Injectable agarose hydrogels and doxorubicin-encapsulated iron-gallic acid nanoparticles for chemodynamic-photothermal synergistic therapy against osteosarcoma.

Osteosarcoma is a malignant bone cancer that usually occurs in children and adolescents. Although chemotherapy, radiotherapy and other methods have been used to treat osteosarcoma, these therapeutic regimens fail to cure this disease completely. Herein, doxorubicin-encapsulated iron-gallic acid (FeGA-DOX) nanoparticles (NPs) were fused with agarose hydrogels (AG) for synergistic therapy of osteosarcoma. Under near-infrared laser irradiation, the local temperature of FeGA-DOX NPs was increased. Therefore, tumour cells were killed using photothermal therapy, and AG dissolved to release FeGA-DOX into the cells. Doxorubicin generates hydrogen peroxide, which is then converted to reactive oxygen species (ROS) via FeGA-DOX by the Fenton reaction, inducing tumour cell apoptosis. ROS induced by chemodynamic therapy compensates for the incomplete cure of osteosarcoma cells. The AG-encapsulated NPs could mediate synergistic chemodynamic and photothermal therapy with self-sufficient H2O2, providing a novel therapeutic strategy for osteosarcoma.

The anti-osteosarcoma property of ailanthone through regulation of miR-126/VEGF-A axis.

Background: Despite the characters of the resistance of tumour of ailanthone (AIL) were found in various tumour cells, its effect on osteosarcoma is still unclear. Herein, we attempted to see the effects of AIL on an osteosarcoma cell line MG63. Methods: MG63 cells were treated by AIL, following which CCK-8 assay, BrdU assay, Transwell assay and Western blot were utilized to detect cell proliferation, migration, invasion and apoptosis. miR-126 expression in osteosarcoma tissues and cell lines was measured by qRT-PCR. Further, the target of miR-126 and the downstream signalling for AIL were studied. Results: Treating MG63 cells with 1.5 µM AIL for 24 h significantly suppressed proliferation, migration, invasion and induced apoptosis. Meanwhile, AIL inhibited PI3K/AKT pathway and up-regulated miR-126 expression. miR-126 of osteosarcoma tissues and cell lines was low expressed, as relative to paracancerous tissues and normal osteoblast. The anti-tumour effects of AIL were attenuated by miR-126 silencing. Further, VEGF-A was a target of miR-126. Conclusions: This study demonstrated that AIL was effective in inhibiting MG63 cells growth, migration and invasion. The anti-tumour properties may be via up-regulating miR-126 and thereby degradation of VEGF-A.

PCAF regulates H3 phosphorylation and promotes autophagy in osteosarcoma cells.

BACKGROUND: Osteosarcoma is one of malignant cancer. Histone phosphorylation is common in tumors. We explored the effects of p300-CBP-associated factor (PCAF) and phosphorylation of H3S28 in osteosarcoma cancer cell autophagy. METHODS: Osteosarcoma cancer cell lines were collected and/or transfected with full length PCAF or interference miRNAs to mimic or silence of PCAF expression. Immunoprecipitation assay and GST pull down was used to target targeting PCAF or H3S28ph. H3-/- SNU-C1 cells were transfected with H3WT- or H3S28F-expressing or enhanced green fluorescent protein (EGFP)-tagged LC3 plasmids, in which H3 was tagged with HA. An in vitro kinase activity assay was performed to test whether recombinant full-length PCAF could phosphorylate H3 in the site of S28. The functions on autophagy was detected by number of autophagosomes, number of EGFP-LC3, LC3-II/I, percentage of degradation and expression of autophagy associated gene (ATG). RESULTS: PCAF positively regulated H3S28ph in osteosarcoma cancer cells; Immunoprecipitation assay and GST pull down demonstrated that PCAF could interact directly with H3 in osteosarcoma cancer cells. In addition, silence of PCAF inhibited the number of autophagosomes, number of EGFP-LC3, LC3-II/I, percentage of degradation and expression of ATG. Moreover, H3S28A (H3S28 mutation) impaired the promoting autophagy effects of PCAF. The PCAF-H3S28ph axis promoted osteosarcoma cancer autophagy viatranscriptional regulation of ATG genes. CONCLUSION: PCAF regulated H3S28 phosphorylation and their axis promotes autophagy in osteosarcoma cancer cells viatargeting ATG5 and ATG7.

Sirt1 modulates H3 phosphorylation and facilitates osteosarcoma cell autophagy.

