Human Osteosarcoma 143B Cells Research Articles

Pyropheophorbide-α methyl ester-mediated photodynamic therapy triggers pyroptosis in osteosarcoma cells via the ROS/caspase-3/GSDME pathway

BackgroundPyropheophorbide-α methyl ester-mediated photodynamic therapy(MPPa-PDT) is a candidate treatment for solid tumors, including osteosarcoma. Pyroptosis has garnered significant attention in cancer research due to its pro-inflammatory and immunomodulatory nature. This study investigated the mechanism and role of MPPa-PDT-induced pyroptosis in osteosarcoma cells. MethodsWe treated human osteosarcoma 143b and HOS cells with MPPa at concentrations of 0.5 μM and 0.25 μM, respectively, then irradiated the cells with LED light at 630 nm wavelength with an energy density of 4.8 J/cm2. Cell viability and apoptosis ratio were detected using CCK-8 and Annexin V–Propidium Iodide staining, respectively. Intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential (MtΔψ) were assessed using 2ʹ,7ʹ-Dichlorofluorescin diacetate, and JC-1 staining kits, respectively. Scanning Electron Microscopy (SEM) was utilized to examine cell ultrastructure. The morphological changes of the cells were observed by an inverted microscope. Western blotting analysis was conducted to measure protein levels. To elucidate the mechanism and role, we re-evaluated relevant parameters after pretreating with NAC,Si caspase-3, and Si GSDME. ResultsMPPa-PDT inhibited the activity of osteosarcoma 143b and HOS cells and induced pyroptosis with mitochondrial damage, ROS aggregation, and activation of Caspase-3 and GSDME. The effects of MPPa-PDT on the activity and apoptosis of osteosarcoma cells were partially reversed after pretreating with Si GSDME. After NAC pretreatment, the activation of pyroptosis and Caspase-3 induced by MPPa-PDT was partially reversed. After Si Caspase-3 pretreatment, the pyroptosis induced by MPPa-PDT was partially reversed. ConclusionMPPa-PDT can induce pyroptosis in osteosarcoma cells, which has the effect of enhancing apoptotic processes. Mitochondrial damage and ROS/caspase-3/GSDME pathway are the possible mechanisms of pyroptosis induced by MPPa-PDT.

Reduced Malignancy of Super Methotrexate-resistant Osteosarcoma Cells With Dihydrofolate Reductase Amplification Despite Paradoxical Gain of Oncogenic PI3K/AKT/mTOR and c-MYC expression.

Methotrexate (MTX) resistance in osteosarcoma leads to a very poor prognosis. In the present study, in order to further understand the basis and ramifications of MTX resistance in osteosarcoma, we selected an osteosarcoma cell line that has a 5,500-fold-increased MTX IC50 Materials and Methods: The super MTX-resistant 143B osteosarcoma cells (143B-MTXSR) were selected from MTX-sensitive parental human 143B osteosarcoma cells (143B-P) by continuous culture with step-wise increased amounts of MTX. To compare the malignancy of 143B-MTXSR and 143B-P, colony-formation capacity was compared with clonogenic assays on plastic and in soft agar. In addition, tumor growth was compared with orthotopic xenograft mouse models of osteosarcoma. Expression of dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and myelocytomatosis oncogene (MYC) was examined with western immunoblotting and compared in 143B-MTXSR and 143B-P cells. 143B-MTXSR had a 5,500-fold increase in the MTX IC50 compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTXSR compared to 143B-P (p<0.01). 143B-MTXSR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (p<0.01) compared to 143B-P and reduced tumor growth in orthotopic xenograft mouse models (p<0.001). These results demonstrate that 143B-MTXSR had reduced malignancy. 143B-MTXSR also showed an increased expression of PI3K (p<0.01), phosphorylated (activated) AKT (p=0.031), phosphorylated mTOR (p=0.043), and c-MYC (p=0.024) compared to 143B-P. The present study demonstrates that the increased expression of DHFR, PI3K/AKT/mTOR and c-MYC appears to be linked to super MTX resistance and, paradoxically, to reduced malignancy. The present results suggest that DHFR may be a powerful tumor suppressor when highly amplified.

