Anti-osteosarcoma Effect And Correlated Mechanisms Of Bone-like Selenium-doped Hydroxyapatite Nanoparticles

Objective: To investigate the inhibitory effects of biomimetic bone-like selenium-doped hydroxyapatite nanoparticles(BBL-Se-HAN)on osteosarcoma cells,explore the potential mechanism of anti-proliferative effect by Se HAN-induced autophagy and apoptosis,and confirm that BBL-Se-HAN have better anti-tumor functions than common morphology materials.Methods:(1)We prepared three kind of selenium-doped hydroxyapatite nanoparticles with different morphologies and used CCK8 assay to assess their inhibitory effects on the cell viability of MNNG/HOS and MG63 cells.(2)Detection of apoptosis: Hoechst 33342 staining and Annexin-V/PI double-staining were carried out to observe the apoptosis,further the levels of apoptotic-related proteins such as cleaved caspase-8,-9,-3 and PARP proteins were measured via western blot.(3)Detection of autophagy: a tandem fluorescent-tagged LC3(m RFP-GFP-LC3)and transmission electron microscopy were used to assess autophagy,in addition,autophagy-related proteins LC3 B,Beclin-1 and P62 were measure via western blot.(4)Cross-talk between autophagy and apoptosis: We performed CCK-8 assay to monitor the cell viability when a specific autophagy inhibitor 3-methyladenine(3-MA)or caspase inhibitor z-VAD alone,or 3-MA and z-VAD in combination were applied.(5)Signal pathway research: Western Blot was used to detect the expression of AKT,p-AKT,m TOR,p-m TOR,JNK,p-JNK protein of osteosarcoma cells after intervention with different morphology Se-HANs;we used AKT / m TOR pathway inhibitor MK-2206 and rapamycin,ROS scavenger Mn TMPy P,and JNK pathway inhibitor SP600125,than used Western Blot to detect the expression of LC3 B,cleaved caspase3 protein,and p-AKT,p-m TOR,and p-JNK protein in tumor cells after treatment of BBL-Se-HAN.;(6)Influences of migration and invasion: wound healing assay and transwell assay were performed to evaluate the influences of migration and invasion abilities to MNNG/HOS cells caused by different Se-HANs.(7)We established an orthotopic osteosarcoma tibial model,then recorded tumor volume and body weight of mice.Additionally,X-ray,micro-CT graphics,Hematoxylin-Eosin(HE)staining,Masson staining and immunohistochemical(IHC)staining of nude mice tibia were used to investigate the antineoplastic efficacy of different Se-HANs.Results:(1)Se-HANs induced apoptosis and autophagy and inhibited the proliferation of osteosarcoma MNNG/HOS and MG63 cells.In addition,BBL-Se-HAN exhibited a better efficacy to inhibit osteosarcoma cells proliferation and promote apoptosis and autophagy than the other two types of non-biomimetic Se HANs.(2)The presence of 3-MA or z-VAD did not increase the tumor cell viability much compared with that without inhibitors.However,cell death was significantly inhibited when 3MA and z-VAD were used in combination.The results verified that the autophagy induced by BBL-Se-HAN in MNNG/HOS cells contributed to the suppression of cells viability,alone with apoptosis.(3)BBL-Se-HAN promoted autophagy and apoptosis of osteosarcoma MNNG/HOS cells via inhibition of AKT/m TOR pathway and promotion of JNK pathway mediated by excessive ROS.(4)The migration and invasion abilities of MNNH/HOS cells were inhibited by Se-HANs,among which BBL-Se-HAN had the most remarkable inhibition capacity.In addition,Se-HANs inhibitted the migration and invasion abilities of MNNH/HOS cells by reducing the expression of MMP9 and MMP3 proteins.(5)Se-HANs especially BBL-Se-HAN are capable of suppressing tumor growth and protect bone structure in vivo.