Combined application of high-throughput sequencing and LC-MS/MS-based metabolomics to evaluate the formation of Zn-protoporphyrin in Nuodeng ham.

In order to deepen the understanding of the formation mechanism of Zn-protoporphyrin (ZnPP), Nuodeng ham was used as the research object, and the effects of bacterial communities and small molecule metabolites on the formation of ZnPP during the processing of Nuodeng ham were analyzed by high-throughput sequencing and LC-MS/MS-based metabolomics. With the prolongation of processing time, the ZnPP content increased significantly, while the heme content decreased significantly. Compound curing agent significantly inhibited the formation of ZnPP and significantly decreased the heme content. The bacterial communities changed dynamically and the five bacterial genera were significantly positively correlated with ZnPP content. Forty-seven differential metabolites were obtained through screening, of which seventeen differential metabolites were significantly positively correlated with ZnPP content. Correlation analysis showed a strongly positive correlation between Staphylococcus, Delftia, Acinetobacter and these seventeen differential metabolites. These findings can provide a theoretical basis for further establishing the color control measures of Nuodeng ham.

Screening for MicroRNA combination with engineered exosomes as a new tool against osteosarcoma in elderly patients.

The most common primary malignant bone sarcoma is Osteogenic sarcoma (OS) which has a bimodal age distribution. Unfortunately, the treatment of OS was less effective for elderly patients than for younger ones. The study aimed to explore a new microRNA (miRNA) which can bind to combining engineered exosomes for treatment of older OS patients. Based on GSE65071 and miRNet 2.0, two up-regulated miRNAs (miR-328, miR-107) and seven down-regulated miRNAs (miR-133b, miR-206, miR-1-3p, miR-133a, miR-449a, miR-181daysay, miR-134) were selected. Next, we used FunRich software to predict the up-stream transcription factors (TFs) of differentially expressed miRNAs (DE-miRNAs). By comparing target genes predicted from DE-miRNAs with differentially expressed genes, we identified 12 down-regulated and 310 up-regulated mRNAs. For KEGG analysis, the most enriched KEGG pathway was Cell cycle, Spliceosome, and Protein digestion and absorption. By using protein-protein interactions network, topological analysis algorithm and GEPIA database, miR-449a /CCNB1 axis was identified. Experiments in vitro were conducted to confirm the results too. MiRNA-449a is down-regulated in osteosarcoma and suppresses cell proliferation by targeting CCNB1. Our findings not only reveal a novel mechanism of miR-449a /CCNB1 in OS but also had laid the groundwork for further investigation and analysis in the field of exosome engineering.

Development of sea urchin type silica stabilised zirconia nanospheres with enhanced antimicrobial and osteoactivity properties.

Zirconia based ceramics are giving new hope in hard tissues replacement and implants application. Among the three forms of zirconia (ZrO2), tetragonal form (t-ZrO2) possess high mechanical stability in comparison with the other two which makes it suitable for fabricating biomedical implants with enhanced osteo activity. Here, tetragonal phase nanospheres consisting of silica stabilised zirconia (1:1) were prepared via sol gel method. The nanospheres exhibit sea urchin type morphology as observed from FESEM analysis. XRD patterns confirm the formation of t -SiO2-ZrO2 binary phase after high temperature calcination at 650°C. The immersion studies in SBF help in the formation of a layer of apatite in a gradual manner over the pallets for the period of 7, 14, 21 and 28 days which was confirmed by XRD, FTIR analysis. Moreover, t- SiO2 - ZrO2 samples were subjected to cytotoxicity tests through MTT assay on MG-63 cell lines. Antibacterial properties were investigated quantitatively using colony forming unit method against both gram positive as well as gram-negative bacteria.

Integrated Bioinformatics and Experimental Analysis Identified TRIM28 a Potential Prognostic Biomarker and Correlated with Immune Infiltrates in Liver Hepatocellular Carcinoma.

