To Study The Effect Of Hyperthermia On The Biological Behavior Of Oral Squamous Cell Carcinoma Cells By Regulating Ferroptosis Pathways

BACKGROUND AND OBJECTThe most frequent malignant tumor in the oral and maxillofacial area is oral squamous cell carcinoma（OSCC）,which has a high incidence of metastasis and mortality.Chemotherapy and radiation are currently used in addition to surgery as the primary form of treatment.However,radiotherapy and chemotherapy’s toxic and side effects have had a significant negative impact on the quality of life for people with oral squamous cell carcinoma.In recent years,clinical applications of hyperthermia（HT）,a noninvasive therapy with few side effects,have garnered extensive study.These findings show that hyperthermia successfully boosts the effectiveness of chemotherapy and radiotherapy and encourages tumor cell death.Currently,cell death is primarily divided into two categories:accidental cell death（ACD）and regulated cell death（RCD）or programmed cell death（PCD）.Thermotherapy（heating temperatures between 41 and 45°C）and thermal ablation（heating temperatures above 55°C）are the two main categories for hyperthermia.According to studies,when the temperature is greater,ACD is the primary cause of cell death,and when the temperature is lower,PCD is the primary cause.One of the variables that has received attention is ferroptosis.An instance of iron-dependent PCD is ferroptosis.Reactive oxygen species（ROS）-induced lipid peroxidation is a recognized biochemical indicator of iron mortality.According to studies,activating the p53/Tf R1 axis can control the amount of intracellular iron,which in turn controls the iron death process.p53 is a crucial tumor suppressor gene that cannot function as a tumor suppressor due to its extreme mutation in a number of cancers,particularly head and neck squamous cell carcinoma.Transferrin receptor 1（Tf R1）,which has a high level of expression in tumors and is thought to be a new tumor therapeutic target,is frequently linked to a poor prognosis.Through the use of molecular biological techniques,this study aims to investigate the regulatory impact of hyperthermia（HT）on OSCC iron death and associated mechanisms.It also aims to further examine the impact of iron death caused by hyperthermia on the biological behavior of OSCC and offer a new therapeutic target for the clinical treatment of oral squamous cell carcinoma.METHODS1.The protein interaction network related to ferroptosis was obtained by searching the bioinformatics database,and the expression differences of related factors in cancer and adjacent tissues（0.5cm of tumor edge）were analyzed,and the related pathways were verified.2.The protein interaction network related to ferroptosis was obtained by searching the bioinformatics database,and the differential expression of related factors in cancer and adjacent tissues（0.5cm of tumor edge）was analyzed,and the related pathways were verified.3.Immunohistochemical staining was used to detect the expression of Tf R1 and HSPA8 in OSCC and adjacent normal tissues..4.CCK-8 assay was used to detect the 50%inhibitory concentration(IC₅₀)of ferroptosis inhibitor Ferrostain 1（Fer-1）.5.Iron ion concentration was detected by iron ion concentration detection kit.6.q RT-PCR and WB were used to detect the regulatory effect of HT on cell ferroptosis pathway in each treatment group.7.Cell scratch test was used to detect the migration ability of cells in each group.8.Flow cytometry（Annexin V FITC/PI double staining）apoptosis kit was used to detect the apoptosis of cells in each group.8.Immunohistochemical staining was used to detect the expression of Tf R1 and HSPA8 in OSCC and adjacent normal tissues.RESULTS1.STRING database search found p53/Tf R1/HSPA8 protein interaction network,and TIMER database search found that Tf R1 and HSPA8 were significantly overexpressed in head and neck squamous cell carcinoma,while p53 had no significant difference.2.IHC results demonstrated that OSCC tissues had significantly higher levels of HSPA8 and Tf R1 expression than the nearby normal tissues.3.The CCK8 method was used to determine the IC₅₀ value of Fer-1 in the Cal-27 cell line,which was 1.184 mol/L.4.A ROS detection kit was used to measure the expression of ROS in each group,and the results revealed that the HT group had significantly higher ROS levels（P<0.01）.ROS were significantly reduced in the HT+Fer-1 group compared to the HT group（P<0.01）.5.The results of iron ion concentration detection kit showed that the iron ion concentration in HT group was significantly higher than that in Control group（P<0.01）.The HT+Fer-1 group had a significantly lower iron concentration than the HT group（P<0.001）.6.q RT-PCR and WB were used to identify the expression of ferroptosis-related genes,target signaling pathway genes,and target proteins in each group.The results demonstrated that the expression of the ferroptosis-inhibiting genes GPX4 and SLC7A11 decreased in the HT group（P<0.05）,and the m RNA and protein expressions of the ferroptosis-related signaling pathway molecules p53,Tf R1,and HSPA8 were up-regulated（P<0.05）;The expressions of the ferroptosis inhibitor genes GPX4 and SLC7A11 in the HT+Fer-1 group.7.In a wound healing assay,oral squamous cell carcinoma cells’ability to migrate was significantly reduced by HT（P<0.05）,but it was restored in the HT+Fer-1 group（P<0.05）.8.Flow cytometry revealed that the HT group’s apoptosis rate was significantly higher（P<0.05）.The apoptosis rate in the HT+Fer-1 group was significantly lower than that in the HT group（P<0.05）.CONCLUSIONS1.The expressions of Tf R1 and HSPA8 in human OSCC tissues were significantly higher than those in adjacent tissues,which may be related to tumor formation.2.HT at 43℃can promote the ferroptosis of CAL-27 by up-regulating intracellular ROS and related cytokines.3.Hyperthermia at 43℃can induce ferroptosis in CAL-27 cells by activating p53/Tf R1/HSPA8 pathway.4.Hyperthermia at 4.43℃may inhibit the migration ability of CAL-27 cells and promote their apoptosis by inducing ferroptosis.