| IntroductionOral squamous cell cancer(OSCC)is the most common oral malignant tumor,which generally accounts for 90%of oral malignancies.It has a high recurrence and metastasis rate,and its overall prognosis is poor.So it is still a serious global public health problem.At present,the treatment of oral squamous cell carcinoma mostly adopts a comprehensive sequence of treatments based on surgery,radiotherapy and chemotherapy.Chemotherapy is still widely regarded as one of the effective clinical treatments for patients with oral squamous cell carcinoma.Platinum-based chemotherapeutics have always been the focus of research and have very important clinical significance.Oxaliplatin is a third-generation platinum-based chemotherapeutic agent.It is a stable water-soluble alkylating agent and can produce alkanes to treat tumors.Oxaliplatin is widely used in the treatment of various tumors,but the specific mechanism in the treatment of oral squamous cell carcinoma has not been fully elucidated.In particular,a variety of new cell death mechanisms have been discovered continuously,which provides a new opportunity for in-depth exploration of the regulation and molecular mechanism of oxaliplatin on the biological behavior of oral squamous cell carcinoma cells.Parhtanatos is mainly caused by the excessive activation of PARP1.It is a unique mode of regulatory cell death and plays an important role in various physiological and pathological processes.PARP1 is an important ribozyme involved in DNA repair.Mildly activated PARP1 can repair DNA damage and cause tumors to resist chemotherapeutics;however,the latest research has found that excessive reactive oxygen species(ROS)and DNA damage caused by alkylating agents can cause excessive activation of PARP1,which in turn triggers the nuclear translocation of apoptosis inducing factor mitochondria associated 1(AIFM1/AIF)and macrophage migration inhibitory factor(MIF).Finally,the DNA is cut and cell death is induced,which is the main mechanism of Parthanatos.Studies have shown that Parthanatos is involved in the process of tumor cell death induced by a variety of alkylating chemical drugs.This new type of cell death has attracted widespread attention in tumor treatment.It is not clear whether oxaliplatin,as an alkylating agent,can trigger Parthanatos.In summary,oxaliplatin is an important platinum-based chemotherapeutic drug,and the therapeutic effect and mechanism of oxaliplatin on oral squamous cell carcinoma are relatively few.Therefore,this study aimed to explore the regulatory effect of oxaliplatin on the biological behavior of oral squamous cell carcinoma cells,to clarify whether there are Parthanatos in the death of oral squamous cell carcinoma cells induced by oxaliplatin,and to explore its specific molecular mechanism.This experiment tested the inhibitory effect of oxaliplatin treatment on oral squamous cell carcinoma through in vivo tumorigenesis experiments in nude mice and in vitro cell experiments.Subsequently,it was confirmed by various experiments that oxaliplatin induced Parthanatoso in oral squamous cell carcinoma cells,and the production of reactive oxygen species plays an important role in the initiation of Parthanatos.Finally,using bioinformatics analysis methods,the expression characteristics of PARP1,the key protein of Parthanatos,in oral squamous cell carcinoma and its correlation with clinical characteristics were analyzed.This study is expected to explore the potential of oxaliplatin in the treatment of oral squamous cell carcinoma from a new perspective,and to provide a certain theoretical basis for further promoting the development of chemotherapeutics.Materials and methods1.The effect of oxaliplatin on the biological behavior of oral squamous cell carcinoma cells(1)In this study,oral squamous cell carcinoma cell lines CAL27 and SCC25 were selected as the research objects.The CCK-8 method was used to detect the effect of 0300 ?M oxaliplatin on the proliferation activities of CAL27 and SCC25 cells,then the IC50 was calculated.(2)Oxaliplatin was used to treat CAL27 and SCC25 cells separately,and the effect of oxaliplatin on the cloning ability of CAL27 and SCC25 cells was detected.(3)Oxaliplatin was used to treat CAL27 and SCC25 cells separately,and the wound healing assay was used to detect the effect of oxaliplatin on the migration ability of CAL27 and SCC25 cells.(4)Oxaliplatin was used to treat CAL27 and SCC25 cells separately,and the toxic effect of oxaliplatin on the two kinds of cells was detected by the lactate dehydrogenase(LDH)release experiment,and the cell death ratio was calculated.(5)After constructing a tumor xenograft model of oral squamous cell carcinoma in nude mice,oxaliplatin or PBS was inject into the intraperitoneal cavity of mice to detect the effect of oxaliplatin on tumor growth by measuring tumor volume and tumor weight.2.The role and molecular mechanism of Parthanatos in the death of oral squamous cell carcinoma cells induced by oxaliplatin(1)Using immunohistochemical staining to analyze the difference of PARP1 expression in tumor tissues of oral squamous cell carcinoma between the oxaliplatin treatment group and the PBS control group.