PODN is a prognostic biomarker and correlated with immune infiltrates in osteosarcoma.

BACKGROUND: Osteosarcoma was the most common primary bone malignancy in children and adolescents. It was imperative to identify effective prognostic biomarkers for this cancer. This study was aimed to identify potential crucial genes of osteosarcoma by integrated bioinformatics analysis. METHODS: Identification of differentially expressed genes from public data gene expression profiles (GSE42352), functional and pathway enrichment analysis, protein-protein interaction (PPI) network construction and module analysis, Cox regression and survival analysis was conducted. RESULTS: Totally 17 co-differential genes were found to be differentially expressed. These genes were enriched in biological processes, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) pathway of inflammatory immune response. PPI network was constructed with 63 differentially expressed genes that co-existed between the test set and the validation set. The area under the receiver operating characteristic curve (AUC value) was 0.855, which indicated that the expression of PODN had a good diagnostic value for osteosarcoma. Furthermore, Cox regression and survival analysis revealed 5 genes were statistically significant. CONCLUSIONS: PODN was regarded as a potential biomarker for the diagnosis and prognosis of osteosarcoma, ACTA2, COL6A1, FAP, OLFML2B and COL6A3, can be used as potential prognostic indicators for osteosarcoma.

miR-143 is implicated in growth plate injury by targeting IHH in precartilaginous stem cells.

Precartilaginous stem cells (PCSCs) are able to initiate chondrocyte and bone development. The present study aimed to investigate the role of miR-143 and the underlying mechanisms involved in PCSC proliferation. In a rat growth plate injury model, tissue from the injury site was collected and the expression of miR-143 and its potential targets was determined. PCSCs were isolated from the rabbits' distal epiphyseal growth plate. Cell viability, DNA synthesis, and apoptosis were determined with MTT, BrdU, and flow cytometric analysis, respectively. Real time PCR and western blot were performed to detect the mRNA and protein expression of the indicated genes. Indian hedgehog (IHH) was identified as a target gene for miR-143 with luciferase reporter assay. Decreased expression of miR-143 and increased expression of IHH gene were observed in the growth plate after injury. miR-143 mimics decreased cell viability and DNA synthesis and promoted apoptosis of PCSCs. Conversely, siRNA-mediated inhibition of miR-143 led to increased growth and suppressed apoptosis of PCSCs. Transfection of miR-143 decreased luciferase activity of wild-type IHH but had no effect when the 3'-UTR of IHH was mutated. Furthermore, the effect of miR-143 overexpression was neutralized by overexpression of IHH. Our study showed that miR-143 is involved in growth plate behavior and regulates PCSC growth by targeting IHH, suggesting that miR-143 may serve as a novel target for PCSC-related diseases.

Treatment Choice of Complete Distal Forearm Fractures in 8 to 14 Years Old Children.

BACKGROUND: New surgical techniques have challenged traditional guidelines for nonsurgical treatment in pediatric and adolescent distal forearm fractures. This study was performed to compare outcomes and costs between closed reduction with percutaneous pinning (CRPP) and closed reduction with casting in the treatment of complete distal forearm fractures in children 8 to 14 years old. METHODS: A retrospective cohort study was performed of 175 displaced distal forearm fractures treated with 2 different methods in the emergency department of a children's trauma center. One hundred and fourteen children were managed using CRPP. The remaining 61 were treated with closed reduction and casting. All patients had initial follow-up radiographs. The quality of reduction and the residual angulation in both the coronal and sagittal planes were recorded. Outcomes included the angulation after reduction, residual angulation at final follow-up, radiation exposure, total immobilization time, days absent from school, total costs, and postoperative complications. RESULTS: The postreduction sagittal plane angulation was significantly lower in the CRPP group (P=0.037). While residual deformity between the groups at the 6-month final follow-up was not significantly different in either the sagittal or coronal planes (P=0.486, 0.726), patients in the nonoperative group received greater radiation than those in the operative group (P<0.001). Patients in the nonoperative group missed fewer classes and sustained lower costs (P<0.001, <0.001). The mean immobilization time in each group was not significantly different (31.4±4.4 vs. 32.8±5.9 d; P=0.227). CONCLUSIONS: Although the postreduction quality was a little better and radiation exposure was less in the CRPP group, there was no difference between the 2 groups in angulation, total immobilization time, or complication rates after 6 months. The cost and time absent from school of patients in the nonoperative group was significantly lower than in the operative group. There is no clear advantage to CRPP treatment on outcomes. Therefore, closed reduction and casting is recommended in complete distal forearm fractures of children 8 to 14 years old. LEVEL OF EVIDENCE: Level III-therapeutic study.

