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The relationship between metastasis-associated protein 2 (MTA2) overexpression and tumor growth and metastasis has been extensively studied in a variety of tumor cells but not in human osteosarcoma cells. This study aims to elucidate the clinical significance, underlying molecular mechanisms, and biological functions of MTA2 in human osteosarcoma in vitro and in vivo . Our results show that MTA2 was elevated in osteosarcoma cell lines and osteosarcoma tissues and was associated with tumor stage and overall survival of osteosarcoma patients. Knockdown of MTA2 inhibited osteosarcoma cell migration and invasion by reducing the expression of urokinase-type plasminogen activator (uPA). Bioinformatic analysis demonstrated that high levels of uPA in human osteosarcoma tissues correlated positively with MTA2 expression. Furthermore, treatment with recombinant human uPA (Rh-uPA) caused significant restoration of OS cell migration and invasion in MTA2 knockdown osteosarcoma cells. We found that ERK1/2 depletion increased the expression of uPA, facilitating osteosarcoma cell migration and invasion. Finally, MTA2 depletion significantly reduced tumor metastasis and the formation of lung nodules in vivo . Overall, our study suggests that MTA2 knockdown suppresses osteosarcoma cell metastasis by decreasing uPA expression via ERK signaling. This finding provides new insight into potential treatment strategies against osteosarcoma metastasis by targeting MTA2.

Osteosarcoma is the most prevalent form of primary malignant bone tumor, primarily affecting children and adolescents. The nerve growth factors (NGF) referred to as neurotrophins have been associated with cancer-induced bone pain; however, the role of NGF in osteosarcoma has yet to be elucidated. In osteosarcoma samples from the Genomic Data Commons data portal, we detected higher levels of NGF and M2 macrophage markers, but not M1 macrophage markers. In cellular experiments, NGF-stimulated osteosarcoma conditional medium was shown to facilitate macrophage polarization from the M0 to the M2 phenotype. NGF also enhanced VCAM-1-dependent monocyte adhesion within the osteosarcoma microenvironment by down-regulating miR-513c-5p levels through the FAK and c-Src cascades. In in vivo xenograft models, the overexpression of NGF was shown to enhance tumor growth, while the oral administration of the TrK inhibitor larotrectinib markedly antagonized NGF-promoted M2 macrophage expression and tumor progression. These results suggest that larotrectinib could potentially be used as a therapeutic agent aimed at mitigating NGF-mediated osteosarcoma progression., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Osteosarcoma, a highly aggressive bone cancer, often develops resistance to conventional chemotherapeutics, leading to poor prognosis and survival rates. The malignancy and chemoresistance of osteosarcoma pose significant challenges in its treatment, highlighting the critical need for novel therapeutic approaches. Bruton's tyrosine kinase (BTK) plays a pivotal role in B-cell development and has been linked to various cancers, including breast, lung, and oral cancers, where it contributes to tumor growth and chemoresistance. Despite its established importance in these malignancies, the impact of BTK on osteosarcoma remains unexplored. Our study delves into the expression levels of BTK in osteosarcoma tissues by data from the GEO and TCGA database, revealing a marked increase in BTK expression compared with primary osteoblasts and a potential correlation with primary site progression. Through our investigations, we identified a subset of osteosarcoma cells, named cis-HOS, which exhibited resistance to cisplatin. These cells displayed characteristics of cancer stem cells (CSCs), demonstrated a higher angiogenesis effect, and had an increased migration ability. Notably, an upregulation of BTK was observed in these cisplatin-resistant cells. The application of ibrutinib, a BTK inhibitor, significantly mitigated these aggressive traits. Our study demonstrates that BTK plays a crucial role in conferring chemoresistance in osteosarcoma. The upregulation of BTK in cisplatin-resistant cells was effectively countered by ibrutinib. These findings underscore the potential of targeting BTK as an effective strategy to overcome chemoresistance in osteosarcoma treatment., (© 2024 Wiley Periodicals LLC.)

