Combined Use Of β2-AR Blocker And COX-2 Inhibitor To Suppress Development Of Oral Squamous Cell Carcinoma

Purpose:To explore the synergistic inhibitory effect of cyclooxygenase-2(COX-2)inhibitor etodolac andβ2-adrenergic receptor(β2-AR)blocker ICI118,551 on invasion and metastasis of oral squamous cell carcinoma(OSCC)in vitro and in vivo.Methods:Investigated the expression of COX-2 in head and neck squamous cell carcinoma(HNSCC)in TCGA database,and 30 cases of OSCC tissues and 15cases of normal oral mucosal epithelial tissues were collected to detect the expression of COX-2 by immunohistochemistry(IHC).Two kinds of OSCC cell lines(Cal27 and SCC9)and the primary OSCC cell that isolated in the previous studies were selected,and the effect of different concentrations of etodolac(0μmol/L,0.1μmol/L,0.5μmol/L,1μmol/L,2.5μmol/L,5μmol/L,7.5μmol/L,10μmol/L)on the migration and invasion of OSCC cells were detected by wound healing assay and transwell invasion assay.The migration and invasion ability of three OSCC cells treated with etodolac(5μmol/L)or ICI118,551(5μmol/L)alone and the combination of two drugs were detected at the same time.In the previous experiment,we carried out high-throughput sequencing to detect the expression changes of genes in primary OSCC cell after treated with ICI118,551,the result showed some genes related to invasion and metastasis were down-regulated,including EGFR,TGF-β1,IL-1β.RT-qPCR was carried to detect the mRNA expression of EGFR,TGF-β1,IL-1βand VEGF-A,MMP2,related to progression of OSCC,in three OSCC cells after treated with the above four methods.ELISA was carried to detect the concentration changes of IL-1β,and Western blot was used to detect the protein expression levels of EGFR,TGF-β1,VEGF-A and MMP2.The orthotopic transplantation tumor model of oral squamous cell carcinoma was established by Cal27 cell line in female Balb/c nude mice(n=24).50μL cell suspension was injected into the tongue of each Balb/c nude mouse,and the cell density was 1×10~7 cells/m L.Balb/c nude mice were randomly divided into control group,ICI118551 treatment group,etodolac treatment group and combined treatment group.6 mice in each group and oral drugs was administered throughout the treatment period.The differences of survival time,tumor size and submandibular lymph node metastasis among the four groups were compared.When the mice died,tongue tumor and submandibular lymph node samples were collected.Part of the tumor tissue and submandibular lymph node tissue were used to determine whether the tongue tumor tissue was squamous cell carcinoma by hematoxylin-eosin staining(H&E staining),and whether there was submandibular lymph node metastasis.The expression changes of EGFR,TGF-β1,IL-1β,VEGFA and MMP2 in tongue tumors were detected by IHC.Western blot was carried to detect the protein expression levels of EGFR,TGF-β1,VEGF-A and MMP2.The serums of mice in four groups were collected and the concentrations of IL-1βwas detected by ELISA assay.Results:TCGA database showed that the expression of COX-2 in HNSCC was higher than normal tissues.The results of IHC showed that the expression of COX-2 in OSCC tissues was significantly higher than that in normal oral mucosal epithelium.Combined with the previous studies,the expression ofβ2-AR and COX-2 in OSCC tissues were both higher than that in normal oral mucosal epithelium.The results of wound healing assay and transwell invasion assay showed that the migration and invasion abilities of three OSCC cells decreased with the increase concentration of etodolac.At the same time,compared with the combination group,the results showed that ICI118,551 or etodolac alone could inhibit the migration and invasion of OSCC cells,but the inhibitory effect was stronger when the two drugs were used in combination.The results of RT-qPCR showed that EGFR,TGF-β1,IL-1βand MMP2 were significantly down-regulated in Cal27,SCC9 cell line and primary oral squamous cell carcinoma cells after combined treatment,and the down-regulation degree was significantly higher than that of ICI118,551 or etodolac alone.However,VEGF-A was significantly down-regulated only in primary OSCC cells,but not in the other two OSCC lines.Western blot results showed that EGFR,TGF-β1 and MMP2 were significantly down-regulated in three OSCC cells treated with two drugs,which were consistent with the results of RT-qPCR.The results of ELISA showed the concentration of IL-1βin combined treatment was also significantly decreased.In vivo assay,orthotopic transplantation tumor models were successfully established in all groups.The results showed that ICI118,551 or etodolac alone could inhibit the sizes of tumors,while the combination of the two drugs could more significantly inhibit the growth of tumors.The survival time of each group was calculated from the day when the cell suspension was injected into the tongue of Balb/c nude mice.The combined treatment group could significantly prolong the survival time of mice compared with the other three groups.The submandibular lymph node metastasis was not found in the combination group.The results of IHC showed that the above genes were significantly down-regulated,which were consistent with the results of Western blot and ELISA assay.Conclusion:The combined treatment ofβ2-AR blocker ICI118,551 and COX-2inhibitor etodolac significantly inhibited the progression of OSCC and prolonged the survival time of Balb/c nude mice with orthotopic implanted tumor.Meanwhile,EGFR,TGF-β1,IL-1β,MMP2 and VEGF-A,which are related to invasion,metastasis and progression of OSCC,were down-regulated by the combined treatment,the expression change of EGFR was the most significant.The combination of the two drugs is expected to be a potential adjuvant therapy for OSCC.