(2)After treating CAL27 and SCC25 cells with corresponding concentrations of oxaliplatin,and the JC-1 probe was used to detect the mitochondrial membrane potential of cells.Then the ratio of monomeric JC-1/aggregate JC-1 was analyzed through fluorescence microscope.(3)Based on the IC50 screened by CCK-8,CAL27 and SCC25 cells were treated with different concentrations of oxaliplatin and for different time gradients,and Western Blot was used to detect the expression levels of Parthanatos-related key proteins such as PARP1,AIF and MIF in the cytoplasm and nucleus;CAL27 and SCC25 cells were treated with corresponding concentrations of oxaliplatin,then the AIF immunofluorescence staining was performed to analyze the nuclear translocation of AIF.(4)CAL27 and SCC25 cells was pretreated with the PARP1 inhibitor Rucaparib,then the experiment is divided into control group,oxaliplatin group,and Rucaparib+oxaliplatin group,then the cell death ratio was analyzed by LDH release experiment;the expression levels of Parthanatos-related key proteins including PARP1,AIF and MIF in the cytoplasm and nucleus were detected by Western Blot;the nuclear translocation of AIF was detected by cell immunofluorescence staining.(5)PARP1 was silenced by siRNA,and then the experiment was divided into interference control(siNC)group,oxaliplatin group,interference PARP1+oxaliplatin(siPARPl+oxaliplatin)group.Then the LDH release experiment was used to analyze the cell death ratio;Western Blot was used to detect the expression levels of Parthanatos-related key proteins PARP1,AIF,MIF,etc.in the cytoplasm and nucleus;cell immunofluorescence staining was used to detect the nuclear translocation of AIF.(6)Oxaliplatin was used to treat CAL27 and SCC25 cells separately to detect the changes in the concentration of reduced glutathione(GSH)and superoxide dismutase(SOD)activity in the cells;after setting up a control group,Oxaliplatin group,antioxidant NAC+oxaliplatin group,DCFH-DA probe was used to detect the level of reactive oxygen species(ROS)in cells.(7)CAL27 and SCC25 cells were pretreated with antioxidant NAC,then the experiment was divided into control group,oxaliplatin group,and NAC+oxaliplatin group.LDH release experiment was used to analyze cell death ratio;Western Blot was used to detect the expression levels of Parthanatos-related key proteins PARP1,AIF and MIF in the cytoplasm and nucleus;cell immunofluorescence staining was used to detect the nuclear translocation of AIF.3.Bioinformatics analysis of Parthanatos key protein PARP1 in oral squamous cell carcinoma(1)Through the oral squamous cell carcinoma tissue gene expression data set of the TCGA public database,the differences of PARP1 expression level between oral squamous cell carcinoma tissues and normal tissues were compared.(2)Kaplan-Meier survival analysis was performed on the high and low expression level groups of PARP1 to evaluate whether the expression of PARP1 was related to the prognosis of patients with oral squamous cell carcinoma.(3)The expression differences of PARP1 in the samples with different ages(?60 years old,>60 years old),gender(male,female),tumor grade(G1,G2,G3),tumor stage(stage ?,stage ?,stage ?,stage ?)were analyzed.(4)The expression differences of genes related with cell proliferation,cell cycle,and apoptosis between the groups with high expression level of PARP1 and groups with low expression level of PARP1 were compared.(5)GSVA analysis method was used to analyze the Hallmark and KEGG enrichment of genes in groups with high and low expression level of PARP1.Results1.The regulation of oxaliplatin on the biological behavior of oral squamous cell carcinoma cells(1)CCK-8 results showed that oxaliplatin had an inhibitory effect on proliferation activity of CAL27 and SCC25 cells,and the inhibitory effect enhanced with the increase of drug concentration and the extension of the action time.The IC50 of CAL27 and SCC25 cells were 125 ?M and 100 ?M,respectively.(2)After been treated with oxaliplatin,the number of clones of CAL27 and SCC25 cells decreased in a concentration-dependent manner,that is,oxaliplatin inhibited the cloning ability of CAL27 and SCC25.(3)Oxaliplatin had an inhibitory effect on the migration ability of CAL27 and SCC25 cells,and the inhibitory efect increased as the concentration increased.(4)The results of the LDH release experiment showed that the treatment of CAL27 and SCC25 cells with oxaliplatin caused significant cell death,and the cell death ratio increased with the prolonged action time increased.