Background: Since the 1970s, liver hepatocellular carcinoma (LIHC) has experienced a constant rise in incidence and mortality rates, making the identification of LIHC biomarkers very important. Tripartite Motif-Containing 28 (TRIM28) is a protein-coding gene which encodes the tripartite motif-containing proteins (TRIMs) family and is associated with specific chromatin regions. TRIM28 expression and its prognostic value and impact on the immune system in LIHC patients are being investigated for the first time. Methods: The TRIM28 expression data from TCGA database was used to analyze TRIM28 expression, clinicopathological information, gene enrichment, and immune infiltration and conduct additional bioinformatics analysis. R language was used for statistical analysis. TIMER, CIBERSORT, and ssGSEA were used to assess immune responses of TRIM28 in LIHC. Next, the results were validated using GEPIA, ROC analysis, and immunohistochemical staining pictures from the THPA. GSE14520, GSE63898, and GSE87630 datasets were analyzed using ROC analysis to further evaluate TRIM28's diagnostic value. To ultimately determine TRIM28 expression, we performed qRT-PCR (quantitative real-time polymerase chain reaction). Results: High TRIM28 expression level was associated with T classification, pathologic stage, histologic grade, and serum AFP levels. In patients with LIHC, TRIM28 was an independent risk factor for a poor prognosis. The pathways ligand-receptor interaction, which is critical in LIHC patients, were closely associated with TRIM28 expression, and the function of DC could be suppressed by overexpression of TRIM28. As a final step, our results were validated by GEO data and qRT-PCR. Conclusions: TRIM28 will shed new light on LIHC mechanisms. As an effective diagnostic and intervention tool, this gene will be able to diagnose and treat LIHC at an early stage.

Electroplating of HAp-brushite coating on metallic bioimplants with advanced hemocompatibility and osteocompatibility properties.

In cases of severe bone tissue injuries, the use of metallic bioimplants is quite widespread due to their high strength, high fracture toughness, hardness, and corrosion resistance. However, they lack adequate biocompatibility and show poor metal-tissue integration during the post-operative phase. To mitigate this drawback, it is beneficial to add a biocompatible polymer layer to ensure a quick growth of cell or tissue over the surface of metallic bioimplant material. Furthermore, this additional layer should possess good adherence with the underlying material and also accompany a rapid bonding between the tissue and the implant material, in order to reduce the recovery time for the patient. Therefore, in this work, we report a novel green electroplating route for growing porous hydroxyapatite-brushite coatings on a stainless steel surface. The malic acid used for the production of hydroxyapatite-brushite coatings has been obtained from an extract of locally available apple fruit (Malus domestica). We demonstrate the effect of electroplating parameters on the structural morphology of the electroplated composite layer via XRD, SEM with EDS, and FTIR characterization techniques and report an optimized set of electroplating parameters that will yield the best composite coating in terms of thickness, adherence to substrate and speed. The hemocompatibility and osteocompatibility studies on the electroplated composites coating show this technology's effectiveness and potential applicability in biomedical applications. Compared to other routes reported in the literature, this electroplating route is quicker and yields better composite coatings with faster bone tissue growth potential.

CD137 Regulates Bone Loss via the p53 Wnt/ß-Catenin Signaling Pathways in Aged Mice.

Senile osteoporosis is a chronic skeletal disease, leading to increased bone brittleness and risk of fragile fractures. With the acceleration of population aging, osteoporosis has gradually become one of the most serious and prevalent problems worldwide. Bone formation is highly dependent on the proper osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in the bone marrow microenvironment, which is generated by the functional relationship among different cell types, including osteoblasts, adipogenic cells, and bone marrow stromal cells in the bone marrow. It is still not clear how osteoporosis is caused by its molecular mechanism. With aging, bone marrow is able to restrain osteogenesis. Discovering the underlying signals that oppose BMSC osteogenic differentiation from the bone marrow microenvironment and identifying the unusual changes in BMSCs with aging is important to elucidate possible mechanisms of senile osteoporosis. We used 3 gene expression profiles (GSE35956, GSE35957, and GSE35959) associated with osteoporosis. And a protein-protein interaction (PPI) network was also built to identify the promising gene CD137. After that, we performed in vivo experiments to verify its function and mechanism. In this experiment, we found that significant bone loss was observed in aged (18-month-old) mice compared with young (6-month-old) mice. The adipose tissue in bone marrow cavity from aged mice reached above 10 times more than young mice. Combining bioinformatics analysis and vivo experiments, we inferred that CD137 might be involved in the p53 and canonical Wnt/ß-catenin signaling pathways and thereby influenced bone mass through regulation of marrow adipogenesis. Importantly, osteoporosis can be rescued by blocking CD137 signaling in vivo. Our research will contribute to our understanding not only of the pathogenesis of age-related bone loss but also to the identification of new targets for treating senile osteoporosis.