Whole genome sequencing of pairwise human subjects reveals DNA mutations specific to developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a common congenital malformation characterized by mismatch in shape between the femoral head and acetabulum, and leads to hip dysplasia. To date, the pathogenesis of DDH is poorly understood and may involve multiple factors, including genetic predisposition. However, comprehensive genetic analysis has not been applied to investigate a genetic component of DDH. In the present study, 10 pairs of healthy fathers and DDH daughters were enrolled to identify genetic hallmarks of DDH using high throughput whole genome sequencing. The DDH-specific DNA mutations were found in each patient. Overall 1344 genes contained DDH-specific mutations. Functional enrichment analysis showed that these genes played important roles in the cytoskeleton, microtubule cytoskeleton, sarcoplasm and microtubule associated complex. These functions affected osteoblast and osteoclast development. Therefore, we proposed that the DDH-specific mutations might affect bone development, and caused DDH. Our pairwise high throughput sequencing results comprehensively delineated genetic hallmarks of DDH. Further research into the biological impact of these mutations may inform the development of DDH diagnostic tools and allow neonatal gene screening.

Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation.

Dexamethasone (DEX) exerts potent cytotoxicity against cultured human osteoblasts. The current study examined the role of the circular RNA HIPK3 (circHIPK3) in the mechanism of cell death. We found that circHIPK3 expression was downregulated in DEX-treated human osteoblasts and circHIPK3 levels decreased in human necrotic femoral head tissues. In OB-6 osteoblastic cells and primary human osteoblasts ectopic overexpression of circHIPK3 potently suppressed DEX-induced apoptosis and programmed necrosis. Conversely, knockdown of circHIPK3by targeted siRNAs enhanced DEX-induced cytotoxicity in human osteoblasts. We further observed that microRNA-124 (miR-124), a key miRNA sponged by circHIPK3, accumulated following DEX treatment in OB-6 cells and primary osteoblasts. Confirming the role of miR-124 in DEX-induced cytotoxicity, miR-124 inhibitor attenuated cell death in human osteoblasts. Conversely, forced overexpression of miR-124 mimicked DEX-induced actions and induced cytotoxicity in human osteoblasts. We conclude that DEX-induced cytotoxicity in human osteoblasts is associated with circHIPK3 downregulation.

P53 dependent mitochondrial permeability transition pore opening is required for dexamethasone-induced death of osteoblasts.

Prolonged or overdose glucocorticoids (GCs) usage is the common cause of osteoporosis. In the present study, we studied the cellular mechanism of dexamethasone (Dex)-induce osteoblast cell death by focusing on the role of mitochondrial permeability transition pore (mPTP). In cultured osteoblastic MC3T3-E1 cells, Dex-induced mPTP opening, which was demonstrated by mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) mitochondrial complexation and cytochrome C (cyto-C) release. The mPTP inhibitor sanglifehrin A (SfA) dramatically inhibited Dex-induced MPP loss, cyto-C release and MC3T3-E1 cell death. Dex-induced cell death requires mPTP composing protein CyPD, as CyPD inhibitor cyclosporin A (CsA) and CyPD siRNA knockdown inhibited Dex-induced MC3T3-E1 cell death, while CyPD overexpression aggravated Dex's cytotoxic effect. We found that Dex induced P53 phosphorylation and translocation to mitochondria, where it formed a complex with CyPD. Glucocorticoid receptor (GR) siRNA knockdown, or P53 inhibition (by its inhibitor pifithrin-α or shRNA silencing) suppressed Dex-induced CyPD-P53 mitochondrial association and subsequent MC3T3-E1 cell death. Finally, in primary cultured osteoblasts, Dex-induced cell death was inhibited by CsA, SfA or pifithrin-α. Together, our data suggest that Dex-induced osteoblast cell death is associated with GR-P53-regulated mPTP opening.

DNA-PKcs-SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation.

Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1Gy) induced phosphorylation of DNA-PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA-PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA-PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA-PKcs-SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA-PKcs-SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

Bone and joint infection complicated with sepsis in neonates and infants under three months of age.

