| Background: Esophageal cancer is one of the most common malignancies and the sixth leading cause of cancer-related death worldwide.The pathology of esophageal cancer is divided into esophageal squamous cell carcinoma and adenocarcinoma.Adenocarcinoma is more common in North America and Western Europe,while esophageal squamous cell carcinoma is the most common pathological type in Asia and has a poor prognosis.Current treatments mainly include surgery,radiotherapy,chemotherapy.The early symptoms of esophageal cancer are not obvious,and about 30-50% of patients lose the chance of surgery when they are diagnosed,and most of them take comprehensive treatment based on radiotherapy.With the progress of radiotherapy technology and the development of drugs,the survival of esophageal cancer has improved.Although the overall 5-year survival rate is still low,the main reasons for failure are local uncontrolled and recurrence(30-50%).Radiation resistance of tumor cells is one of the main biological factors affecting the therapeutic effect of radiotherapy.Therefore,it has become an urgent task to find new treatment methods or drugs,especially radiosensitizers,to improve the radiosensitivity of ESCC and the prognosis of ESCC,and it is also a research hotspot in the field of radiotherapy.Radiation induces apoptosis and necrosis of tumor cells by damaging DNA,which is one of the main mechanisms of radiation killing tumor cells.However,radiation resistance occurs due to activation of DNA repair pathway and apoptosis resistance.Therefore,sensitizing cancer cells to radiation through other non-apoptotic death pathways is of great value in tumor therapy.Ferroptosis,a regulated death characterized by iron-dependent,lethal lipid peroxide accumulation and depletion of PUFA or PUFA phospholipids on the plasma membrane,can be induced by GPX4,Nrf2,TFR1,SLC7A11 and other core gene regulation.Previous studies have found that ferroptosis is an important form of radiation-induced death in HT1080 and lung adenocarcinoma cell lines.However,the molecular mechanism of radiation-induced ferroptosis in these two tumors is completely different.In HT1080 cells,IR inhibits the expression of SLC7A11 by activating ATM,limiting tumor cystine uptake,reducing glutathione,and inducing ferroptosis by increasing lipid peroxide.In lung cancer cell lines H460,A549,the mechanism is that IR-induced ACSL4 expression increases lipid peroxide accumulation to promote ferroptosis,and SLC7A11,GPX4 expression increased after IR,negative feedback to repair cell survival.At the same time,the study found that MCF-7 and UMRC6 cell lines did not detect the increase in the expression of ferroptosis marker gene PTGS2 after IR.Using ferroptosis inducers in 860 tumor cell lines,and it was found that the sensitivity of different cells to ferroptosis inducers varied widely.SLC7A11 is a member of the solute carrier family.As a transmembrane protein,it mediates cystine entry into cells,participates in GSH synthesis,and protects cells from oxidative stress damage.SLC7A11 is a key gene in the ferroptosis pathway,which regulates ferroptosis by participating in GSH synthesis and redox of lipid peroxides.Inhibition of SLC7A11 can trigger ferroptosis.SLC7A11 is widely and highly expressed in lung cancer,liver cancer,breast cancer and other tumors,and plays an important role in the occurrence,development,invasion and metastasis of tumors.ATF3,a member of the ATF/CREB transcription factor family,is a known oxidative stress response gene.Its expression can be rapidly induced by a variety of cellular stresses,including DNA damage,oxidative stress,and cellular damage.At present,only in human fibrosarcoma HT1080 cells,ATF3 was found to inhibit the SLC7A11 promoter by competing with transcriptional activators for the binding of BS-1/BS-2,inhibit the expression of SLC7A11,and play a role in Erastin-induced ferroptosis in HT1080 cells.At present,the role of ferroptosis in radiation-mediated ESCC cell death is unclear,and the specific molecular mechanism is unclear.By targeting the ferroptosis pathway,it has great research value to increase the radiosensitivity of ESCC cells and improve the prognosis of esophageal cancer.This study will verify that radiation can induce ferroptosis in ESCC from the cellular,metabolic and molecular levels,through a series of experiments to explore and prove that radiation inhibits the expression of SLC7A11 by up-regulating the expression of ATF3 and mediates ferroptosis