A Prognosis Marker Dynein Cytoplasmic 1 Heavy Chain 1 Correlates with EMT and Immune Signature in Liver Hepatocellular Carcinoma by Bioinformatics and Experimental Analysis.

Background: Liver hepatocellular carcinoma (LIHC) has had a continuous increase in incidence and mortality rates over the last 40 years. Dynein Cytoplasmic 1 Heavy Chain 1 (DYNC1H1) is a protein coding gene which encodes the cytoplasmic dynein heavy chain family. This is the first investigation into the expression of DYNC1H1 and its mechanisms of action in LIHC patients. Methods: Based on the DYNC1H1 expression data from the TCGA database, we performed the DYNC1H1 expression, clinicopathological data, gene enrichment, and immune infiltration analysis. TIMER and CIBERSORT were used to assess immune responses of DYNC1H1 in LIHC. GEPIA, K-M survival analysis, and immunohistochemical staining pictures from the THPA were used to validate the results. In order to evaluate the diagnostic value of DYNC1H1, GEO datasets were analyzed by using ROC analysis. And quantitative real-time polymerase chain reaction was also carried out to evaluate the expression of DYNC1H1. Results: DYNC1H1 expression levels were associated with T classification, pathologic stage, histologic grade, and serum AFP levels. DYNC1H1 is an independent factor for a poor prognosis in patients with LIHC. Further study showed that high expression of DYNC1H1 was enriched in epithelial-mesenchymal transition (EMT) and the TGF ß signaling pathway by GSEA analysis enrichment, indicating that DYNC1H1 might play a key role in the progression of CRC through EMT and immune response, which also had been validated by the experimental assays. Conclusions: DYNC1H1 will provide a novel and important perspective for the mechanisms of LIHC by regulating EMT. This gene will be able to act as an efficacious tool for the early diagnosis and effective intervention of LIHC.

Activation of 4-1BB signaling in bone marrow stromal cells triggers bone loss via the p-38 MAPK-DKK1 axis in aged mice.

Senile osteoporosis can cause bone fragility and increased fracture risks and has been one of the most prevalent and severe diseases affecting the elderly population. Bone formation depends on the proper osteogenic differentiation of bone marrow stromal cells (BMSCs) in the bone marrow microenvironment, which is generated by the functional relationship among different cell types in the bone marrow. With aging, bone marrow provides signals that repress osteogenesis. Finding the signals that oppose BMSC osteogenic differentiation from the bone marrow microenvironment and identifying the abnormal changes in BMSCs with aging are key to elucidating the mechanisms of senile osteoporosis. In a pilot experiment, we found that 4-1BBL and 4-1BB were more abundant in bone marrow from aged (18-month-old) mice than young (6-month-old) mice. Meanwhile, significant bone loss was observed in aged mice compared with young mice. However, very little data have been generated regarding whether high-level 4-1BB/4-1BBL in bone marrow was associated with bone loss in aged mice. In the current study, we found upregulation of 4-1BB in the BMSCs of aged mice, which resulted in the attenuation of the osteogenic differentiation potential of BMSCs from aged mice via the p38 MAPK-Dkk1 pathway. More importantly, bone loss of aged mice could be rescued through the blockade of 4-1BB signaling in vivo. Our study will benefit not only our understanding of the pathogenesis of age-related trabecular bone loss but also the search for new targets to treat senile osteoporosis.

The lncRNA LINC00691Functions as a ceRNA for miRNA-1256 to Suppress Osteosarcoma by Regulating the Expression of ST5.

INTRODUCTION: Osteosarcoma is the most common primary malignant tumor in children and young patients. Although neoadjuvant chemotherapy and surgery could improve the prognosis of these patients, treatment outcomes are poor because of its low early diagnosis rate and high degree of malignancy as well as its tendency for early metastasis. In the field of osteosarcoma, lncRNAs have become a hot spot for studying the molecular mechanisms driving malignant biological characteristics and exploring effective treatment methods. An lncRNA is a long noncoding RNA lacking protein-encoding ability, and in its RNA form, it regulates various gene expression processes, such as epigenetic regulation, transcriptional regulation, and posttranscriptional regulation. LncRNAs play an important role in tumorigenesis and metastasis. METHODS: We used bioinformatics software to analyze the data in geo database. CCK-8 and Transwell were used to detect the effect of lncRNA LINC00691 on the proliferation and migration of osteosarcoma cells. The target gene of LINC00691 was detected by bioinformatics analysis and RNA pull down. RESULTS: In this study, we identified the lncRNA LINC00691 and confirmed its expression in osteosarcoma cells through GEO database analysis. Expression analysis showed that the levels of lncRNA LINC00691 in osteosarcoma cells were decreased compared to those of control cells. Overexpression of LINC00691 could inhibit the proliferation, migration, invasion, and induction of G1 cell cycle arrest in osteosarcoma cells, which was shown through in vitro and in vivo studies. Using bioinformatics analysis, RNA pull down experiments and luciferase reporter gene detection assays, we found that LINC00691 regulated ST5 expression by binding miR-1256. LINC00691 overexpression inhibited EMT by promoting the expression of E-cadherin and increasing the expression of ZEB1, Snail, and Fibronectin. CONCLUSION: These results suggested that overexpressed LINC00691 promoted the expression of ST5 by regulating the function of miR-1256 through a ceRNA mechanism. The LINC00691/miR-1256/ST5 pathway plays an important role in the progression and metastasis of osteosarcoma and represents a good therapeutic target.