Melittin Inhibits Growth of Human Osteosarcoma 143B Cells through Induction of Apoptosis via Suppressing the Wnt/β-catenin Signaling Pathway.

Osteosarcoma is the most commonly seen type of primary malignant bone tumors in children and adolescents. Partial patients with osteosarcoma cannot tolerate the side effects of chemotherapy drugs. Hence, it is urgent to find anti-osteosarcoma drugs with low side effects. Melittin is an anti-tumor Traditional Chinese Medicine with low side effects. The purpose of this study was to explore the anti-osteosarcoma effect of melittin and its possible molecular mechanisms. The effects of melittin on cell growth were detected by CCK-8, clonal formation, and flow cytometry. The related molecules were also investigated by Real-time PCR and Western blot. A xenograft model in nude mice was established to observe the effects of melittin on tumor growth and the related molecular expression was detected by immunohistochemistry. Melittin can inhibit the proliferation of osteosarcoma 143B cells, reduce colony formation, and induce apoptosis while significantly up-regulating the expression of Bax and Caspase-3 and down-regulating the expression of Bcl-2 proteins. Moreover, treatment with melittin significantly reduced the mRNA and protein levels of β-catenin and Wnt/β- catenin related genes (LRP5, c-Myc, and Survivin) in osteosarcoma 143B cells in vitro. The xenograft model found that melittin significantly inhibited tumor growth and decreased the protein expression levels of β-catenin and Wnt/β- catenin related genes in vivo. These findings show that melittin could inhibit the growth of osteosarcoma 143B cells, which may be related to the inhibition of Wnt/β-catenin signaling pathway activity and induce apoptosis by up-regulating the ratio of Bax/Bcl-2 in osteosarcoma 143B cells. Therefore, melittin is a promising anti-tumor drug for the treatment of osteosarcoma.

A Selective Reduction of Osteosarcoma by Mitochondrial Apoptosis Using Hydroxyapatite Nanoparticles.

BackgroundIn recent years, using hydroxyapatite nanoparticles (HANPs) for tumor therapy attracted increasing attention because HANPs were found to selectively suppress the growth of tumor cells but exhibit ignorable toxicity to normal cells.PurposeThis study aimed to investigate the capacities of HANPs with different morphologies and particle sizes against two kinds of osteosarcoma (OS) cells, human OS 143B cells and rat OS UMR106 cells.MethodsSix kinds of HANPs with different morphologies and particle sizes were prepared by wet chemical method. Then, the antitumor effect of these nanoparticles was characterized by means of in vitro cell experiments and in vivo tumor-bearing mice model. The underlying antitumor mechanism involving mitochondrial apoptosis was also investigated by analysis of intracellular calcium, expression of apoptosis-related genes, reactive oxygen species (ROS), and the endocytosis efficiency of the particles in tumor cells.ResultsBoth in vitro cell experiments and in vivo mice model evaluation revealed the anti-OS performance of HANPs depended on the concentration, morphology, and particle size of the nanoparticles, as well as the OS cell lines. Among the six HANPs, rod-like HANPs (R-HANPs) showed the best inhibitory activity on 143B cells, while needle-like HANPs (N-HANPs) inhibited the growth of UMR106 cells most efficiently. We further demonstrated that HANPs induced mitochondrial apoptosis by selectively raising intracellular Ca2+ and the gene expression levels of mitochondrial apoptosis-related molecules, and depolarizing mitochondrial membrane potential in tumor cells but not in MC3T3-E1, a mouse pre-osteoblast line. Additionally, the anti-OS activity of HANPs also linked with the endocytosis efficiency of the particles in the tumor cells, and their ability to drive oxidative damage and immunogenic cell death (ICD).ConclusionThe current study provides an effective strategy for OS therapy where the effectiveness was associated with the particle morphology and cell line.