OBJECTIVE: Studies focusing on bone and joint infections (BJIs) in young infants are rare. Some cases of BJI are accompanied by sepsis. This study aimed to identify the clinical and bacteriological features of sepsis in neonates and young infants with BJIs. METHODS: Neonates and infants younger than 3 months diagnosed with BJI in the present institution from 2014 to 2021 were retrospectively reviewed. Patient characteristics, clinical data, and outcomes were documented and compared between those with and without sepsis. RESULTS: Twenty-five patients with a mean age of 34.8 days were included. Nine BJI cases had concomitant sepsis (group A), and 16 had BJI without sepsis (group B). Within group A, staphylococcus aureus was the major pathogenic germ (5 cases, of which 4 were of the methicillin-resistant staphylococcus aureus (MRSA) type). There was no statistical difference in male-to-female ratio, age, history of hospitalization, anemia, birth asphyxia, peripheral leukocyte counts, C-reactive protein on admission, and sequelae between groups. Univariate analyses indicated a significant difference in the incidence of septic arthritis (SA) combined with osteomyelitis (OM) (88.9% vs 37.5%), congenital deformities (44.4% vs 0%), and mean duration of symptoms (2.83 days vs 9.21 days) in comparisons between groups A and B. CONCLUSION: Staphylococcus aureus is the main pathogenic bacteria in BJI cases complicated with sepsis in neonates and young infants. Among infants younger than 3 months diagnosed with BJI, those with concurrent SA and OM, MRSA infection, or congenital deformities are more likely to develop sepsis.

Efficacy of short-term splint immobilization in the treatment of pediatric discoid lateral meniscus after saucerization management.

There are no universal guidelines for rehabilitation after saucerization for children with discoid lateral meniscus. This study determined if short-term knee splint immobilization and delayed rehabilitation produces the same benefit as early rehabilitation after saucerization in children, in terms of knee function and pain intensity. A retrospective review was performed by categorizing patients into 2 groups depending on whether a splint immobilization was adopted postoperatively: for group A, rehabilitation began early without splint immobilization after surgery, and for group B, a knee splint was immobilized for 2 weeks. Numerical rating scale scores were collected in patients 1, 3, and 7 days, Lysholm scores were measured at 4 and 8 weeks postoperatively, and the gradual return to normal activities was documented. Forty-eight patients and 53 knees were included: group A had 30 patients with 31 knees, and group B had 18 patients with 22 knees. There was no improvement in numerical rating scale scores on the 1st (P=.519), 3rd (P=.421), and 7th (P=.295) postoperative days in group B. The Lysholm scores of group A (62.94 ±â€…8.68) was higher than that of group B (46.68 ±â€…9.82) measured 4 weeks following surgery, but there was no difference at 8 weeks (P=.237), and both groups had similar time to return to normal activities (P=.363). For discoid lateral meniscus patients who underwent isolated saucerization, short-term splint immobilization did not significantly help relieve postoperative pain. There was a comparable time-course for return to normal activities in both study groups.

Open Reduction of Displaced Radial Neck Fractures in Children by Internal Fixation Techniques: Comparison of Percutaneous Kirschner Wiring and Elastic Stable Intramedullary Nailing.

Background: Although most paediatric radial neck fractures can be treated with closed reduction, some severely displaced fractures require open reduction. The purpose of this study is to compare the effects of ESIN and KW fixation in open reduction of radial neck fracture in children. Methods: Twenty-four patients with mean age of 8.5 years were included. Four of the patients had a Judet type III fracture and 20 had a Judet type IV fracture. Ten patients who underwent percutaneous KW fixation were assigned to group A, while 14 patients who underwent ESIN fixation were assigned to group B. Variables of interest included age, sex, fracture type, associated lesions, surgical time, fracture reduction, cost, follow-up, healing time, X-rays, clinical outcomes, and complications. Results: There were no significant between-group differences in sex, age, additional injuries, fracture type, and quality of reduction. Costs were significantly lower in Group A. Fracture healing was achieved in 23 of 24 patients (10/10 in group A and 13/14 in group B). In a postoperative elbow function assessment based on the Steele and Graham classification, 80% of patients in group A had a score of excellent or good, compared to 78.6% of patients in group B. Two cases of nail shifting and joint protrusion were observed in group B, one of which also presented with nonunion during follow-up. Conclusions: Both KW and ESIN may achieve good clinical outcomes, but KW is associated with lower costs, easier implant removal (without the need for a secondary surgery), and lower iatrogenic complications.