in ESCC cells.And verify that the ferroptosis inducer targeting SLC7A11 has a radio-sensitizing effect on ESCC cells.This study will provide an in-depth understanding of the mechanism of radiation-induced ferroptosis,provides a theoretical basis for targeting ferroptosis to increase the radiosensitivity of ESCC,and promotes individualized and precise treatment of tumors.Objects: 1.To verify that radiation can induce ferroptosis in ESCC cells.2.To demonstrate that radiation inhibits SLC7A11 expression by upregulating ATF3 expression,mediating ferroptosis in ESCC cells.3.To validate that ferroptosis inducers targeting SLC7A11 can act as potential radiosensitizers.4.To verify the correlation between the expression of ATF3 and SLC7A11 in the pathological samples of esophageal squamous cell carcinoma,and the relationship with the prognosis of radiotherapy.Methods: 1.Add ferroptosis inhibitors(Ferr-1,Lipr-1,DFO),apoptosis inhibitors(V-ZAD),and necrosis inhibitors(Necr-1)to the culture wells containing ESCC cell lines KYSE150 and TE1 cells.After irradiation and culturing for a certain period of time,CCK-8 was used to detect cell viability,and the effects of different death inhibitors on cell viability after IR were calculated,reflecting the proportion of ferroptosis,apoptosis and necrosis in cell death after IR;The clonogenic assay of death inhibitor combined with IR was confirmed again.2.The changes of lipid ROS levels in cells were detected by C11-Bodipy staining through flow cytometry,and the changes of total GSH levels in cells with gene interference with ATF3 and SLC7A11 expression before and after irradiation were detected by glutathione detection kit.3.Real-time PCR and Western blot were used to detect the changes of ferroptosis key genes PTGS2,SLC7A11,GPX4,ACSL4,LPCAT3 in ESCC cells KYSE150 and TE1 after IR,as well as the upstream transcription factors of SLC7A11,Nrf2,ATF4,TP53,ATF3 gene changes.,to explore the underlying molecular mechanism of IR-induced ferroptosis in ESCC.4.SLC7A11 over-expression KYSE150 cells and SLC7A11 knock-down TE1 cells were constructed by lentivirus interfering.Western blot was used to verify the protein level and the IR-induced DNA damage repair(γ-H2AX(p-s139))effect.The change of ferroptosis ratio after radiation in SLC7A11 over-expression KYSE150 cells and SLC7A11 knock-down TE1 cells were detected by CCK8 and ferroptosis inhibitor,and re-verified with clone formation assay.Thus.to prove that SLC7A11 is the important factor on ferroptosis in esophageal squamous cell cancers after radiation-therapy.5.Using small interfering RNA to interfere with the expression of ATF3 in esophageal squamous cell carcinoma KYSE150 and TE1 cell lines,and the efficiency of ATF3 interference and its effect on the expression of SLC7A11 were verified by Western blot after 72 hours.Then,the cell lines that interfered with the expression of ATF3 were irradiated,and Western blot was used to detect the changes in the expression of ATF3 and SLC7A11 after irradiation compared with the NC group,and the effect on DNA damage repair.Using CCK8 and ferroptosis inhibitors,to detect changes in the ratio of ferroptosis after irradiation in cells that interfere with ATF3 expression,and to revalidate with clone formation assay.Thus,to prove the regulation of ATF3 on SLC7A11 protein expression and its effect on ferroptosis in ESCC cells after IR.6.Using CCK8 to detect drug toxicity to calculate the IC50 concentrations of ferroptosis inducers Erastin and SAS targeting SLC7A11 after 24 h,48h,and 72 h function in KYSE150 and TE1 cell lines.The appropriate drug concentrations were selected,combined with radiation for clonal formation to verify that ferroptosis inducers can be used as potential radiosensitizer for esophagus squamous cell carcinoma.7.The clinical data and follow-up information of patients with esophageal squamous cell carcinoma who received radical radiotherapy in the First Hospital of China Medical University from January 2014 to July 2018 were collected.The expression of ATF3 and SLC7A11 in the pathological tissue of esophageal squamous cell carcinoma before radiotherapy was detected by SP immunohistochemical method.The high and low expression groups were divided according to the staining score,and the correlation and prognosis were analyzed.Results: 1.Increased lipid ROS levels in ESCC cells after irradiation The lipid peroxide levels in ESCC cell lines KYSE150 and TE1 cells after IR increased with the increase