(5)The results of an oral squamous cell carcinoma xenograft model experiment in nude mice showed that the average volume and average weight of tumors after treatment with oxaliplatin were significantly lower than that of the PBS control group.2.The role and molecular mechanism of Parthanatos in the death of oral squamous cell carcinoma cells induced by oxaliplatin(1)In an oral squamous cell carcinoma tumor model in nude mice,the expression level of PARP1 in the oxaliplatin treatment group was significantly higher than that in the PBS control group.(2)After CAL27 and SCC25 cells were treated with oxaliplatin,the ratio of monomeric JC-1/aggregate JC-1 increased significantly compared with the blank control group,indicating the depolarization of mitochondrial membrane potential.(3)Western Blot results showed that after CAL27 and SCC25 cells were treated with oxaliplatin with different drug concentration and action time,the expression levels of the key proteins related to Parthanatos including PARP1,AIF,and MIF in the nucleus were increased;the immunofluorescence staining results of AIF showed that the nuclear translocation ratio of AIF increased in CAL27 and SCC25 cells after the treatment of oxaliplatin.(4)After pretreatment with the PARP1 inhibitor Rucaparib,the LDH release test results showed that the cell death rate of the Rucaparib+oxaliplatin group was significantly lower than that of the oxaliplatin group;Western Blot results showed that the expression levels of PARP1,AIF and MIF in the nucleus in the Rucaparib+oxaliplatin group were significantly lower than those in the oxaliplatin group;the immunofluorescence staining results showed that the AIF nuclear translocation ratio of the Rucaparib+oxaliplatin group was significantly lower than that in the oxaliplatin group.(5)After oxaliplatin treatment of CAL27 and SCC25 cells transfected with siPARP1,the LDH test results showed that the cell death rate of the siPARP1+oxaliplatin group was significantly lower than that of the siNC+oxaliplatin group;the Western Blot results showed that,the expression levels of PARP1,AIF,and MIF in the nucleus of the siPARPl+oxaliplatin group were significantly lower than those in the siNC+oxaliplatin group;the immunofluorescence staining results showed that the AIF nuclear translocation ratio of the siPARP1+oxaliplatin group was significantly lower than that in the siNC+oxaliplatin group.(6)Compared with the control group,the GSH concentration and SOD activity in CAL27 and SCC25 cells decreased after they were treated with oxaliplatin;DCFH-DA probe detection showed that the ROS level of the oxaliplatin group was higher than that of the control group.However,the ROS level of the NAC+oxaliplatin group was lower than that of the oxaliplatin group.(7)After pretreatment of CAL27 and SCC25 cells with antioxidant NAC,the results of LDH release experiment showed that the cell death rate of NAC+oxaliplatin group was lower than that of oxaliplatin group;Western Blot results showed that the expression levels of PARP1,AIF and MIF in the nucleus of the NAC+oxaliplatin group were lower than those of the oxaliplatin group;the results of cellular immunofluorescence showed that the nuclear translocation ratio of AIF in the NAC+oxaliplatin group was lower than that of the oxaliplatin group.3.Bioinformatics analysis of Parthanatos related key protein PARP1 in oral squamous cell carcinoma(1)The expression level of PARP1 in oral squamous cell carcinoma tissues was higher than that in normal tissues.(2)Compared with the PARP1 low expression group,the PARP1 high expression group had a lower survival rate,shorter survival time,and worse prognosis.(3)The stage and grade of oral squamous cell carcinoma were related to the expression of PARP1,that is,the higher the tumor grade and stage,the higher the expression level of PARP1.(4)The expression levels of cell proliferation,cell cycle,and apoptosis related genes in oral squamous cell carcinoma groupps with high expression level of PARP1 were higher than those in the PARP1 low expression group.(5)GSVA analysis showed that the proliferation score of oral squamous cell carcinoma in the PARP1 high expression group was significantly higher than that in the low PARP1 expression group,indicating that PARP1 was a positive regulator of OSCC cell proliferation.Conclusions1.Oxaliplatin has a significant effect on the biological behavior of oral squamous cell carcinoma cells.It can inhibit the proliferation and migration of oral squamous cell carcinoma cells in vitro,promote cell death,and inhibit the tumor growth in nude mouse tumorigenesis models in vivo.2.Oxaliplatin can cause the overexpression of PARP1 in oral squamous cell carcinoma cells,depolarization of mitochondrial membrane potential,and nuclear translocation of AIF,then promotes PARP1-mediated Parthanatos.And Parthanatos induced by oxaliplatin may be caused by increasing the production of ROS.3.The expression level of PARP1 in oral squamous cell carcinoma is higher than that in normal tissues.The higher the expression level of PARP1,the worse the prognosis of patients.There are two sides to the role of PARP1 in oral squamous cell carcinoma. |
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