New therapeutic approaches are needed for metastatic osteosarcoma (OS), as survival rates remain low despite surgery and chemotherapy. Epigenetic changes, such as histone H3 methylation, play key roles in many cancers including OS, although the underlying mechanisms are not clear. In this study, human OS tissue and OS cell lines displayed lower levels of histone H3 lysine trimethylation compared with normal bone tissue and osteoblast cells. Treating OS cells with the histone lysine demethylase inhibitor 5-carboxy-8-hydroxyquinoline (IOX-1) dose-dependently increased histone H3 methylation and inhibited cellular migratory and invasive capabilities, suppressed matrix metalloproteinase expression, reversed epithelial-to-mesenchymal transition by increasing levels of epithelial markers E-cadherin and ZO-1 and decreasing the expression of mesenchymal markers N-cadherin, vimentin, and TWIST, and also reduced stemness properties. An analysis of cultivated MG63 cisplatin-resistant (MG63-CR) cells revealed lower histone H3 lysine trimethylation levels compared with levels in MG63 cells. Exposing MG63-CR cells to IOX-1 increased histone H3 trimethylation and ATP-binding cassette transporter expression, potentially sensitizing MG63-CR cells to cisplatin. In conclusion, our study suggests that histone H3 lysine trimethylation is associated with metastatic OS and that IOX-1 or other epigenetic modulators present promising strategies to inhibit metastatic OS progression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Osteosarcoma is a highly mortal bone tumor, with a high metastatic potential, promoted in part by the enzyme procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2). Increasing level of PLOD2 in osteosarcoma tissue correlates with lymphatic and distant metastasis. The adipokine apelin (APLN) is also found in different cancers and APLN upregulation promotes angiogenesis and metastasis, but its effects on osteosarcoma metastasis are uncertain. We explored APLN functioning in metastatic osteosarcoma. An analysis of records from the Gene Expression Omnibus (GEO) database showed higher levels of APLN expression in osteosarcoma tissue than in normal tissue. Similarly, levels of APLN and PLOD2 mRNA synthesis were upregulated in osteosarcoma tissue. Levels of APLN and PLOD2 protein correlated positively with osteosarcoma clinical stages. APLN increased PLOD2 expression in human osteosarcoma cell lines and cell migration via the mammalian Sterile 20-like kinase 1 (MST1), monopolar spindle-one-binder protein (MOB)1, and YAP cascades, and through hsa_circ_0000004 functioning as a sponge of miR-1303. We also found that knockdown of APLN antagonized lung metastasis in mice with osteosarcoma. APLN may be a therapeutic target in osteosarcoma metastasis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Osteosarcoma is the most common type of primary malignant bone cancer, and it is associated with high rates of pulmonary metastasis. Integrin αvβ3 is critical for osteosarcoma cell migratory and invasive abilities. Chemokine (C-C motif) ligand 4 (CCL4) has diverse effects on different cancer cells through its interaction with its specific receptor, C-C chemokine receptor type 5 (CCR5). Analysis of mRNA expression in human osteosarcoma tissue identified upregulated levels of CCL4, integrin αv and β3 expression. Similarly, an analysis of records from the Gene Expression Omnibus (GEO) dataset showed that CCL4 was upregulated in human osteosarcoma tissue. Importantly, the expression of both CCL4 and integrin αvβ3 correlated positively with osteosarcoma clinical stages and lung metastasis. Analysis of osteosarcoma cell lines identified that CCL4 promotes integrin αvβ3 expression and cell migration by activating the focal adhesion kinase (FAK), protein kinase B (AKT), and hypoxia inducible factor 1 subunit alpha (HIF-1α) signaling pathways, which can downregulate microRNA-3927-3p expression. Pharmacological inhibition of CCR5 by maraviroc (MVC) prevented increases in integrin αvβ3 expression and cell migration. This study is the first to implicate CCL4 as a potential target in the treatment of metastatic osteosarcoma.

Aims: Insulin-like growth factor (IGF) signaling has been documented in several human malignancies and is thought to contribute to cellular differentiation and migration, as well as malignant progression. A major binding molecule of IGF, IGF-binding protein 3 (IGFBP-3), regulates multiple IGF effects. Here, we focused on the effect of IGFBP-3 in the motility of osteosarcoma cells and examined signaling regulation., Materials and Methods: Using a human osteosarcoma tissue array, immunohistochemical staining determined levels of IGFBP-3 expression in osteosarcoma tissue and in normal tissue. The wound healing migration assay, Transwell migration assay, luciferase reporter assay, immunofluorescence staining, Western blot and real-time quantitative PCR were performed to examine whether IGFBP-3 facilitates VCAM-1-dependent migration of osteosarcoma cells., Key Findings: In this study, we found significantly higher IGFBP-3 levels in osteosarcoma tissue compared with normal healthy tissue. IGFBP-3 treatment of two human osteosarcoma cell lines promoted cell migration and upregulated levels of VCAM-1 expression via PI3K/Akt and AP-1 signaling., Significance: IGFBP-3 appears to be a novel therapeutic target in metastatic osteosarcoma., (Copyright © 2020. Published by Elsevier Inc.)