ADCK1 is a potential therapeutic target of osteosarcoma.

We here showed that ADCK1 (AarF domain-containing kinase 1), a mitochondrial protein, is upregulated in human osteosarcoma (OS) tissues and OS cells. In primary and established OS cells, ADCK1 shRNA or CRISPR/Cas9-induced ADCK1 knockout (KO) remarkably inhibited cell viability, proliferation and migration, and provoked apoptosis activation. Conversely, ectopic ADCK1 overexpression exerted pro-cancerous activity by promoting OS cell proliferation and migration. ADCK1 depletion disrupted mitochondrial functions in OS cells and induced mitochondrial membrane potential reduction, ATP depletion, reactive oxygen species production. Significantly, ADCK1 silencing augmented doxorubicin-induced apoptosis in primary OS cells. mTOR activation is important for ADCK1 expression in OS cells. The mTOR inhibitors, rapamycin and AZD2014, as well as mTOR shRNA, potently decreased ADCK1 expression in primary OS cells. In nude mice, the growth of subcutaneous pOS-1 xenografts was largely inhibited when bearing ADCK1 shRNA or ADCK1 KO construct. Moreover, ADCK1 KO largely inhibited pOS-1 xenograft in situ growth in proximal tibia of nude mice. ADCK1 depletion, apoptosis activation and ATP reduction were detected in pOS-1 xenografts bearing ADCK1 shRNA or ADCK1 KO construct. Together, the mitochondrial protein ADCK1 is required for OS cell growth and is a novel therapeutic target of OS.

Open reduction and Herbert screw fixation of Pipkin type IV femoral head fracture in an adolescent: A case report.

BACKGROUND: Femoral head fracture is extremely rare in children. This may be the youngest patient with femoral head fracture ever reported in the literature. There are few pediatric studies that focus on cases treated with open reduction via the modified Hardinge approach. CASE SUMMARY: A 14-year-old female adolescent suffered a serious traffic accident when she was sitting on the back seat of a motorcycle. A pelvic radiograph and computed tomography revealed a proximal femoral fracture and slight acetabular rim fracture. This was diagnosed as a Pipkin type IV femoral head fracture. An open reduction and Herbert screw fixation was performed via a modified Hardinge approach. After 1-year follow-up, the patient could walk without aid and participate in physical activities. The X-ray results showed that the fractures healed well with no evidence of complications. CONCLUSION: Open reduction and Herbert screw fixation is an available therapy to treat Pipkin type IV femoral head fractures in children.

MAFG-driven osteosarcoma cell progression is inhibited by a novel miRNA miR-4660.

Osteosarcoma (OS) is the most common primary bone malignancy in the adolescent population. MAFG (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) forms a heterodimer with Nrf2 (NF-E2-related factor 2), binding to antioxidant response element (ARE), which is required for Nrf2 signaling activation. We found that MAFG mRNA and protein expression is significantly elevated in human OS tissues as well as in established and primary human OS cells. In human OS cells, MAGF silencing or knockout (KO) largely inhibited OS cell growth, proliferation, and migration, simultaneously inducing oxidative injury and apoptosis activation. Conversely, ectopic overexpression of MAFG augmented OS cell progression in vitro. MicroRNA-4660 (miR-4660) directly binds the 3' untranslated region (UTR) of MAFG mRNA in the cytoplasm of OS cells. MAFG 3' UTR luciferase activity and expression as well as OS cell growth were largely inhibited with forced miR-4660 overexpression but augmented with miR-4660 inhibition. In vivo, MAGF short hairpin RNA (shRNA) or forced overexpression of miR-4660 inhibited subcutaneous OS xenograft growth in severe combined immunodeficient mice. Furthermore, MAFG silencing or miR-4660 overexpression inhibited OS xenograft in situ growth in proximal tibia of the nude mice. In summary, MAFG overexpression-driven OS cell progression is inhibited by miR-4660. The miR-4660-MAFG axis could be novel therapeutic target for human OS.

MiR-132/212 promotes the growth of precartilaginous stem cells (PCSCs) by regulating Ihh/PTHrP signaling pathway.

Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was involved in the proliferation of PCSCs via Hedgehog signaling pathway. PCSCs were isolated and purified with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Cell viability, DNA synthesis and apoptosis were measured using MTT, BrdU and flow cytometric analysis. The mRNA and protein expression were detected by real-time PCR and Western blot, respectively. The target gene for miR-132/212 was validated by luciferase reporter assay. Results showed that transfection with miR-132/212 mimic significantly increased cell viability and DNA synthesis, and inhibited apoptosis of PCSCs. By contrast, miR-132/212 inhibitor could suppress growth and promote apoptosis of PCSCs. Luciferase reporter assays indicated that transfection of miR-132/212 led to a marked reduction of luciferase activity, but had no effect on PTCH1 3'-UTR mutated fragment, suggesting that Patched1 (PTCH1) is a target of miR-132/212. Furthermore, treatment with miR-132/212 mimics obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster might serve as a novel target for bone diseases.

Bone and joint infection complicated with sepsis in neonates and infants under three months of age

Abstract Objective: Studies focusing on bone and joint infections (BJIs) in young infants are rare. Some cases of BJI are accompanied by sepsis. This study aimed to identify the clinical and bacteriological features of sepsis in neonates and young infants with BJIs. Methods: Neonates and infants younger than 3 months diagnosed with BJI in the present institution from 2014 to 2021 were retrospectively reviewed. Patient characteristics, clinical data, and outcomes were documented and compared between those with and without sepsis. Results: Twenty-five patients with a mean age of 34.8 days were included. Nine BJI cases had concomitant sepsis (group A), and 16 had BJI without sepsis (group B). Within group A, staphylococcus aureus was the major pathogenic germ (5 cases, of which 4 were of the methicillin-resistant staphylococcus aureus (MRSA) type). There was no statistical difference in male-to-female ratio, age, history of hospitalization, anemia, birth asphyxia, peripheral leukocyte counts, C-reactive protein on admission, and sequelae between groups. Univariate analyses indicated a significant difference in the incidence of septic arthritis (SA) combined with osteomyelitis (OM) (88.9% vs 37.5%), congenital deformities (44.4% vs 0%), and mean duration of symptoms (2.83 days vs 9.21 days) in comparisons between groups A and B. Conclusion: Staphylococcus aureus is the main pathogenic bacteria in BJI cases complicated with sepsis in neonates and young infants. Among infants younger than 3 months diagnosed with BJI, those with concurrent SA and OM, MRSA infection, or congenital deformities are more likely to develop sepsis.

Treatment of Severely Displaced Radial Neck Fractures in Children With Percutaneous K-wire Leverage and Closed Intramedullary Pinning.

To evaluate the efficacy and safety of percutaneous K-wire leverage (PKWL) reduction and closed intramedullary pinning (CIMP) for the treatment of pediatric radial neck fractures. From June 2010 to December 2013, a total of 50 children with Judet III and IV radial neck fractures were treated at our hospital. Manual closed reduction was first attempted to reduce the radial neck fractures. Upon successful closed reduction or the radial neck-shaft angle was reduced to <45°, radial intramedullary pinning or CIMP was performed for fixation. Unsuccessful manual reduction was corrected using percutaneous K-wire leverage and CIMP. The injured arm was fixed at the functional position using plaster for 4 to 6 weeks. Sixteen patients were treated with manual closed reduction and CIMP (group A). Percutaneous K-wire leverage and CIMP were performed for 30 patients (group B). Another 4 patients were treated with open reduction and CIMP (group C). Groups B and C showed no significant difference in the radial neck-shaft angle, fracture displacement, and angle/displace ratio (P > 0.05), but were significantly larger than group A in the radial neck-shaft angle and fracture displacement (P < 0.05). Group A and B had significantly shorter operation time than group C (58.4 ± 14.5 minutes, 55.2 ± 11.2 minutes, versus 81.4 ± 7.5 minutes, P < 0.05). Forty-five patients were followed up for a mean of 2 years. Bone union was achieved in all patients within a mean time of 4.1 months. The patients treated with manual reduction or percutaneous leverage reduction showed excellent results. Three patients, however, treated with open reduction showed 10 to 20° limitation in range of motion of the elbow. No other complications were seen. Percutaneous K-wire leverage and CIMP are safe and effective for the treatment of pediatric Judet III and IV radial neck fractures.

miR-135b expression downregulates Ppm1e to activate AMPK signaling and protect osteoblastic cells from dexamethasone.