of irradiation dose.Compared with the control group,the lipid peroxide levels in the 8Gy/72 h group in KYSE150 cells increased by 1.322 times,TE1 cells increased by 1.559 times,P<0.05,the difference was statistically significant.2.Ferroptosis inhibitors partially reverse radiation-induced cell death The results of CCK8 detection showed that compared with the simple irradiation group,using the ferroptosis inhibitor Ferr-1 and DFO both increased the viability of KYSE150 and TE1 cells after irradiation(P<0.05).The colony formation assay showed that in the two cell lines,the SF fraction of the Ferr-1+IR group and the lipr-1+IR group was higher than that of the pure IR group,suggesting that ferroptosis inhibitors could partially reverse radiation-induced cell death.3.The expression of intracellular ferroptosis marker gene PTGS2 increased after irradiation Real-time PCR was used to detect the changes of ferroptosis marker gene PTGS2 in ESCC cell lines KYSE150 and TE1.The results showed that PTGS2 m RNA levels in KYSE150 and TE1 cells were significantly increased after irradiation(P<0.05).4.The expression level of SLC7A11 in ESCC cells decreased after irradiation,and the level of intracellular GSH decreased Real-time PCR and Western blot techniques were used to detect the expression changes of key ferroptosis pathway genes SLC7A11,ACSL4,GPX4 in KYSE150 and TE1 cells after irradiation.The results showed that the expression of SLC7A11 decreased,while ACSL4 and GPX4 were not detected in the two cell lines.Variety.The results of GSH detection showed that the level of GSH in KYSE150 and TE1 cells decreased gradually after 8Gy irradiation(P<0.05).It is suggested that the decreased level of SLC7A11 inhibited by radiation may be a key factor of ferroptosis in ESCC cells.5.Silencing SLC7A11 expression increases radiation-induced ferroptosis in ESCC cells,and overexpression of SLC7A11 partially reverses radiation-induced ferroptosis in ESCC cells 5.1 Using lentivirus to interfere with SLC7A11 expression,KYSE150-overexpressing SLC7A11 and TE1-silencing SLC7A11 cell lines were successfully constructed.The detection of GSH and lipid ROS levels showed that,compared with the NC group,the intracellular GSH level increased and the lipid ROS level decreased after irradiation after overexpression of SLC7A11,and the opposite results occurred after SLC7A11 was silenced.5.2 After overexpression of SLC7A11,the colony formation rate of cells after irradiation increased(P<0.05),and the proportion of cell death induced by ferroptosis inhibitor rescue irradiation decreased(P<0.05).It was suggested that overexpression of SLC7A11 partially reversed cell ferroptosis after KYSE150 radiotherapy.After silencing SLC7A11,the cell proliferation ability decreased,the colony formation rate decreased after irradiation,and the radiosensitivity of TE1 cells increased(P<0.05).The results of CCK8 assay showed that silencing SLC7A11 could reduce the cell viability after irradiation,and the ferroptosis inhibitor rescued the increase in the proportion of cell death caused by irradiation,P<0.05.Suggest that silencing SLC7A11 increases radiation-induced ferroptosis.5.3 Overexpression of SLC7A11 did not affect γ-H2AX(p-s139)protein levels after irradiation,that is,DNA damage repair was not affected.The level of γ-H2AX(p-s139)increased after irradiation after silencing SLC7A11,suggesting a decreased level of DNA damage repair.It is suggested that silencing SLC7A11 increases radiosensitivity by affecting cell proliferation,ferroptosis and DNA damage repair,and is a potential regulatory target.6.The expression of ATF3 in KYSE150 and TE1 cells increased after irradiation.In this study,the post-radiation m RNA levels of the upstream transcripts TP53,NRF2,ATF3,and ATF4 that regulate the expression of SLC7A11 were detected.The results showed that TP53 and NRF2 did not change significantly,and the changes of ATF4 were inconsistent in the two ESCC cell lines.Suggest they are not the key factor influencing the changes in SLC7A11.while ATF3 showed a consistent marked increase in m RNA levels after irradiation in both ESCC cell lines.Western blotting at the protein level showed that ATF3 protein expression was significantly increased after irradiation,which was opposite to that of SLC7A11 protein.It is suggested that the increased expression of ATF3 in ESCC cells after irradiation may be an upstream factor that inhibits the expression of SLC7A11.7.Down-regulation of ATF3 expression