Abnormal histone modifications have been recognized as an important contributing factor to the initiation and progression of osteosarcoma. Sirtuin 1 (Sirt1) up-regulation has been discovered in osteosarcoma cells. This study tested the influence of Sirt1 on histone H3 phosphorylation at threonine 3 (H3T3ph) in osteosarcoma cells, along with Sirt1-H3T3ph axis on osteosarcoma cell autophagy. Plasmids or si-RNAs transfection was carried out to alter Sirt1 or H3T3ph expressions. Co-immunoprecipitation analysis and GST pull-down assay were done to probe the relationship between Sirt1 and H3T3ph. Phosphoryltransferase activity of Sirt1 was tested by in vitro kinase activity assay. Cell autophagy was measured by a number of autophagosome, conversion of LC3-I to LC3-II, degradation of long-lived protein and ATG protein expressions. We found that Sirt1 directly interacted with H3 and phosphorylate H3T3 at threonine 3 in osteosarcoma cells. Moreover, Sirt1 facilitated osteosarcoma cell autophagy under starvation condition. H3T3ph took part in the Sirt1-facilitated osteosarcoma cell autophagy under starvation condition. Besides, Sirt1-H3T3ph axis facilitated osteosarcoma cell autophagy might be achieved through transcriptional activation of ATG genes. Sirt1 promoted osteosarcoma initiation and progression might be via phosphorylate H3T3 and then facilitate osteosarcoma cell autophagy through activating ATG genes transcription under starvation condition.

Green tea polyphenols attenuate LPS-induced inflammation through upregulating microRNA-9 in murine chondrogenic ATDC5 cells.

BACKGROUND: Osteoarthritis (OA), a universal chronic musculoskeletal disorder, is closely related to inflammation. More effective drugs for improving OA outcome are definitely needed. Herein, we attempted to verify the protective role of green tea polyphenols (GTP) after treatment with murine in ATDC5 cells to reveal the regulatory mechanism. METHODS: ATDC5 cells were stimulated with lipopolysaccharide (LPS) to mimic an inflammatory response during OA. Cell activity, apoptosis, levels of relative proteins, and prophlogistic factors were tested via a Cell Counting Kit-8 experiment, a flow cytometry experiment, western blot, and RT-qPCR (ELISA and Western blot), separately. miR-9 level was detected by RT-qPCR and altered via miR-9 mimic and inhibitor transfection. We finally studied MAPK and NF-κB pathways in GTP-related modulations using western blot. RESULTS: LPS caused inflammatory cell damage in ATDC5 cells, showing decreased cell activity, enhanced apoptosis, and increased levels of pro-inflammatory cytokines. GTP pretreatments could significantly attenuate LPS-induced alterations. In addition, LPS-induced miR-9 upregulation was further positively regulated in ATDC5 cells. The effects of GTP pretreatments in LPS-caused ATDC5 cells were enhanced via miR-9 upregulation, whereas they were reduced via miR-9 suppression. Finally, we found that GTP pretreatments could suppress the MAPK and NF-κB pathways through miR-9 regulation. CONCLUSION: GTP pretreatments attenuated LPS-induced inflammatory response accompanied by the suppression of the MAPK and NF-κB pathways via positively regulating miR-9 in ATDC5 cells.

Long non-coding RNA activated by transforming growth factor beta alleviates lipopolysaccharide-induced inflammatory injury via regulating microRNA-223 in ATDC5 cells.

Osteoarthritis (OA) is a conversant joint disease, which seriously threatens the health of the elderly, and even leads to disability. Long non-coding RNA-activated by transforming growth factor beta (lncRNA-ATB) has been reported in diverse cancers. However, the functions of lncRNA-ATB in OA remain uninvestigated. The current study aimed to explore the impacts of lncRNA-ATB on lipopolysaccharide (LPS)-induced inflammatory injury in ATDC5 cells and to uncover the underlying mechanism. LPS-induced ATDC5 cell injury model was constructed, and the effects of lncRNA-ATB on LPS-injured cells were explored via analyzing cell viability, apoptosis, iNOS, COX-2, and inflammatory cytokines (IL-6 and TNF-α). Subsequently, the relationship between lncRNA-ATB and microRNA (miR)-223 was detected, and whether miR-223 was involved in modulating LPS-induced cells injury in ATDC5 cells was investigated. Finally, MyD88/NF-κB and p38MAPK pathways were assessed to explore the underlying mechanism. Results showed that LPS repressed cell viability, induced apoptosis, and promoted iNOS, COX-2, IL-6 and TNF-α expression. Additionally, we observed that lncRNA-ATB expression was down-regulated in LPS-injured cells, and lncRNA-ATB overexpression significantly alleviated LPS-induced inflammatory injury in ATDC5 cells. Interesting results revealed that miR-223 expression was down-regulated by lncRNA-ATB and miR-223 overexpression declined the protective effect of lncRNA-ATB on LPS-injured ATDC5 cells. Further, the signaling pathway experiments showed that lncRNA-ATB inhibited MyD88/NF-κB and p38MAPK pathways by down-regulating miR-223 in LPS-injured cells. These data demonstrated that lncRNA-ATB protected ATDC5 cells against LPS-induced inflammatory injury by repressing MyD88/NF-κB and p38MAPK pathways, which was mediated by down-regulation of miR-223.