Reduction of invasion and cell stemness and induction of apoptotic cell death by Cinnamomum cassia extracts on human osteosarcoma cells.

Cinnamomum cassia possesses antioxidative activity and induces the apoptotic properties of various cancer types. However, its effect on osteosarcoma invasion and cancer stemness remains ambiguous. Here, we examined the molecular evidence of the anti-invasive effects of ethanoic C. cassia extracts (CCE). Invasion and migration were obviously suppressed after the expression of urokinase-type plasminogen activator and matrix metalloprotein 2 in human osteosarcoma 143B cells were downregulated. CCE reversed epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor β1 and downregulated mesenchymal markers, such as snail-1 and RhoA. CCE suppressed self-renewal property and the expression of stemness genes (aldehyde dehydrogenase, Nanog, and CD44) in the 143B cells. CCE suppressed cell viability, reduced the colony formation of osteosarcoma cancer cells, and induced apoptotic cell death in the 143B cells, as indicated by caspase-9 activation. The xenograft tumor model of immunodeficient BALB/c nude mice showed that CCE administered in vivo through oral gavage inhibited the growth of implanted 143B cells. These findings indicated that CCE inhibited the invasion, migration, and cancer stemness of the 143B cells. CCE reduced proliferation of 143B cell possibly because of the activation of caspase-9 and the consequent apoptosis, suggesting that CCE is a potential anticancer supplement for osteosarcoma.

GO-Y078, a Curcumin Analog, Induces Both Apoptotic Pathways in Human Osteosarcoma Cells via Activation of JNK and p38 Signaling.

Osteosarcoma is the most common primary bone malignancy in teenagers and continues to confer a generally poor prognosis due to its highly metastatic potential. Poor solubility in water and instability of curcumin limits its bioavailability for use in the adjuvant situation to improve the prognosis and the long-term survival of patients with osteosarcoma. To further obtain information regarding the apoptosis induced by a new curcumin analog, GO-Y078, in human osteosarcoma cells, flow cytometric analysis, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. GO-Y078 dose-dependently decreased viabilities of human osteosarcoma U2OS, MG-63, 143B, and Saos-2 cells and induced sub-G1 fraction arrest and apoptosis in U2OS and 143B cells. In addition to the effector caspase 3 and poly adenosine diphosphate-ribose polymerase, GO-Y078 significantly activated both initiators of extrinsic caspase 8 and intrinsic caspase 9, whereas cellular inhibitors of apoptosis 1 (cIAP-1) and X-chromosome-linked IAP (XIAP) in U2OS and 143B cells were significantly repressed. Moreover, GO-Y078 increased phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2, and p38 in U2OS and 143B cells. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), GO-Y078′s increases in cleaved caspases 8, 9, and 3 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Altogether, GO-Y078 simultaneously induces both apoptotic pathways and cell arrest in U2OS and 143B cells through activating JNK and p38 signaling and repressing IAPs. These findings contribute to a better understanding of the mechanisms responsible for GO-Y078′s apoptotic effects on human osteosarcoma cells.

MiR‐124‐3p signaling in the control of metastasis‐related cytoskeleton organization, cell junctions and adhesion

Our recent studies using novel bioengineered miR-124-3p molecules have demonstrated the functions of miR-124-3p in the inhibition of human osteosarcoma cell invasiveness in vitro and lung metastasis in vivo. This study is to investigate the molecular and cellular mechanisms by which miR-124-3p controls tumor metastasis. LC-MS-based proteomics study identified a set of proteins selectively and significantly downregulated by bioengineered miR-124-3p, which were assembled into multiple biological pathways critical for cancer, such as cell junctions and focal adhesion. Among these proteins, we identified and verified plectin (PLEC) as a new direct target for miR-124-3p that links cytoskeleton to intercellular junctions. Associated with significantly lower levels of PLEC, ITGB1, IQGAP1 and CDH2 proteins in miR-124-3p-treated human osteosarcoma 143B and MG63 cells as well as NSCLC A549 cells, cytoskeleton was remodeled and cell-cell junctions were sharply inhibited. Furthermore, miR-124-3p decreased the ability of cells to form focal adhesion complexes and plaques, leading to much lower levels of cell adhesion capacity. Together, our results connect miR-124-3p-PLEC to other known elements in the control of cytoskeleton, cell adhesion and junctions essential for cancer cell invasion and extravasation towards metastasis.

