| Background:Osteosarcoma（OS）is one of the most common primary bone malignancies in children and adolescents,mainly seen in the metaphysis of long tubular bones.Although the standard treatments for OS therapy,such as surgery,chemotherapy,and radiotherapy,have significantly reduced patient mortality in recent years,these treatment strategies failed to prolong the overall survival rate of the affected patients.Therefore,it is required to find innovative therapeutic modalities to not only significantly improve their performance efficacy against OS but also the quality of patient’s life.Levobupivacaine is one of the commonly used local anesthetics in clinical practice,which has been proven to affect the progression of numerous malignant tumors,such as gastric and breast cancers.However,the functional roles and relevant mechanistic views of levobupivacaine in the progression of OS have yet remained to be explored.Therefore,this study intends to explore the in-depth effects of levobupivacaine on the progression of OS and lay a theoretical foundation for clarifying the occurrence and development of OS toward finding new therapeutic targets.Objective:This study is aimed to observe the effect of levobupivacaine on the biological behaviors of cells in OS,such as proliferation,migration,invasion,and apoptosis.In addition,we explore the regulation of levobupivacaine on OS-specific molecular mechanisms relevant to the malignant behavior of cells.Methods:Part I: To investigate the effect of levobupivacaine on the malignant behavior of OS cells,MG63 and U2 OS cells were initially treated with 0 or 2 m M of levobupivacaine and then subjected to Ed U assay and plate clone formation assay.Further,the changes in the proliferation ability of U2 OS cells and the apoptosis ability of MG63 and U2 OS cells were detected by flow cytometry.Moreover,the altered migration and invasion abilities of MG63 and U2 OS cell lines were detected by scratch healing and Transwell assays,respectively.In addition,the changes in the MG63 and U2 OS cell lines towards the formation of tumorigenic spheroids were detected.In this context,the effect of levobupivacaine on the stemness of MG63 and U2 OS cells was preliminarily detected.The effect of levobupivacaine on the expression levels of stemness marker proteins in the MG63 and U2 OS cell lines was further detected by Western blot analysis.Finally,to explore the in vivo investigations,MG63 and U2 OS cells were applied to build a nude mouse osteosarcoma tumor-bearing model.Further,the interventions of levobupivacaine were observed in terms of tumor growth in nude mice to confirm the effects of levobupivacaine in nude mice.Part II: To explore the specific molecular mechanisms by which levobupivacaine affects the malignant biological behavior of OS（MG63 and U2OS）cells were initially treated with 0 or 2 m M of levobupivacaine and detected the changes in the expression of MAFB by q RT-PCR experiments.To determine the mechanistic views at the molecular level of Levobupivacaine affecting the progress of the OS disease,the regulatory relationship between KAT5 and MAFB by Ch IP assay was initially detected,and further observed the effect of KAT5 knockdown and KAT5 overexpression on MAFB expression levels in MG63 and U2 OS cells by q RT-PCR analysis.Then,the expression of KAT5 was knocked down in MG63 and U2 OS cells by RNA interference technology.The Ed U assay was utilized to detect the effect of knockdown of KAT5 on the proliferation ability of MG63 and U2 OS cells.In addition,the flow cytometry analysis was used to determine the knockdown effect of KAT5 on the apoptosis rates of MG63 and U2 OS cell lines.Moreover,the effect of KAT5 knockdown on the stemness of MG63 and U2 OS cells was detected by tumor spheroid formation assay.Finally,KAT5 was overexpressed while intervening in the MG63 and U2 OS cells with levobupivacaine to confirm the role of levobupivacaine in OS disease progression through the KAT5/MAFB axis.Further,the changes in the MAFB expression by q RT-PCR,the altered proliferation using the Ed U assay,apoptosis levels by flow cytometry,stemness using the spheroid formation ability,and Western blot assays of MG63 and U2 OS cells were performed.Further,the intervention of Levobupivacaine and its effects on the proliferation,apoptosis levels,and stemness changes of MG63 and U2 OS cells were detected using various assays systematically.Results:Part I:1.After levobupivacaine treatment,the Edu staining and plate colony formation results showed that,the positive staining rate and the ability of clone colony formation of MG63 and U2 OS cells were significantly