| Background:Iron is one of the most abundant and essential trace elements in human body,and plays an important role in DNA synthesis,electron transport,oxygen transport and other metabolic processes.Normal cell iron metabolism is in a dynamic balance of continuous absorption,utilization,storage and circulation,namely iron homeostasis,which plays an important role in maintaining normal physiological functions of cells.When iron homeostasis in the cell is disrupted,the cell becomes overloaded with iron.Reactive oxygen species(ROS)produced by Fenton reaction can induce a programmed cell death--iron death,which is different from apoptosis,autophagy and necrosis.The mechanism is the depletion of intracellular glutathione(GSH),the decline of glutathione peroxidase 4(GPX4)activity,lipid oxides can not be metabolized through GPX4-catalyzed glutathione reduction reaction,and then iron divalent ion oxidizes lipid in the way of Fenton reaction and produces a large number of reactive oxygen species,promoting iron death of cells.In order to meet the needs of continuous growth and reproduction,tumor cells need to synthesize and accumulate a large number of enzymes related to DNA synthesis,DNA replication and DNA repair during the division cycle.Iron,as the active center component of DNA polymerase and repair enzyme related to chromosome stability,is the main rate-limiting factor in DNA synthesis.High iron metabolism is one of the major manifestations of tumor cells,so iron has great potential significance for tumor growth,survival and metastasis.Radiotherapy is one of the main treatment methods for malignant tumor.Some studies have proposed the mechanism that radiation may induce iron death in tumor cells:radiation can activate ATM and synergically inhibit SLC7A11,the unit of glutamic-cystine transporter Xc~-,resulting in reduced cystine uptake,enhanced tumor lipid oxidation and iron death,thus achieving the purpose of killing tumor cells.Subsequently,studies have shown that radiation can induce lipid peroxidation and iron death by inducing the expression of ACSL4,a lipid metabolism enzyme required for iron death.Down-regulation of ACSL4 expression can inhibit iron death and lead to radiation resistance of tumor cells.In addition,the expression of SLC7A11 and GPX4(glutathione peroxidase 4)was induced by radiation,and the expression of SLC7A11 or GPX4 was down-regulated by FINs with iron death inducer(FINs)to improve the radiosensitivity of radioresistant tumor cells and xenograft tumors.In addition,iron death was associated with better outcomes in patients receiving radiation therapy.These results indicate that iron death may be an important way for radiation to kill tumor cells,and inducing iron death can be a new direction for improving the efficacy of radiotherapy.However,the mechanism of iron death and the mechanism pathway of iron death induced by radiation remain to be explored.Esophageal squamous cell carcinoma is one of the most common malignant tumors.Radiation therapy is one of the main treatment methods for esophageal cancer,but the prognosis is poor.How to improve the radiation sensitivity of esophageal cancer has always been a key field of radiation therapy for esophageal cancer.Studies have found that the positive expression rate of HSPB1 in esophageal squamous cell carcinoma tissues is higher than that in normal esophageal tissues.The expression of HSPB1 was associated with lymph node metastasis.It is concluded that HSPB1 plays a role in the occurrence and development of esophageal squamous cell carcinoma and can be used as a prognostic factor of ESCC.As a new direction of cancer treatment in recent years,the relationship between iron death and esophageal cancer is unclear.It was found that overexpression of DNAJB6a in esophageal cancer tissue showed tumor suppressive effect in vitro and in vivo.The mechanism may be that DNAJB6a inhibits the expression of Gpx4 and phosphorylated AKT(p-AKT)proteins,thus promoting the occurrence of iron death.It is concluded that DNAJB6 plays an important role in the development of iron death in esophageal cancer.Therefore,this study aims to investigate whether the radiosensitivity of esophageal cancer can be upregulated by inducing iron death in esophageal cancer cells and explore the related mechanisms.Heat shock protein family B member 1(HSPB1,mouse HSP25,human HSPB1)is a member of HSPs constitutively expressed in multiple cells or tissues,and is highly expressed in a variety of tumors.It is an ATP-independent molecular chaperone induced by heat shock or other chemical stimuli.HSPB1 can inhibit programmed