Resveratrol Derivative, Trans-3, 5, 4'-Trimethoxystilbene Sensitizes Osteosarcoma Cells to Apoptosis via ROS-Induced Caspases Activation.

Numerous studies have shown that resveratrol can induce apoptosis in cancer cells. Trans-3, 5, 4′-trimethoxystilbene (TMS), a novel derivative of resveratrol, is a more potent anticancer compound than resveratrol and can induce apoptosis in cancer cells. Herein, we examined the mechanisms involved in TMS-mediated sensitization of human osteosarcoma (143B) cells to TNF-related apoptosis-inducing ligand- (TRAIL-) induced apoptosis. Our results showed that cotreatment with TSM and TRAIL activated caspases and increased PARP-1 cleavage in 143B cells. Decreasing cellular ROS levels using NAC reversed TSM- and TRAIL-induced apoptosis in 143B cells. NAC abolished the upregulated expression of PUMA and p53 induced by treatment with TRAIL and TSM. Silencing the expression of p53 or PUMA using RNA interference attenuated TSM-mediated sensitization of 143B cells to TRAIL-induced apoptosis. Knockdown of Bax also reversed TSM-induced sensitization of 143B cell to TRAIL-mediated apoptotic cell death. These results indicate that cotreatment with TRAIL and TSM evaluated intracellular ROS level, promoted DNA damage, and activated the Bax/PUMA/p53 pathway, leading to activation of both mitochondrial and caspase-mediated apoptosis in 143B cells. Orthotopic implantation of 143B cells in mice also demonstrated that cotreatment with TRAIL and TSM reversed resistance to apoptosis in cells without obvious adverse effects in normal cells.

Calycosin induces apoptosis via p38‑MAPK pathway‑mediated activation of the mitochondrial apoptotic pathway in human osteosarcoma 143B cells.

Previous studies have demonstrated that calycosin is a natural phytoestrogen with a similar structure to estrogen, which can inhibit cell proliferation and induce apoptosis in a variety of tumors. Calycosin exerts potential pharmacological effects on osteosarcoma cells by inducing apoptosis. The aim of the present study was to elucidate the specific molecular mechanism of calycosin-induced apoptosis in osteosarcoma cells. Cell proliferation was determined by an MTT assay. Annexin V/PI and JC-1 staining were used to detect apoptosis and mitochondrial dysfunction, respectively, by flow cytometry. Western blot analysis was used to detect the expression of caspases or mitochondrial proteins. The results revealed that calycosin reduced the cell viability of human osteosarcoma 143B cells, induced apoptosis and increased the loss of mitochondrial membrane potential (MMP). In addition, calycosin increased the expression of the proapoptotic antiapoptotic proteins cleaved caspase-3, cleaved caspase-9, cleaved poly(ADP-ribose) polymerase and Bcl-2-associated X protein (Bax), and decreased the expression of the antiapoptotic proapoptotic protein B-cell lymphoma-2 (Bcl-2), thus altering the Bax/Bcl-2 ratio. In addition, the expression levels of cytochrome c were markedly decreased in the mitochondria and increased in the cytoplasm following calycosin treatment. Furthermore, calycosin treatment induced p38-mitogen-activated protein kinase (MAPK) phosphorylation, whereas the p38-MAPK inhibitor BIRB 796 markedly reversed cell viability, apoptosis and loss of MMP in 143B cells. These results suggested that calycosin inhibited osteosarcoma 143B cell growth via p38-MAPK regulation of mitochondrial-dependent intrinsic apoptotic pathways.

