CXCR4 Blockade Enhances The Sensitivity Of Osteosarcoma To Doxorubicin By Inducing Autophagic Cell Death Via PI3K-Akt-Mtor Pathway Inhibition

Background: Doxorubicin is one of the most frequent used chemotherapy drugs in the treatment of osteosarcoma (OS), but the emergence of chemoresistance often leads to its treatment failure. CXCR4 has been proved to regulate osteosarcoma progression and metastasis, however whether CXCR4 is also implicated in OS chemoresistance and its molecular mechanisms have not yet been fully elucidated. In this study, we investigated CXCR4-mediated autophagy for OS chemotherapy. Methods: : Cell viability, western blot and apoptosis were performed to evaluate doxorubicin resistance after CXCR4 regulation (downregulation or upregulation) and treatment with autophagy modulators (bafilomycin A1 or rapamycin) in two OS cell lines (LM8 and Dunn). Autophagy-related proteins (Beclin1 and LC3B) detected by western blot, autophagosomes and autolysosomes observed by transmission electron microscopy, and autophagic flux monitored by confocal microscopy were utilized to comprehensively assess autophagy level changes. A C3H mouse osteosarcoma orthotopic model was established to determine anti-tumor effect of CXCR4 antagonist AMD3100 and doxorubicin in vivo. Results: : CXCR4 inhibition enhanced doxorubicin-induced apoptosis by reducing p-glycoprotein, while CXCR4 overexpression promoted OS doxorubicin resistance. Moreover, CXCR4 inhibition combined with or without doxorubicin increased Beclin1 and LC3B expression, numbers of autophagosome and autolysosome, and induced autophagic flux activation by suppressing PI3K-Akt-mTOR pathway. In addition, pretreatment with autophagy inhibitor bafilomycin A1 attenuated CXCR4 abrogation-induced cell death. Finally, CXCR4 antagonist AMD3100 synergistically reinforced anti-tumor effect of doxorubicin in orthotopic OS mouse model. Conclusions: : This study revealed CXCR4 inhibition sensitizes OS to doxorubicin by inducing autophagic cell death. Targeting CXCR4/autophagy axis may be a promising therapeutic strategy to overcome OS chemotherapy resistance.