cell death such as apoptosis.In recent years,it has been found that HSPB1 is involved in the process of iron death,but the mechanism of how HSPB1 affects iron death in tumor cells remains unclear.Method:1.A total of 53 patients with esophageal squamous cell carcinoma who received three-dimensional conformal or intensity modulated radiotherapy in the First Affiliated Hospital of China Medical University from 2014 to 2016 were collected.Before treatment,all patients were subjected to fibrous gastroscopy for pathological examination.Tissue specimens were fixed with 10%formalin,embedded in paraffin and sealed,and confirmed by HE staining.The treatment method was linear accelerator6MV-X 3d conformal or intensity modulated radiotherapy,60-66Gy/30-33 times,2Gy/time,5 times per week.Patients were followed up in outpatient clinic or by telephone every 3-6 months to collect their survival,time and cause of death.The cut-off point of follow-up was death,loss of follow-up,or December 2016.Immunohistochemical staining was used to detect the expression of HSPB1 and SLC40A1 in esophageal squamous cell carcinoma biopsy tissue sections.SPSS20.0 statistical software was used for statistical processing.2 test was used to compare the count data,kaplan-Meier method was used to estimate the survival rate,Logrank test was used to test the survival difference,and COX model was used to test the prognostic factors.P<0.05 was statistically significant.2.Esophageal squamous cell carcinoma cell lines KYSE150 and TE1 were cultured in vitro,transfected with sh RNA by lentivirus,and screened by purinomycin to obtain stable HSPB1 silencing esophageal carcinoma cell lines.In vitro cell irradiation method:when cells were routinely cultured in vitro to logarithmic growth phase,6MV X-ray irradiation was adopted,with a dose rate of 300c Gy/min.Different doses were taken according to specific experiments.Culture was continued after irradiation or corresponding treatment was given.Real-time PCR was used to analyze the m RNA expression level of HSPB1 gene in the cells,and the expression of related proteins in the cells was detected by Western blot.The intracellular iron concentration was detected by the iron ion kit.Intracellular lipid ROS levels were detected by C11 Bodipy probe and flow cytometry.The clone formation experiment was used to evaluate the cell clone formation rate under different irradiation doses and different treatments.Graph Pad Prism7 software was used to fit the cell survival curve by clicking the multi-target model Y=1-(1-exp(-k*x))^N,and then k and N values were obtained according to the formula K=1/D0.Ln N=Dq/D0,SER=D0(control group)/D0(experimental group),mean lethal dose(D0),quasi-threshold dose(Dq)and radiosensitization ratio(SER)of cells were obtained.SPSS 20.0 and Graph Pad Prism were used to analyze the experimental data.All results were obtained by repeating the experiment at least three times.The experimental data were presented as Mean±standard deviation(SD).Normality test and homogeneity analysis of variance were carried out first when the mean values of the two groups were compared.For example,the data were normally distributed and homogeneity of variance,the t test was used to statistically compare the differences between the two groups;If variance is not uniform,rank sum test is used.P<0.05indicated that the difference was statistically significant.3.Co-Immunoprecipitation was used to detect protein interaction,immunohistochemical method was used to analyze the pathological sections of esophageal squamous cell carcinoma,and the co-expression of HSPB1 and SLC40A1 was detected.Spearman rank correlation was used to test the correlation.Results:1.The expression levels of HSPB1 and SLC40A1 are correlated with the prognosis of patients with esophageal squamous cell carcinoma after radiotherapyAmong the 53 cases of esophageal squamous cell carcinoma that received radiotherapy alone,21 cases had high expression of HSPB1,32 cases had low expression of HSPB1,8 cases had high expression of SLC40A1,and 45 cases had low expression of SLC40A1.Analysis of the correlation between the expression levels of HSPB1 and SLC40A1 and clinicopathological factors showed that the expression level of HSPB1was correlated with the T stage and clinical stage of esophageal squamous cell carcinoma(P<0.05),and the expression level of SLC40A1 was correlated with the T stage of esophageal squamous cell carcinoma(P<0.05).Kaplan-meier survival analysis showed that patients with high HSPB1 expression and low SLC40A1 expression had poor prognosis.Cox proportional risk regression