| Background: The cure rates for osteosarcoma remain unchanged in the past three decades,especially for patients with pulmonary metastasis.Thus,a novel and effective treatment is urgently needed for metastatic osteosarcoma.Anti-angiogenic targeted drugs have been utilized in the treatment of a variety of tumors including lung cancer,soft tissues sarcomas,and thyroid carcinoma.As a multi-target receptor tyrosine kinase inhibitor,anlotinib has been reported to have anti-tumor effects on advanced osteosarcoma,the refore,improving the efficacy and duration of anlotinib for osteosarcoma could potentially improve the prognosis of patients with osteosarcoma.Autophagy plays an indispensable role in the progression and metastasis of cancer.However,either the effect o f anlotinib on autophagy in osteosarcoma or the mechanism of anlotinib-mediated autophagy in pulmonary metastasis of osteosarcoma are unclear.Thus,how to improve the therapeutic effect of anlotinib on osteosarcoma can be investigated from this aspect.Thi s research aims to investigate the impact of anlotinib on the migration,invasion and autophagy in osteosarcoma cells,and further explore the underlying effect and molecular mechanism of anlotinibmediated autophagy in pulmonary metastasis of osteosarcoma.Methods: CCK-8 assay was used to detect the effect of anlotinib on cell proliferation and determine the proper concentration of anlotinib for further experiment.Transwell,wound healing and phalloidin staining were used to observe the effect of anlotinib on osteosarcoma cell metastasis,and Western blotting was used to detect the expression of EMT-related proteins and cytoskeleton-related proteins.Immunofluorescence,tran smission electron microscopy and Western blotting were used to detect the expression of autophagosomes and autophagy-related proteins in osteosarcoma cells.ATG5-silenced Osteosarcoma cells were constructed by short hairpin RNA(sh RNA)lentivirus transfect ion,and ATG5-overexpressed osteosarcoma cells were constructed by plasmid containing ATG5 c DNA.ATG5 knockout or ATG5 overexpressed osteosarcoma cells were treated with anlotinib and cytoskeletal inhibitor Y-27632,differences of expression level between related proteins were detected by Western blotting,and the changes in autophagy,invasion and migration of osteosarcoma cells were compared by transwell and immunofluorescence.A mouse model of lung metastasis of osteosarcoma was established,and the diff erences of lung metastases and the expression of related proteins were compared by HE staining and IHC.Result: After treatment with anlotinib,the number of invading and migrating 143 B and KHOS cells was significantly reduced,the expression of EMT-related proteins including N-cadherin,vimentin,MMP-9 and Slug was decreased,the expression of E-cadherin was increased,suggesting anlotinib could inhibit the metastasis of osteosarcoma cells by suppressing the epithelial-mesenchymal transition(EMT).However,anlotinib promoted the formation of autophagosomes and promoted the LC3 B and autophagic proteins such as ATG5.After knocking down ATG5,the autophagy level of osteosarcoma cells decreased,the activity of Rho A and the expression of p-LIMK2 and p-cofilin in osteosarcoma cells decreased significantly,which inhibited the rearrangement of cytoskeleton and enhanced the inhibitory effect of amlotinib on the invasion and migration of osteosarcoma cells.In addition,Y-27632 inhibited cytoskeletal rearrangement by induction of a decrease in p-LIMK2 and p-cofilin expression,while overexpression of ATG5 reversed the inhibition of cytoskeletal rearrangement by promoting autophagy and activating Rho A,and also promoted the epithelial-mesenchymal transition in osteosarcoma cells.In animal experiments,the number of metastatic nodules in the lung in the sh ATG5 experimental group decreased significantly compared to the control group.Immunohistochemistry showed that the expression of E-cadherin was increased and the expression of N-cadherin,ATG5,and p-LIMK2 was decreased in metastatic tissues in the sh ATG5 group.These results indicate that by targeting ATG5,anlotinib-induced autopha gy promotes migration and invasion of osteosarcoma via the activaton of EMT and cytoskeletal rearrangement both in vitro and in vivo.Conclusions: Our results demonstrated anlotinib could induce a protective autophagy in osteosarcoma cells,and inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis.Thus,the therapeutic effect of anlotinib treatment can be improved by the combination of autophagy inhibitors,which provided a new direction for the treatment of metastatic osteosarcoma. |
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