Magnesium promotes osteogenesis via increasing OPN expression and activating CaM/CaMKIV/CREB1 pathway.

Magnesium (Mg) based alloy has been used as a biodegradable implant for fracture repair with considerable efficacy, and it has been proved that magnesium ion (Mg2+ ), one of the degradation products, could stimulate osteogenesis. Here, we investigated the osteogenesis property of magnesium both in vitro and in vivo, and to identify the cellular and molecular mechanisms that mediate these effects. Results showed that magnesium exerts a dose-dependent increase in the proliferation of MC3T3 and MG63 cells, and in the expression of osteopontin (OPN), a promising biomarker of osteogenesis. Subsequently, the protein-protein interaction (PPI) network analysis showed the interactions between calmodulin (CaM) and calmodulin-dependent protein kinase (CaMK) and CREB1. The ratio of p-CaMKIV/CaMKIV and p-CREB1/CREB were increased at protein level in MC3T3 and MG63 cells after treatment with Mg2+ . Dual-luciferase reporter gene assay showed that p-CREB1 could directly bind to OPN promoter and up-regulate the transcription of OPN after nuclear entry. Meanwhile, the expression of OPN and p-CREB1, which increased after Mg2+ treatment, was down-regulated by sh-CaMKIV or sh-CREB1. Moreover, the mineralized deposit and expression of OPN were reduced after treatment with an inhibitor of CaMKIV, KN93. In addition, massive cavities in the cortical bone around the Mg screw were showed in vivo after injection of KN93. These data indicated that the osteogenic effect of Mg is related to the activation OPN through CaM/CaMKIV/CREB1 signaling pathway.

Synergistic antitumor efficacy of PD-1-conjugated PTX- and ZSQ-loaded nanoliposomes against multidrug-resistant liver cancers.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor chemotherapeutic efficiency due to multidrug resistance (MDR); it is very important to develop a targeted nanocarrier for the treatment of HCC. In this study, a programmed death ligand 1 (PD-L1)-conjugated nanoliposome was constructed for co-delivery of paclitaxel (PTX) and P-glycoprotein (P-gp) inhibitor zosuquidar (ZSQ) to overcome MDR in human HCC cells and tumors in vivo. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to examine the nanoparticles morphology and size; PD-1-conjugated PTX and ZSQ-loaded nanoliposomes (PD-PZLP) revealed a spherical shape with a size of 139.5 ± 10.7 nm. Then, the physicochemical properties, as well as the drug loading capacity, release profile, cellular uptake, and cytotoxicity of the dual drug-encapsulated nanoliposomes were characterized. PD-PZLP displayed a high drug loading capacity of 20 ~ 30% for both PTX and ZSQ; the drug release of PTX and ZSQ in pH 5.0 was significantly faster than in pH 7.4. Cellular uptake study demonstrated PD-PZLP had higher internalization efficiency than non-targeted PZLP. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and reactive oxygen species (ROS) analysis demonstrated that PD-PZLP triggered an excessive ROS reaction and cell apoptosis compared with that of free PTX or ZSQ, which was also consistent with the cell antiproliferative effects in MTT assay. Furthermore, PD-PZLP could enhance synergistic antitumor effects on 7721/ADM xenograft tumor model, which also significantly alleviated hepatotoxicity as evident from the decreased aspartate transaminase (AST) and alanine transaminase (ALT) levels. Overall, PD-PZLP exhibited high loading capacity, significant synergistic effects, promising antitumor efficacy, and the lowest toxicity, which provide a promising strategy to overcome MDR in HCC.