miR-152 Attenuates Apoptosis in Chondrocytes and Degeneration of Cartilages in Osteoarthritis Rats via TCF-4 Pathway.

INTRODUCTION: Osteoarthritis (OA) is associated with deregulation of various miRNAs (miRs). The present study reported protective effect of miR-152 in osteoarthritis. METHODS: Tissue cartilage tissues of OA and normal subjects were used, rat model of anterior cruciate ligament transection (ACLT) was developed. Cartilage study was done by Safranin O-fast green, histological and immunostaining. The chondrocytes were isolated from tissues and were treated with IL-1ß and infected with miR-152 or TCF-4 cloned lentiviral vectors. MTT assay was done for cell viability, apoptosis by Annexin-V-FITC staining. Expressions of proteins by western blot assay. Collagen-II assay was done by immunofluroscent assay. Luciferase activity by dual luciferase reporter assay. RESULTS: Upregulation of miR-152 improved viability of chondrocytes, decreased apoptosis and balanced the catabolic and anabolic factors of extracellular matrix in vitro. Injecting miR-152 lentivirus in rats improved articular cartilage in osteoarthritis ACLT rats. Bioinformatics analysis suggested TCF-4 as favorable target gene of miR-152, having binding site on the 3'UTR region of TCF-4 mRNA and inhibited the expression of TCF-4. Osteoarthritis tissue cartilage both from humans and rats showed expression of miR-152 inversely linked with expression of TCF-4. CONCLUSION: Present study concludes miR-152 diminished the progression of osteoarthritis partially by inhibiting the expression of TCF-4.

microRNA-26a-5p Promotes Proliferation and Migration of Osteosarcoma Cells by Targeting HOXA5 in vitro and in vivo.

BACKGROUND: Osteosarcoma is the most common primary malignant tumor of bone. However, the underlying pathogenic mechanisms are still unclear. miR-26a was an endogenous non-coding small RNAs that have been showed to play a critical role in regulating varieties of biological and pathological processes. In this study, we will investigate the function of miR-26a-5p in osteosarcoma cells. METHODS: In this study, we explored the role of miR-26a-5p in osteosarcoma cell lines using qPCR, detected the proliferation, cell cycle and cell migration by CCK-8, PI and transwell. RESULTS: We found that compared with noncancerous cells, miR-26a-5p was highly expressed in osteosarcoma cell lines, especially in U2OS cells. Overexpression of miR-26a-5p promotes cell proliferation, cell cycle, and cell migration, but inhibits cell apoptosis. But down-regulation of miR-26a-5p in U2OS cells exhibits opposite effects. We also confirmed that miR-26a-5p directly targets HOXA5 in U2OS cells. Overexpression of HOXA5 reversed the effect of miR-26a-5p on cell proliferation, migration, and apoptosis. Besides, we showed in that knock-down of miR-26a-5p or overexpression of HOXA5 increased cell sensitivity to chemotherapeutic drug paclitaxel. CONCLUSION: These findings indicate that highly expressed miR-26a-5p in osteosarcoma cells, and promotes proliferation and migration, but inhibits apoptosis of osteosarcoma cells by targeting HOXA5 which suggest that miR-26a-5p could serve as a novel therapeutic target for osteosarcoma.

Multimodal Nanoprobe Based on Upconversion Nanoparticles for Monitoring Implanted Stem Cells in Bone Defect of Big Animal.