Activation of AMP-activated protein kinase (AMPK) could potently protect osteoblasts/osteoblastic cells from dexamethasone (Dex). We aim to induce AMPK activation via microRNA ("miRNA") downregulation of its phosphatase Ppm1e. We discovered that microRNA-135b ("miR-135b") targets the 3' untranslated regions (UTRs) of Ppm1e. In human osteoblasticOB-6 cells and hFOB1.19 cells, forced-expression of miR-135b downregulated Ppm1e and activated AMPK signaling. miR-135b also protected osteoblastic cells from Dex. shRNA-induced knockdown of Ppm1e similarly activated AMPK and inhibited Dex-induced damages. Intriguingly, in the Ppm1e-silenced osteoblastic cells, miR-135b expression failed to offer further cytoprotection against Dex. Notably, AMPK knockdown (via shRNA) or dominant negative mutation abolished miR-135b-induced AMPK activation and cytoprotection against Dex. Molecularly, miR-135b, via activating AMPK, increased nicotinamide adenine dinucleotide phosphate (NADPH) activity and inhibited Dex-induced oxidative stress. At last, we found that miR-135b level was increased in human necrotic femoral head tissues, which was correlated with Ppm1e downregulation and AMPK activation. There results suggest that miR-135b expression downregulates Ppm1e to activate AMPK signaling, which protects osteoblastic cells from Dex.

The anti-cancer activity of the mTORC1/2 dual inhibitor XL388 in preclinical osteosarcoma models.

In the present study, we investigated the activity of XL388, a novel mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, in preclinical osteosarcoma (OS) models. XL388 was cytotoxic, cytostatic and pro-apoptotic to multiple established OS cell lines and primary human OS cells. XL388 blocked mTORC1/2 activation and downregulated cyclin D1/B1 expressions in OS cells, leaving AKT Thr-308 phosphorylation un-affected. Intriguingly, AKT1 T308A mutation potentiated XL388-induced cytotoxicity in OS cells. XL388 activated cytoprotective autophagy in OS cells. Autophagy inhibition, either pharmacologically or genetically, augmented XL388-induced anti-OS activity. Further, XL388 oral administration inhibited U2OS xenografts growth in severe combined immuno-deficient (SCID) mice. Such activity was enhanced with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). Similarly, Beclin-1-silenced U2OS xenografts were remarkably more sensitive to XL388. Thus, concurrent blockage of mTORC1/2 with XL388 may have therapeutic value for OS.

Identification of DNA-PKcs as a primary resistance factor of salinomycin in osteosarcoma cells.

Malignant osteosarcoma (OS) is still a deadly disease for many affected patients. The search for the novel anti-OS agent is extremely urgent and important. Our previous study has proposed that salinomycin is a novel anti-OS agent. Here we characterized DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a primary salinomycin resistance factor in OS cells. DNA-PKcs inhibitors (NU7026, NU7441 and LY294002) or DNA-PKcs shRNA knockdown dramatically potentiated salinomycin-induced death and apoptosis of OS cells (U2OS and MG-63 lines). Further, forced-expression of microRNA-101 ("miR-101") downregulated DNA-PKcs and augmented salinomycin's cytotoxicity against OS cells. Reversely, over-expression of DNA-PKcs in OS cells inhibited salinomycin's lethality. For the mechanism study, we show that DNA-PKcs is required for salinomycin-induced pro-survival autophagy activation. DNA-PKcs inhibition (by NU7441), shRNA knockdown or miR-101 expression inhibited salinomycin-induced Beclin-1 expression and autophagy induction. Meanwhile, knockdown of Beclin-1 by shRNA significantly sensitized salinomycin-induced OS cell lethality. In vivo, salinomycin administration suppressed U2OS xenograft tumor growth in severe combined immuno-deficient (SCID) mice, and its anti-tumor activity was dramatically potentiated with co-administration of the DNA-PKcs inhibitor NU7026. Together, these results suggest that DNA-PKcs could be a primary resistance factor of salinomycin in OS cells. DNA-PKcs inhibition or silence may thus significantly increase salinomycin's sensitivity in OS cells.

Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: new implications for osteonecrosis treatment?

Elevated hydrogen peroxide (H2O2) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H2O2 stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H2O2-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H2O2-induced cell damage. These results confirmed that H2O2-activated AMPK is pro-cell survival. We observed that H2O2 induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H2O2-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H2O2 in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H2O2-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H2O2 in these cells. Based on these results, we concluded that H2O2-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.