reduced,suggesting that levobupivacaine could significantly inhibit the proliferation efficacy of OS cells.2.It was observed from the scratch healing assay that the migration abilities of MG63 and U2OS cells were significantly reduced after levobupivacaine treatment.Further,the Transwell experiments showed that the number of MG63 and U2 OS cells passing through the chamber significantly reduced after levobupivacaine treatment.These results indicated that levobupivacaine could significantly inhibit the migration and invasion abilities of the selected OS cell lines.3.The flow cytometry results presented that the early and late apoptosis levels of MG63 and U2 OS cells were significantly increased after levobupivacaine treatment of cells,indicating that levobupivacaine could significantly stimulate the apoptosis of OS cells.4.The tumor spheroid formation experiments presented that the levobupivacaine treatment could significantly inhibition the spheroid formation in terms of the number and the volume of spheroids of MG63 and U2 OS cells.Further,the Western blot analysis displayed that levobupivacaine could significantly inhibit the cell stemness marker proteins Sox-2,Oct3/4,and Nanog.These results suggested that levobupivacaine may have a precise inhibitory effect on the stemness of OS cells.5.Further,the in vivo investigations indicated that levobupivacaine could significantly inhibit the tumor volume,weight and lung metastasis of MG63cells-based tumor-bearing nude mice,suggesting the inhibition of the OS cell growth and metastasis of levobupivacaine in nude mice.Part II:1.RT-qPCR recordings presented that levobupivacaine could significantly inhibit the expression level of MAFB in MG63 and U2 OS cell lines.2.The ChIP experiments showed that KAT5 was enriched with the MAFB promoter.However,the knockdown of KAT5 in MG63 and U2 OS cells resulted in the significant downregulation of the enrichment of MAFB,along with the H3K27 ac and RNA polymerase II in the MAFB promoter region.3.The EdU staining presented that the knockdown of KAT5 could significantly inhibit the proliferation of the OS cells,i.e.,MG63 and U2 OS cell lines.Further confirmations by the flow cytometry results indicated that knockdown of KAT5 could significantly promote the apoptosis of MG63 and U2 OS cells.4.Tumor spheroid formation experiments showed that the KAT5 knockdown could considerably inhibit the spheroid formation ability of both the OS cell lines.A Western blot was performed to explore the protein expression levels to verify this aspect.The Western blot experiments indicated that the KAT5 knockdown significantly inhibited the expression of cell stemness marker proteins（Sox-2,Oct3/4,and Nanog）.5.The RT-qPCR and Western blot results showed that levobupivacaine could significantly inhibit the expression level of MAFB in MG63 and U2 OS cells.6.After simultaneous levobupivacaine treatment and overexpression of KAT5,the Ch IP assay results showed that the KAT5 enrichment level in the MAFB promoter region was significantly decreased.Further,the RT-q PCR and Western blot results showed that the expression of MAFB was increased significantly in MG63 and U2 OS cells.However,the expression level of MAFB was significantly higher than that of the levobupivacaine-treated group.These results suggested that levobupivacaine down-regulated the MAFB expression in MG63 and U2 OS cells by inhibiting the KAT5 expression.7.The levobupivacaine treatment combined with KAT5 overexpression synergistically promoted the proliferation ability,spheroid formation ability,and the expression of stemness marker proteins of MG63 and U2 OS cells.In contrast,the combinatorial treatment inhibited the apoptosis ability of MG63 and U2 OS cells.8.The levobupivacaine treatment combined with MAFB overexpression also significantly promoted the proliferation ability,spheroid formation ability,and the expression of cell stemness marker proteins of MG63 and U2 OS cells.Contrarily,the combinatorial treatment substantially inhibited the apoptosis ability of MG63 and U2 OS cells.9.The Levobupivacaine can reduce the expression level of KAT5/MAFB axis in mouse tumor tissue.Conclusion:1.In summary,the levobupivacaine treatment significantly inhibited the malignant behaviors of OS cells,such as proliferation,clone formation,migration,invasion,cell stemness,tumorigenesis and invasion ability in vivo.2.Moreover,the levobupivacaine antagonized the binding ability of KAT5 and MAFB by inhibiting the KAT5 expression,thereby reducing the expression level of MAFB and finally inhibiting the disease progression of OS. |
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