Effect And Mechanism Of Labile Iron Pool On Radiosensitivity Of Cervical Cancer

Objective: Cervical cancer has seriously threat women's health worldwide,and the incidence of cervical cancer ranks first among malignant tumors of female reproductive system in China.Radiotherapy is one of the most important radical treatment for cervical cancer.Local recurrence and distant metastasis caused by radiotherapy resistance are still one of the most important reasons for the poor prognosis of cervical cancer.Therefore,it is of great significance to explore radiosensitizers of cervical cancer.Ferroptosis is a new form of cell death independent of apoptosis and necrosis,which was first reported in 2012.The feature of ferroptosis is mainly induced by the excessive accumulation of divalent iron-dependent polyunsaturated fatty acid peroxidation.Relevant studies have confirmed that ferroptosis is one of the main mechanisms of radiotherapy to kill tumor cells,suggesting that promoting ferroptosis sensitivity has great potential in promoting the radiosensitivity of malignant tumors,but there is currently a lack of relevant research.Since the labile iron pool is a key factor necessary for ferroptosis,the purpose of this study is to explore whether increasing the level of labile iron pool has a radiosensitizing effect,and explore the related mechanisms.Methods1.We first constructed a radiation resistant cell line for cervical cancer.Finding differentially expressed genes by proteomic sequencing with unirradiated cell lines.And verify that radiotherapy induces ferroptosis in cervical cancer through protein interaction network and enrichment analysis.And we analyze the histological sequencing results of cervical cancer before and after radiotherapy in the GEO database for further verification.2.Verify that radiotherapy induces ferroptosis on cervical cancer cells by detecting the level of lipid peroxidation,using ferroptosis inhibitors,and detecting the expression of iron death-related genes.3.Verify that increasing the level of labile iron pool can promote the radiosensitivity of cervical cancer.1)We use ferrous ammonium sulfate to directly act on cervical cancer cell lines and combined treatment with radiotherapy.And verify its radiosensitization effect by plate clone formation experiment.Then combined with ferroptosis inhibitors for rescue experiments.2)We detect the concentration of labile iron pool after RSL3 effect on cervical cancer cells by flow cytometry.And detect the expression level of NCOA4 and ferritin by western blot.Detect the m RNA level of TFRC by q PCR.Radiotherapy was given at the time point when the concentration of labile iron pool increased,and the plate clone formation experiment was used to verify its radiosensitization effect.And detect lipid peroxidation levels by flow cytometry.And we perform rescue experiments by ferroptosis inhibitor and iron chelator,respectively.3)Detecting the concentration of labile iron pool after erastin effect on cervical cancer cells by flow cytometry.And detect the expression level of NCOA4 and ferritin by western blot.Radiotherapy was given at the time point when the concentration of labile iron pool increased,and the plate clone formation experiment was used to verify its radiosensitization effect.And detect lipid peroxidation levels by flow cytometry.And we perform rescue experiments by ferroptosis inhibitor,NCOA4knock-down or iron chelator respectively.4.We use ROS inhibitor combined with erastin to effect on cervical cancer cell lines,and detect the induction level of ferritinophagy by Western blot.5.Detecting the induction level of ferritinophagy in cervical cancer cells after radiotherapy by Western blot.And detect the level of labile iron pool by fluorescence spectrophotometer.The plate colony formation experiment was performed to detect its effect on radiotherapy.Results:1.Ferroptosis resistant genes are significantly up-regulated in cervical cancer radioresistant cell lines.Ferroptosis resistant genes are up-regulated for cervical cancer tissue after radiotherapy compared with before treatment.Enrichment analysis and protein interaction network suggest that the relevant pathways regulated by iron ions have significant changes.2.Radiotherapy could induce ferroptosis in cervical cancer cell lines,and ferroptosis inhibitors can inhibit radiosensitivity and exceed apoptosis inhibitors.3.Increased the level of labile iron pool promotes the radiosensitivity of cervical cancer.1)Extracellular administration of iron ions can limitedly promote the radiosensitivity of cervical cancer cell lines and can be reversed by ferroptosis inhibitors.However,extracellular iron ions can significantly promote proliferation of cervical cancer cells.2)RSL3 can also promote the labile iron pool concentration of Siha cells,but RSL3 did not induce ferritinophagy in cervical cancer cell lines.The q PCR results showed that the transferrin receptor was significantly up-regulated within 16 hours of RSL3 effect on Siha cells.Radiotherapy at the time point when the labile iron concentration is high after RSL3 effect on Siha cells can significantly improve its radiosensitivity,and promote the accumulation of lipid peroxides in cervical cancer cells.And this effect can be reversed by ferroptosis inhibitors and iron chelator.3)Erastin can induce ferritinophagy in cervical cancer cell lines and lead to an increase of labile iron pool concentration.Combining radiotherapy at the time when the ferritinophagy effect is most significant can significantly increase the radiosensitivity of cervical cancer cell lines.Ferroptosis inhibitors and iron chelators can reverse the radiosensitization effect of erastin on cervical cancer.When NCOA4 is knocked down,it significantly inhibits ferritinophagy and reduces the concentration of labile iron pools,resulting in a significantly weakened ability of erastin to radiosensitize cervical cancer cells.4.Erastin combined with ROS scavenger to effect on cervical cancer cells,and the expression of ferritinophagy-related genes was detected.It was found that ROS scavenger significantly inhibited ferritinophagy.Radiotherapy can induce ferritinophagy and increase the labile iron pool concentration in cervical cancer cells.However,knocking down NCOA4 has no effect on the radiosensitivity of cervical cancer cells.Conclusion:1.Radiotherapy can induce ferroptosis of cervical cancer cells,but the adaptive response after radiotherapy can lead to ferroptosis resistance.2.The increase of labile iron pool promoted by the direct administration of extracellular iron ions,transferrin overexpression and ferritinophagy can significantly promote the radiosensitivity of cervical cancer cell lines.But extracellular iron ions also increase the total iron content of cells,which can promote the proliferation of cervical cancer cells.Therefore,ferritinophagy is a more advantageous strategy for radiosensitization.3.Ion-independent ROS is the upstream mechanism of ferritinophagy induction,and it has been found that ionizing radiation can induce ferritinophagy in cervical cancer,but the application of this mechanism needs to be further explored.