Wnt/β‑catenin signaling modulates piperine‑mediated antitumor effects on human osteosarcoma cells.

The plant extract piperine is used as a traditional Chinese medicine due to its anti-inflammatory effects and efficacy against numerous types of cancer. The aim of the present study was to investigate the antitumor mechanism of piperine in human osteosarcoma U2OS and 143B cell lines. The effects of piperine on cell apoptosis and invasion of human osteosarcoma cells were assessed using flow cytometry and Transwell assays. Moreover, western blotting was used to measure the effects of piperine on the protein expression levels of the metastasis markers matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF). In addition, the involvement of the Wnt/β-catenin signaling pathway in modulating the effects of piperine was examined via western blot analysis. The results of MTT and Transwell invasion assays indicated that piperine treatment dose-dependently reduced U2OS and 143B cell viability and invasion. Furthermore, a significant reduction was identified in MMP-2, VEGF, glycogen synthase kinase-3β and β-catenin protein expression levels, as well as the expression levels of their target proteins cyclooxygenase-2, cyclin D1 and c-myc, in U2OS cells after piperine treatment. In addition, similar results were observed in 143B cells. Therefore, the present study demonstrated the efficacy of piperine in osteosarcoma, and identified that the Wnt/β-catenin signaling pathway may modulate the antitumor effects of piperine on human U2OS and 143B cells.

Mycophenolic acid is a drug with the potential to be repurposed for suppressing tumor growth and metastasis in osteosarcoma treatment.

Our previous review of proteomics data showed that in osteosarcoma, some overexpressed proteins were targets of FDA-approved immunosuppressive and anti-arrhythmic drugs, including mycophenolate mofetil (MMF), ribavirin, leflunomide, azathioprine and digoxin. Here, these drugs were screened for growth inhibitory effects in human osteosarcoma cell lines, including MNNG/HOS, U2OS, SaOS-2, MG-63 and 143B cells. Only mycophenolic acid (MPA), an active metabolite of MMF, efficiently inhibited osteosarcoma cell growth with IC50 values of 0.46-7.3 μM; these values are in the therapeutic range for organ transplant patients. At a therapeutic dose (10 μM), MPA significantly inhibited colony formation, caused cell cycle arrest in the S phase, and induced apoptosis. Moreover, the in vitro invasion of osteosarcoma cells was reduced by MPA by inhibiting cell migration capability. The in vivo antitumor effect of MMF was determined in nude mice harboring 143B cell xenografts. Daily oral administration of 200 mg/kg/day MMF for 2 weeks significantly suppressed tumor growth in treated mice, achieving 57.4 ± 11.1% tumor growth inhibition. Compared with the vehicle group, the MMF group treated with 50-200 mg/kg/day for 3 weeks had a significant reduction in the number of lung metastatic nodules in a tail vein-lung metastasis model of 143B cells. MMF doses of 50, 100 and 200 mg/kg/day are approximately equivalent to the non-toxic doses of 0.25, 0.5 and 1 g/day in humans, respectively. These findings indicate that MPA/MMF can effectively control osteosarcoma tumor growth and metastasis. Thus, the potential to repurpose MPA/MMF for use in osteosarcoma chemotherapy is of great interest.

Targeting the Wnt/β-catenin pathway in human osteosarcoma cells.

