled instrument.The intracellular iron ion content of A2780 cells was significantly increased after GNA treatment（P<0.01）.（5）H2DCF-DA and BODIPY-C11 fluorescent probes were used,flow cytometry showed that GNA could significantly increase the levels of cytoplasmic and lipid ROS in A2780 cells（P<0.01）.（6）Elisa results showed that SOD activity decreased significantly（P<0.01）,MDA content increased significantly（P<0.01）,intracellular GSH content and GPX activity decreased significantly（P<0.01）after GNA treatment with A2780.（7）Western blot assay was used to detect the expression of TFR and Ptgs2.Compared with the control group,the expression of TFR and Ptgs2 was significantly increased after A2780 treatment with GNA（P<0.01）.3 Molecular Mechanisms of GNA Inducing Ferroptosis in A2780 Cells Based on Nrf2/HO-1 Signaling Pathway（1）Western blot results showed that the expression of Nrf2,HO-1 and GPX4 proteins was significantly down-regulated after GNA treatment（P<0.01）.（2）The results of q RT-PCR showed that the expression levels of Nrf2,HO-1 and GPX4m RNA were significantly down-regulated after GNA treatment（P<0.01）.（3）Nrf2 protein was knocked down by lentivirus,and the efficiency of Nrf2 sh RNA transfection was observed by fluorescence microscopy.Compared with the normal group,the expression of Nrf2 protein was significantly inhibited by Western blot（P<0.01）,suggesting that Nrf2 knockdown was successful.（4）After knocking down the Nrf2,GNA inhibited the proliferation of A2780 cells,and there was no obvious protective effect after Fer-1 pretreatment.（5）After knockout of Nrf2,the expression of GPX4 and HO-1 in GNA-damaged cells decreased significantly from gene and protein levels（P<0.01）,while there was no statistical difference between the pre-treatment of fer-1 and the GNA group（P>0.05）.Conclusion:The results showed that GNA damage A2780 cells resulted in an increase in intracellular iron content,which caused a significant decrease in SOD activity,a significant increase in MDA content,a significant decrease in intracellular GSH content and GPX enzyme activity,and induced oxidative stress to induce Ferroptosis.The molecular mechanism of Ferroptosis in GNA-injured A2780 cells may be closely related to the Nrf2/HO-1 pathway.