Effect And Mechanism Of HMGB1in Drug Resistance Of Chemotherapy In Osteosarcoma

Osteosarcoma is the most common malignant cancer in children and adolescents. Unfortunately, treatment failure often occured due to chemoresistence, with which molecular mechanism is still not clearly understood. HMGB1is a DNA binding protein which expressed in several cells and involves in the process of replication, recombination and transcription of DNA.The secreted form of HMGB1also exerts immunoregulatory effects in the development of drug resistance in osteosarcoma.Anti-cancer agents including doxorubicin, cisplatin, and methotrexate each induced HMGB1upregulation in human osteosarcoma cells and RNAi-mediated knockdown of HMGB1restored the chemosensitivity of osteosarcoma cells in vitro and in vivo. Mechanistic investigation revealed that overexpression of HMGB1increased conversion of LC3-â…  to LC3-â…¡ which is wildly chosen for detection of autophagy. To explore whether antophagy mediates the effects of HMGB1-mediated resistance to the apoptotic response after treatment with anticancer agents, we knocked down PI3KC3, Beclin1and Atg7, which are critical autophagic regulators in mammalian cells. Silencing of these genes in MG-63cells inhibited HMGB1overexpression-induced LC3-â…¡ formation and consequently decreased autophagy and increased apoptosis. By Western-blotting and immunoflurescence, we figured out that Knockdown of HMGB1inhibited accumulation of LC3puncta in osteosarcoma cells by LC3antibody or mRFP-GFP-LC3construct. Rapamycin induces autophagy by inhibiting the protein mammalian target of rapamycin (mTOR). However, rapamycin conferred less protection in HMGB1knockdown cells due to diminished autophagic capacity. These findings suggest that HMGB1is an important regulator of autophagy-mediated cell survival.These findings supported the critical role of HMGB1in autophagic process of osteosarcoma cells. To investigate the potential mechism of HMGB1in autophagy, we analysed expression levels of ULK1,mAtg13, FIP200, Beclin1and PI3KC3in osteosarcoma cells. Knockdown of HMGB1does not affect formation of ULK1-mAtgl3-FIP200complex following doxorubicin treatment. However, HMGB1knockdown does influence the formation of the Beclin1-PI3KC3complex, which mediates vesicle nucleation in autophagy. HMGB1formed a complex with Beclinl which was detected by co-immunoprecipitation in osteosarcoma cells. Moreover, knockdown of ULK1or FIP200inhibited the interaction between HMGB1and Beclin1, and increased sensitivity to anti-cancer agent-induced cell apoptosis. These studies suggest that HMGB1is a downstream signal from ULK1-mAtgl3-FIP200complex formation and facilitates autophagy in osteosarcoma cells by interacting with Beclin1. Inhibition of this pathway in autophagy will increase drug sensitivity.In vivo, we inoculated NOD/SCID mice with MG-63tumor cells that had previously been transfected with HMGB1specific shRNA. Beginning at day7, mice were treated with doxorubicin. The growth of HMGB1knockdown tumor cells was significantly inhibited compared to control shRNA transfected tumors after treatment.Apoptosis and autophagy in tumor samples were assayed by TUNEL or LC3stain respectively. We observed that tumor cells transfected with HMGB1specific shRNA demonstrated decreased autophagy and increased apoptosis in vivo in response to therapy compared to control shRNA transfected tumor cells. Together, these results demonstrate that HMGB1is important in modulating drug resistance in osteosarcoma cells in vivo.Therefore, HMGB1is a critical factor in the development of chemoresistance in osteosarcoma cell and offers a novel potential target.