Aberrant activation of Wnt signaling has been implicated in human osteosarcoma, which may provide a genetic vulnerability that can be targeted in osteosarcoma treatment. To test whether Wnt activation is necessary for osteosarcoma growth, colony formation, invasion, and metastasis, we treated human osteosarcoma cells with a small molecule inhibitor of Wnt/β-catenin, PRI-724, which suppresses Wnt/β-catenin-mediated transcription. We found increased protein levels of endogenous active-β-catenin in five human osteosarcoma cell lines. Treatment with PRI-724 was sufficient to inhibit human osteosarcoma 143B and SJSA-1 cell proliferation. Suppressed Wnt signaling was confirmed by decreased protein levels of the Wnt target Cyclin D1. Furthermore, we revealed significant inhibitory effects on cell migration, invasion, and colony formation in the human osteosarcoma cells. Using deposited data from next generation sequencing studies, we analyzed somatic mutations and gene expression of components in the Wnt/β-catenin pathway. We found somatic mutations and upregulated gene expression of many components in the Wnt/ β-catenin pathway, indicating activated Wnt signaling. Taken together, our results illustrate the critical role of Wnt/β-catenin signaling in human osteosarcoma pathogenesis and growth, as well as the therapeutic potential of Wnt inhibitors in the treatment of human osteosarcoma.

HGK-sestrin 2 signaling-mediated autophagy contributes to antitumor efficacy of Tanshinone IIA in human osteosarcoma cells

Tanshinone IIA (TIIA) is a diterpenoid naphthoquinone isolated from the herb Salvia miltiorrhiza with antitumor effects manifested at multiple levels that are mechanistically obscure. In our previous studies, we illustrated that TIIA treatment triggered apoptosis in human osteosarcoma 143B cells both in vitro and in vivo, accompanied with mitochondrial dysfunction. Importantly, the overall survival rate of patients with osteosarcoma who were randomly recruited to S. miltiorrhiza treatment was significantly higher than those without. Pursuing this observation, we evaluated the potential effect of TIIA on autophagy induction in osteosarcoma both in vivo and in vitro. We discovered that TIIA inhibited osteosarcoma cell survival through class I PI3K and Akt signaling pathways. In contrast, expression of class III PI3K required in the early stages of autophagosome generation was predominantly enhanced by TIIA treatment. Our study indicated that treatment of TIIA effectively induced autophagy in human osteosarcoma cells, which contributed to the blockade of anchorage-independent growth of osteosarcoma cells and ameliorated tumor progression in NOD/SCID mice. We demonstrated that TIIA-mediated autophagy occurred in a sestrin 2 (SESN2)-dependent but not Beclin 1-dependent manner. In addition, we defined the activation of HGK (MAP4K4 or mitogen-activated protein kinase kinase kinase kinase)/SAPK/JNK1/Jun kinase pathways in upregulating transcription of SESN2, in which TIIA triggered HGK/JNK1-dependent Jun activation and led to increased Jun recruitment to AP-1-binding site in the SESN2 promoter region. Our results offer novel mechanistic insight into how TIIA inhibits osteosarcoma growth and suggest TIIA as a promising therapeutic agent for the treatment of cancer.

Imaging the interaction of α v integrin‐GFP in osteosarcoma cells with RFP‐expressing host stromal cells and tumor‐scaffold collagen in the primary and metastatic tumor microenvironment

Human osteosarcoma 143B cells were previously stably transfected with an αv integrin green flourescent protein (GFP) vector. 143B cells expressing αv integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of αv integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing αv integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing αv integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing αv integrin-GFP in the lung. The current study demonstrates the importance of αv integrin interaction with stromal elements in osteosarcoma.

Sequence analysis of cell-free DNA derived from cultured human bone osteosarcoma (143B) cells.

The true importance of cell-free DNA in human biology, together with the potential scale of its clinical utility, is tarnished by a lack of understanding of its composition and origin. In investigating the cell-free DNA present in the growth medium of cultured 143B cells, we previously demonstrated that the majority of cell-free DNA is neither a product of apoptosis nor necrosis. In the present study, we investigated the composition and origin of this cell-free DNA population using next-generation sequencing. We found that the cell-free DNA comprises mainly of repetitive DNA, including α-satellite DNA, mini satellites, and transposons that are currently active or exhibit the capacity to become reactivated. A significant portion of these cell-free DNA fragments originates from specific chromosomes, especially chromosomes 1 and 9. In healthy adult somatic cells, the centromeric and pericentromeric regions of these chromosomes are normally densely methylated. However, in many cancer types, these regions are preferentially hypomethylated. This can lead to double-stranded DNA breaks or it can directly impair the formation of proper kinetochore structures. This type of chromosomal instability is a precursor to the formation of nuclear anomalies, including lagging chromosomes and anaphase bridges. DNA fragments derived from these structures can recruit their own nuclear envelope and form secondary nuclear structures known as micronuclei, which can localize to the nuclear periphery and bud out from the membrane. We postulate that the majority of cell-free DNA present in the growth medium of cultured 143B cells originates from these micronuclei.