partially reverses the decreased expression of SLC7A11 after radiation,but has no effect on DNA damage repair ATF3 expression was down-regulated using small interfering RNA,and Western blot assay confirmed that down-regulation of ATF3 expression increased SLC7A11 expression in both cell lines,and previous radiation-induced increases in ATF3 expression and decreased SLC7A11 expression were reversed.Cells with downregulated ATF3 expression did not show an increase in ATF3 expression after irradiation,and the corresponding decrease in SLC7A11 expression partially recovered,and the intracellular γ-H2AX(p-s139)protein level did not change after irradiation,suggesting that the repair of DNA damage after irradiation is not affected.8.Downregulation of ATF3 expression partially reverses ferroptosis after radiation The clone formation assay showed that down-regulation of ATF3 expression had no effect on the proliferation of KYSE150 and TE1 cells.After irradiation,the clone formation rate increased and the rate of radiation-induced cell death decreased(P<0.05).The results of CCK8 assay showed that down-regulation of ATF3 expression increased cell viability after irradiation,and the ability of ferroptosis inhibitors to rescue the cells viability under irradiation decreased.It indicated that the proportion of radiationinduced ferroptosis decreased after down-regulation of ATF3 expression(P<0.05).Compared with the control group,after down-regulation of ATF3 expression,intracellular GSH level increased and lipid ROS level decreased after IR.Similar to the previous results of overexpression of SLC7A11.9.Ferroptosis inducers targeting SLC7A11 increase radiosensitivity of ESCC cells The IC50 concentration of ferroptosis inducers Erastin and SAS targeting SLC7A11 in esophageal squamous cell carcinoma cell line KYSE150 and TE1 cell line at 24 h,48h and 72 h were detected by CCK8.Appropriate drug concentrations(Erastin 5u M,SAS 500 u M)were selected to act on the two cell lines,and the cell clone formation rate was calculated after 0,2,4,6,and 8Gy irradiation.The single-click multi-target model was used to fit the curve and the multiple t test was used to calculate the results.The results showed that Erastin and SAS could increase the radiosensitivity of ESCC cells(P<0.05).10.SLC7A11 is negatively correlated with ATF3 expression,and is an independent prognostic factor in patients with esophageal squamous cell carcinoma after radiotherapy The results of immunohistochemistry showed that the expression of SLC7A11 protein was localized in the cell membrane and cytoplasm,and the expression of ATF3 was mainly localized in the cytoplasm and a few nuclei.The two expressions were negatively correlated,r=-0.1918,P=0.0125,the difference was statistically significant.The protein expression of SLC7A11 and ATF3 was not related to gender,age,tumor location,length,clinical stage,etc.The prognosis of SLC7A11 low expression group was good,and the prognosis of ATF3 low expression group was poor;the combined prediction showed that patients with only high ATF3 expression had the best prognosis,and only high SLC7A11 expression group had the worst prognosis,P<0.05,the difference was statistically significant.COX analysis showed that SLC7A11 expression,ATF3 expression and T stage were independent prognostic factors in patients with ESCC after radiotherapy.Conclusions: 1.Ferroptosis is an essential component of ESCC cell death after IR 2.Decreased expression of SLC7A11 is an important factor in ferroptosis after IR。 3.Down-regulation of SLC7A11 expression increases radiosensitivity by promoting ferroptosis,inhibiting proliferation,and affecting DNA damage repair,which is a potential regulatory target;up-regulating SLC7A11 expression can partially reverse ferroptosis without affecting proliferation and DNA damage repair;4.The expression of ATF3 increased after radiation,and after down-regulation of ATF3,the expression of SLC7A11 increased,partially reversed ferroptosis after radiation,but did not affect proliferation and DNA damage repair;5.Radiation up-regulates ATF3 expression,which inhibits SLC7A11 expression,is the molecular mechanism that mediates ferroptosis in ESCC;6.The ferroptosis inducers targeting SLC7A11 can increase the radiosensitivity of ESCC cells.7.The expression of SLC7A11 and ATF3 in ESCC pathological samples was negatively correlated,and were independent prognostic factors in patients with ESCC after radiotherapy. |
PDF Full Text Request