Monitoring implanted stem cells in bone regeneration and other cell therapies is of great importance to reveal the mechanism of tissue repair and to optimize clinical treatments. However, big challenge still remained in lacking an imaging nanoprobe. Herein, we designed surface modified upconversion nanoparticles (UCNs) with multimodal imaging capabilities of fluorescence, magnetic resonance imaging (MRI) and dual-energy computed tomography (CT). It was found that the UCNs can label stem cells in an efficient (over 200 pg/cell) and long-term (at least 14 days) manner, with almost no influence on the viability, cell cycle, apoptosis, and multilineage differentiation. Thus, clinical dual-energy CT and MRI were successfully applied to observe the migration of labeled cells on a bone-defect model of rabbit for at least 14 days. The results visualized the gathering of stem cells at the defect site of cortical bone, and the in vivo images were well-correlated with the in vitro fluorescence observation without extra staining. Therefore, a potentially translatable nanoprobe was developed for noninvasive and real-time tracking of cells, which may be meaningful for understanding the bone regeneration in clinic and shed light on the visualization of cells in other cell therapies.

Baicalin induces apoptosis in human osteosarcoma cell through ROS-mediated mitochondrial pathway.

Baicalin is extracted from a traditional Chinese herb, Scutellaria baicalensis. In this study, the anticancer activity and underlying mechanisms of baicalin towards human osteosarcoma cell (HOS) were investigated. Baicalin could inhibit HOS cell proliferation in a concentration-dependent manner. Mitochondrial membrane potential decreased obviously after treated with different concentration of baicalin by flow cytometry assay and revealed that baicalin triggered a significant generation of reactive oxygen species (ROS). Western blotting assay further revealed that baicalin-induced cell apoptosis by suppressing Bcl-2 level, then activating caspase-9 and caspase-3. In vivo experiment, baicalin significantly suppressed tumour growth in female BALB/C nude mice bearing HOS tumours. In addition, baicalin did show toxicity to treated animal by comparing the body weight increase and mortality. In general, the present results demonstrated that baicalin-induced apoptosis in human osteosarcoma cell via a ROS-mediated mitochondrial pathway. The paper indicated that baicalin is a promising candidate for the treatment of HOS.

Geraniin inhibits migration and invasion of human osteosarcoma cancer cells through regulation of PI3K/Akt and ERK1/2 signaling pathways.

Geraniin, an active compound isolated from Geranium sibiricum, was found to inhibit proliferation and induce apoptosis of tumor cells. However, the antimetastatic effects of geraniin remain elusive. Our study found the potential antitumor mechanisms of geraniin through inhibiting the migration and invasion of human osteosarcoma U2OS cells. The western blot, gelatin zymography, and reversed transcription-PCR analysis showed that geraniin suppressed matrix metalloproteinase-9 (MMP-9) expression in a concentration-dependent manner. Geraniin potently suppressed the phosphorylation of extracellular signal regulating kinase (ERK)1/2, phosphatidylinositide-3-kinase (PI3K), and Akt, but did not affect phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. Furthermore, when transforming growth factor-ß1 (TGF-ß1) was used as an agonist, geraniin inhibited TGF-ß1-mediated cell invasion and upregulation of MMP-9. These results suggested that geraniin inhibited U2OS cell migration and invasion by reducing the expression of MMP-9 through the PI3K/Akt and ERK1/2 signaling pathways.

Gold Nanoparticles as a Potential Cellular Probe for Tracking of Stem Cells in Bone Regeneration Using Dual-Energy Computed Tomography.

Transplant of bone marrow mesenchymal stem cells (BMSCs) has attracted considerable interest for bone regeneration. However, noninvasive and real-time tracking of location and concentration of the implanted BMSCs remains a big challenge. Herein we designed a novel approach involving the surface modification of gold nanoparticles (AuNPs) with silica layers and DNA Transfectin 3000 (TS) to improve biocompatibility and to enhance the uptake by BMSCs, hence rendering the ability of tracking BMSCs with dual-energy computer tomography (DECT). Results showed that the endocytosis of AuNPs@SiO2-TS by BMSCs was as high as ∼255 pg/cell after one-day incubation and did not obviously decrease after 14 days. Meanwhile, the AuNPs@SiO2-TS had no influence on the viability, cell cycle, and capabilities on osteogenic, chondrogenic, and adipogenic differentiation of BMSCs. Under a bone-defect rabbit model, the DECT images showed the migration of BMSCs toward a cortical bone defect without variation in volume. This study demonstrated that AuNPs@SiO2-TS could be a potential cellular probe for noninvasive and real-time tracking of BMSCs in bone tissue repairs using clinical CT or DECT techniques. It provided a novel and intuitive methodology for observing and investigating the bone regeneration in clinic.