model showed that HSPB1 and SLC40A1expressions were correlated with prognosis(P<0.05),and were independent prognostic factors of esophageal squamous cell carcinoma patients receiving radiotherapy.2.Iron death level of esophageal squamous cell carcinoma increased after silencing HSPB1Through the detection of intracellular iron ion concentration and lipid ROS level,after the irradiation dose of 8Gy,the iron ion concentration and lipid ROS level of HSPB1 gene silenced cells were significantly higher than those of NC group,with statistical difference(P<0.05),suggesting that the down-regulation of HSPB1 expression can improve the iron death level in esophageal squamous cell carcinoma cells.3.Radiotherapy sensitivity of esophageal squamous cell carcinoma was increased after silencing HSPB1Clonal formation experiments showed that radiotherapy sensitivity of HSPB1 gene silenced cells increased after irradiation with different doses of radiation(P<0.05).By clicking the multi-target model Y=1-(1-exp(-k*x))^N and fitting the cell survival curve and related parameters,it was found that the cell survival rate of HSPB1 gene silenced cells was significantly decreased and the quasi-threshold dose was significantly increased when the cells were irradiated with 2Gy(P<0.05).This suggests that the radiotherapy sensitivity of esophageal squamous cell carcinoma increased after silencing HSPB1.4.HSPB1 targeted drug KRIBB3 can increase the radiotherapy sensitivity by increasing the iron death level of esophageal squamous cell carcinoma cellsThe iron ion concentration and lipid ROS level in cells were detected.After irradiation dose of 8Gy,the iron ion concentration and lipid ROS level of Kribb3-treated cells were significantly increased,with statistical difference(P<0.05),indicating that HSPB1 targeting KRIBB3 can improve the iron death level in esophageal squamous cell carcinoma cells.The clone formation experiment showed that the radiotherapy sensitivity of kribb3-treated cells increased after irradiation with different doses of radiation(P<0.05).By clicking the multi-target model Y=1-(1-exp(-k*x))^N,fitting the cell survival curve and related parameters,it can be found that the cell survival rate of Kribb3-treated cells is significantly decreased when 2Gy irradiation is carried out,and the quasi-threshold dose is also significantly increased(P<0.05).These results suggest that HSPB1 targeting KRIBB3 can increase the radiotherapy sensitivity of esophageal squamous cell carcinoma cells.5.HSPB1 regulates iron death by regulating SLC40A1 in esophageal squamous cell carcinoma cellsWestern blot analysis showed that the iron-heavy chain protein and SLC40A1protein in esophageal squamous cell carcinoma cells decreased after the silencing of HSPB1,suggesting that HSPB1 may regulate the intracellular iron level by regulating the expression of SLC40A1,and thus regulate iron death.SLC40A1+plasmid overexpression recovery experiment showed that overexpression of SLC40A1 after silencing HSPB1 could down-regulate the iron death level of esophageal cancer cells to a certain extent.6.Immunoprecipitation confirmed the direct effect of HSPB1 and SLC40A1 in cellsCOIP results showed that SLC40A1 protein could also be detected by Western blot when HSPB1 protein was pulled down to precipitate by magnetic beads in esophageal squamous cell carcinoma cell lines,indicating physiological interaction between HSPB1and SLC40A1.7.HSPB1 and SLC40A1 were positively correlated in esophageal squamous cell carcinomaHistological diagnosis of esophageal squamous cell carcinoma was collected,and immunohistochemical staining of paraffin sections and grading criteria were used.Spearman rank correlation test showed that there was a significant positive correlation between HSPB1 and SLC40A1 expression levels(P<0.05).Conclusion:1.The expression of HSPB1 is correlated with the prognosis of patients with esophageal squamous cell carcinoma receiving radiotherapy,and the patients with high expression of HSPB1 have a poor prognosis after radiotherapy.HSPB1 can be an independent prognostic factor in patients with esophageal squamous cell carcinoma.2.Down-regulation of HSPB1 expression increased the iron death level of esophageal squamous cell carcinoma cells and increased the radiosensitivity.3.HSPB1 directly interacts with SLC40A1 protein to affect iron metabolism and participate in iron death process.4.HSPB1 may be a potential sensitization target for radiotherapy of esophageal cancer,and HSPB1 targeting inhibitor KRIBB3 can be used as a potential drug for radiotherapy of esophageal cancer. |
PDF Full Text Request