Sensitive electrochemical cytosensor for highly specific detection of osteosarcoma 143B cells based on graphene-3D gold nanocomposites

A novel electrochemical cytosensor was developed for detection of human osteosarcoma 143B cells based on graphene-three dimensional nanostructure gold nanocomposites (G-3D Au) using facile and rapid one-step electrochemical co-reduction technique. Characterizations by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that the gold nanoparticles in the scale of about 5–80 nm were deposited on the graphene sheets - showing pinecone-like 3D gold nanostructures. The proposed cytosensor, due to the increasing surface area and enhanced electron transfer rate by the nanocomposite, provided a highly stable and efficient matrix for the convenient conjugation of target CXCR4 antibody and the following incubation of cells. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) showed that the developed cytosensor was able to detect 143B cells ranging from 5.0 × 103 to 5 × 106 cells mL−1 with a lower detection limit of 1292 cells mL−1. This electrochemical cytosensor showed a fast response, high sensitivity and selectivity, which could be used in various applications such as cancer diagnosis, drug screening and cell adhesion studies.

Study on the inhibitory effect of rhodioside on osteosarcoma 143 cells invasion, migration and potential mechanism

Objective To investigate the effect of rhodioside on the proliferation, invasion and migration of human osteosarcoma 143B cells and its possible mechanism. Methods Osteosarcoma 143B cells were cultured in dulbeeeo modified eagle medium (DMEM) medium (including 10% serum and 1% double resistance). Osteosarcoma 143B cells were divided into control group, 30 and 60 μmol/L rhodioside treatment group. Cell proliferation was tested by methyl thiazol tetrazolium (MTT) assay at 24, 36, 72 h, Transwell method was used to detect three groups of cell invasion; migration ability were detected by cell scratch assay. After 24 h treatment, calculation of cell migration rate. After 24 h treatment, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the relative expression level of matrix metalloproteinase (MMP)-2 and MMP-9 mRNA. The protein expression level of MMP-2 and MMP-9 in each group was detected by Western blotting. The autophagy after 24 h was detected by transmission electron microscopy. Results After the treatment of 24 h, the absorbance value of 60 mol/L group (0.038±0.002) and 30 mol/L group (0.054±0.002) 143B cells was lower than that of the control group. Compared with the control group, rhodioside with concentration of 30 and 60 mol/L at 48 h and 72 h inhibited the proliferation of 143B cells, respectively, with a dose-dependent and time dependent manner. The number of transmembrane of 143B cells in 60 μmol/L (48.7±8.2) and 30 μmol/L (76.8±12.8) rhodioside group was less than that of the control group (94.5±11.9), t1=8.056, P=0.000; t=2.472, P=0.038, respectively. The migration rates of 143B cells in 60 μmol/L [(52.9±6.8)%] and 30 μmol/L [(82.6±3.7)%] rhodioside group were lower than those of the control group [(98.7±0.3)%, t=12.576, P=0.000, t=9.794, P=0.000], respectively. The relative expression of protein and mRNA of MMP-2 and MMP-9 in 60 mol/L and 30 mol/L rhodioside treatment group was lower than that of the control group. The number of autophage in the 60 mol/L rhodioside group was (2.6±0.5) more than that of the control group (1.3±0.4, t=3.517, P=0.025). Conclusion Rhodioside inhibits the proliferation, migration and invasion of osteosarcoma cells, and its mechanism may be related to the induction of autophagy and the inhibition of the expression of MMP family protein. Key words: Osteosarcoma; Rhodioside; Migration; Invasion; Autophagy

SK-216, a Novel Inhibitor of Plasminogen Activator Inhibitor-1, Suppresses Lung Metastasis of Human Osteosarcoma.