Dryofragin inhibits the migration and invasion of human osteosarcoma U2OS cells by suppressing MMP-2/9 and elevating TIMP-1/2 through PI3K/AKT and p38 MAPK signaling pathways.

Dryofragin, a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott, was found to inhibit proliferation and induce apoptosis of tumor cells. However, the mechanism involved in the suppression of cancer cell metastasis by dryofragin remains unclear. Our study investigated the mechanisms for the antitumor properties of dryofragin on the migration and invasion of human osteosarcoma U2OS cells. Dryofragin suppressed the migration and invasive ability of U2OS cells, and it decreased the expression of MMP-2 and MMP-9 and elevated the expression of TIMP-1 and TIMP-2. Western blotting assays indicated that dryofragin was effective in suppressing the phosphorylation of phosphatidylinositide-3 kinase (PI3K), Akt, and p38 MAPK. These results suggest that dryofragin inhibited U2OS cell migration and invasion by reducing the expression of MMP-2 and MMP-9 and elevating the expression of TIMP-1 and TIMP-2 through the PI3K/AKT and p38 MAPK signaling pathways. Above all, we conclude that dryofragin represents an anti-invasive agent and may potentially be applicable in osteosarcoma therapy.

Notoginsenoside R1 stimulates osteogenic function in primary osteoblasts via estrogen receptor signaling.

Notoginsenoside R1 (NGR1), a novel phytoestrogen isolated from Panax notoginseng, has been widely used in the treatment of microcirculatory diseases in Asian countries. Here we investigated the effect of NGR1 on osteoblast differentiation and mineralization process. Furthermore, we also evaluated NGR1's estrogenic properties, especially its effects on estrogen receptors (ERs). NGR1 activated the transcriptional activity of phosphorylated estrogen response element (pERE)-luciferase (Luc) and induced ERα phosphorylation in hBMSC. In addition, ER activation correlated with induction and was associated with osteoblast differentiation biomarkers including alkaline phosphatase activity and transcription of osteoblastic genes, e.g., type I collagen (COL1), osteonectin, osteocalcin (OC), runt related protein 2 (Runx2), and osterix. NGR1 also promoted the mineralization process of osteoblasts. The NGR1-induced effects were confirmed to be mediated by the ER by the observation that pretreatment of the osteoblasts with the ER antagonist, ICI 182,780 fully blocked the effects. Our results showed that NGR1 stimulates osteogenic differentiation of cultured osteoblasts by activating ER signaling and in turn might be a potential therapeutic alternative for the prevention and treatment of osteoporosis.

Cell cycle arrest and apoptosis induced by aspidin PB through the p53/p21 and mitochondria-dependent pathways in human osteosarcoma cells.

Aspidin PB is a natural product extracted from Dryopteris fragrans (L.) Schott, which has been characterized for its various biological activities. We reported that aspidin PB induced cell cycle arrest and apoptosis through the p53/p21 and mitochondria-dependent pathways in human osteosarcoma cells. Aspidin PB inhibited the proliferation of Saos-2, U2OS, and HOS cells in a dose-dependent and time-dependent manner. Aspidin PB induced changes in the cell cycle regulators (cyclin A, pRb, CDK2, p53, and p21), which caused cell cycle arrest in the S phase. We also explored the role of siRNA targeted to p53; it led to a dose-dependent attenuation of aspidin PB-induced apoptosis signaling. Moreover, after treatment with aspidin PB, the p21-silenced cells decreased significantly at the S phase. Aspidin PB increased the percentage of cells with mitochondrial membrane potential disruption. Western blot analysis showed that aspidin PB inhibited Bcl-2 expression and induced Bax expression to disintegrate the outer mitochondrial membrane and caused cytochrome C release. Mitochondrial cytochrome C release was associated with the activation of caspase-9 and caspase-3 cascades. Furthermore, the double-stranded DNA breaks and reactive oxygen species signaling were both involved in aspidin PB-induced DNA damage. In addition, aspidin PB inhibited tumor growth significantly in U2OS xenografts. Above all, we conclude that aspidin PB represents a valuable natural source and may potentially be applicable in osteosarcoma therapy.