Procaine Inhibits Proliferation and Migration and Promotes Cell Apoptosis in Osteosarcoma Cells by Upregulation of MicroRNA-133b.

Procaine (PCA) is a conventional chemotherapeutic agent for osteosarcoma. Recent studies have proposed that the growth-inhibitory effect of PCA is through regulation of microRNAs (miRNAs). miR-133b has been proven to be a tumor suppressor in osteosarcoma, but whether it is involved in the antitumor effects of PCA on osteosarcoma has not been investigated. In this study, we aimed to explore the effects of PCA on osteosarcoma MG63 cells by regulation of miR-133b, as well as its underlying mechanisms. MG63 cells were treated with different concentrations of PCA, and cell viability, apoptosis, and miR-133b expression were then detected by MTT, flow cytometry, and qRT-PCR, respectively. Cells were then transfected with the miR-133b inhibitor and treated with 2 µM PCA. Thereafter, cell viability, migration, and apoptosis were detected. Analysis of signaling pathways was detected by Western blot. Our results showed that PCA significantly inhibited cell viability and promoted apoptosis and the expression level of miR-133b in a dose-dependent manner (p < 0.05 or p < 0.01). Moreover, we observed that PCA + miR-133b inhibitor dramatically reversed the effects of PCA on cell viability, apoptosis, and migration (p < 0.05 or p < 0.01). In addition, PCA significantly decreased the levels of p/t-AKT (p308 or p473), p/t-ERK, and p/t-S6, whereas PCA + miR-133b inhibitor rescued these effects. Our results suggest that PCA inhibits proliferation and migration but promotes apoptosis in osteosarcoma cells by upregulation of miR-133b. These effects may be achieved by inactivation of the AKT/ERK pathways.

Interleukin 1ß and tumor necrosis factor α promote hFOB1.19 cell viability via activating AP1.

Bone trauma healing is a complex physiological process, which may involve the function of various inflammatory cytokines. Our study aimed to explore the roles of inflammatory cytokines in bone trauma healing and reveal the potential mechanism. Concentrations of interleukin (IL)-6, IL-1ß and tumor necrosis factor alpha (TNF-α) in peripheral blood serum of bone trauma patients after surgery were determined by ELISA. The human osteoblast hFOB1.19 cell line was cultured to determine the effect of these cytokines in cell viability using MTT assay. In addition, luciferase reporter assay was performed to investigate the activator protein 1 (AP1) transcriptional activity, and small interfering RNA was transfected to inhibit FOS, a component of AP1 molecule. IL-6, IL-1ß and TNF-α exhibited higher level in patients with more severe bone traumas after surgery. IL-1ß and TNF-α, but not IL-6, induced a significant increase of hFOB1.19 viability after three days of treatment (P < 0.05). IL-1ß and TNF-α could activate AP1 transcriptional activity in hFOB1.19 cells (P < 0.001), but the activation was inhibited when cells were pretreated with inhibitor of JNKs, SP600125 (P < 0.001). Besides, the effect of IL-1ß and TNF-α on promoting viability was significantly inhibited after knockdown of FOS. These findings indicated that IL-1ß and TNF-α played an important role in promoting osteoblast viability via the activation of AP1 transcriptional activity, which was likely to involve the JNK/MAPK signaling pathway. Modulating inflammatory cytokines is a potential strategy for improving the outcome of bone trauma healing.

[Radiation exposure to spine surgeon: a comparison of computer-assisted navigation and conventional technique].

To analyze the radiation exposure of surgeon in spine surgery and compare computer-assisted navigation and conventinal technique. While performing spine surgery, the surgeon is exposed to a significant amount of radiation. Spinal surgeons should be considered as workers of radiational occupation accordingly. Methods of reducing radiation exposure should be strongly recommended. Comparing with conventional fluoroscopic technique,the computer-assisted navigtion can reduce surgical time, radiation exposure, and has become an increasingly accepted and practiced from of intraoperative spinal navigation.