Lung metastasis constitutes the leading cause of the death in patients with osteosarcoma. We have previously reported that plasminogen activator inhibitor-1 (PAI-1) regulates the invasion and lung metastasis of osteosarcoma cells in a mouse model and as well as in clinical samples. In the present study, we examined the anti-metastatic effect of SK-216, a small compound PAI-1 inhibitor, in human 143B osteosarcoma cells. An in vitro study showed that SK-216 treatment suppressed invasion activity by inhibiting PAI-1 expression in 143B cells, but had no influence on their proliferation or migration. 143B cells treated with SK-216 exhibited reduced matrix metalloproteinase-13 (MMP-13) secretion in a dose-dependent manner. Moreover, intraperitoneal injection of SK-216 into mouse models resulted in downregulation of PAI-1 expression levels in the primary tumors and showed suppression of lung metastases without influencing the proliferative activity of the tumor cells in the primary lesions. These results indicate that SK-216, a PAI-1 inhibitor, may serve as a novel drug to prevent lung metastasis in human osteosarcoma.

Loss of the Mitochondrial Fatty Acid \u03b2-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function

Medium-chain acyl-Coenzyme A dehydrogenase (MCAD) is involved in the initial step of mitochondrial fatty acid β-oxidation (FAO). Loss of function results in MCAD deficiency, a disorder that usually presents in childhood with hypoketotic hypoglycemia, vomiting and lethargy. While the disruption of mitochondrial fatty acid metabolism is the primary metabolic defect, secondary defects in mitochondrial oxidative phosphorylation (OXPHOS) may also contribute to disease pathogenesis. Therefore, we examined OXPHOS activity and stability in MCAD-deficient patient fibroblasts that have no detectable MCAD protein. We found a deficit in mitochondrial oxygen consumption, with reduced steady-state levels of OXPHOS complexes I, III and IV, as well as the OXPHOS supercomplex. To examine the mechanisms involved, we generated an MCAD knockout (KO) using human 143B osteosarcoma cells. These cells also exhibited defects in OXPHOS complex function and steady-state levels, as well as disrupted biogenesis of newly-translated OXPHOS subunits. Overall, our findings suggest that the loss of MCAD is associated with a reduction in steady-state OXPHOS complex levels, resulting in secondary defects in OXPHOS function which may contribute to the pathology of MCAD deficiency.

Interleukin-24 inhibits osteosarcoma cell migration and invasion via the JNK/c-Jun signaling pathways.

Approximately 25% of osteosarcoma patients present with clinically detectable metastatic disease at the time of initial diagnosis. High-dose chemotherapy and/or surgery for the treatment of primary metastatic osteosarcoma is ineffective, and <20% of patients will survive 5 years from diagnosis. Therefore, the treatment of metastases is critical for the improvement of the prognosis of primary metastatic osteosarcoma patients. We have previously observed that overexpression of interleukin-24 (IL-24) inhibits neuroblastoma cell proliferation, migration and invasion in vitro. The present study investigated whether IL-24 may be a novel agent for osteosarcoma metastasis-suppressive treatment. It was observed that IL-24 is able to inhibit migration and invasion in spontaneously metastasizing human 143B osteosarcoma cells via the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway. IL-24 was effective in inhibiting JNK and c-Jun phosphorylation to downregulate matrix metalloproteinase (MMP)-2 and MMP-9, which contributed to the suppression of cell migration and invasion. It was concluded that IL-24 may be a potent agent in the inhibition of highly metastatic 143B osteosarcoma cells, and IL-24 may have translational potential as an effective therapeutic agent for the treatment of metastatic osteosarcoma.