Osteosarcoma is the most common primary tumor of the skeletal system and is well-known to have an aggressive clinical outcome and high metastatic potential. The chemokine (C-X-C motif) ligand 13 (CXCL13) plays a vital role in the development of several cancers. However, the effect of CXCL13 in the motility of osteosarcoma cells remains uncertain. Here, we found that CXCL13 increases the migration and invasion potential of three osteosarcoma cell lines. In addition, CXCL13 expression was upregulated in migration-prone MG-63 cells. Vascular cell adhesion molecule 1 (VCAM-1) siRNA and antibody demonstrated that CXCL13 promotes migration via increasing VCAM-1 production. We also show that CXCR5 receptor controls CXCL13-mediated VCAM-1 expression and cell migration. Our study identified that CXCL13/CXCR5 axis facilitate VCAM-1 production and cell migration in human osteosarcoma via the phospholipase C beta (PLCβ), protein kinase C α (PKCα), c-Src, and nuclear factor-κB (NF-κB) signaling pathways. CXCL13 and CXCR5 appear to be a novel therapeutic target in metastatic osteosarcoma.

Over the last two decades, there have been no significant changes in patient outcomes in relation to the treatment of osteosarcoma, an aggressive malignant neoplasm. It is known that vascular endothelial growth factor-A (VEGF-A) plays a crucial role in angiogenesis and in osteosarcoma. Moreover, VEGF-A expression correlates with clinical stages of osteosarcoma. The adipokine resistin exhibits proinflammatory, proangiogenic and metastatic properties, and evidence suggests that resistin may serve as a prognostic biomarker linking obesity and inflammation to cancer. However, whether resistin has a role in osteosarcoma angiogenesis is unclear. This investigation shows that resistin promotes VEGF-A expression in human osteosarcoma cells and activates the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 signaling pathways, while ERK, JNK, and p38 inhibitors or their small interfering RNAs (siRNAs) inhibit resistin-induced VEGF-A expression as well as endothelial progenitor cell (EPC) migration and tube formation. We also found that resistin upregulates VEGF-A expression by enhancing activation of the transcription factor nuclear factor-kappa B (NF-κB). Finally, resistin promotes angiogenesis in the chick chorioallantoic membrane (CAM) model. Resistin appears to be a promising target for human osteosarcoma.

In recent years, osteosarcoma survival rates have failed to improve significantly with conventional treatment modalities because of the development of chemotherapeutic resistance. The human breast cancer resistance protein/ATP binding cassette subfamily G member 2 (BCRP/ABCG2), a member of the ATP-binding cassette family, uses ATP hydrolysis to expel xenobiotics and chemotherapeutics from cells. CCN family member 2 (CCN2) is a secreted protein that modulates the biological function of cancer cells, enhanced ABCG2 protein expression and activation in this study via the α6β1 integrin receptor and increased osteosarcoma cell viability. CCN2 treatment downregulated miR-519d expression, which promoted ABCG2 expression. In a mouse xenograft model, knockdown of CCN2 expression increased the therapeutic effect of doxorubicin, which was reversed by ABCG2 overexpression. Our data show that CCN2 increases ABCG2 expression and promotes drug resistance through the α6β1 integrin receptor, whereas CCN2 downregulates miR-519d. CCN2 inhibition may represent a new therapeutic concept in osteosarcoma., (© 2018 Wiley Periodicals, Inc.)

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.

In recent years, much research has focused on the role of angiogenesis in osteosarcoma, which occurs predominantly in adolescents and young adults. The vascular endothelial growth factor-A (VEGF-A) pathway is the key regulator of angiogenesis and in osteosarcoma. VEGF-A expression has been recognized as a prognostic marker in angiogenesis. Aberrant WNT1-inducible signaling pathway protein-1 (WISP-1) expression is associated with various cancers. However, the function of WISP-1 in osteosarcoma angiogenesis is poorly understood. We demonstrate a positive correlation between WISP-1 and VEGF-A expression in human osteosarcoma. Moreover, we show that WISP-1 promotes VEGF-A expression in human osteosarcoma cells, subsequently inducing human endothelial progenitor cell (EPC) migration and tube formation. The focal adhesion kinase (FAK), Jun amino-terminal kinase (JNK), and hypoxia-inducible factor (HIF)-1α signaling pathways were activated after WISP-1 stimulation, while FAK, JNK, and HIF-1α inhibitors or small interfering RNA (siRNA) abolished WISP-1-induced VEGF-A expression and angiogenesis. In vitro and in vivo studies revealed down-regulation of microRNA-381 (miR-381) in WISP-1-induced VEGF-A expression and angiogenesis. Our findings reveal that WISP-1 enhances VEGF-A expression and angiogenesis through the FAK/JNK/HIF-1α signaling pathways, as well as via down-regulation of miR-381 expression. WISP-1 may be a promising target in osteosarcoma angiogenesis.

Osteosarcoma is the most common primary solid tumor of bone. It has a high metastatic potential and occurs predominantly in adolescents and young adults. Angiopoietin 2 (Angpt2) is a key regulator in tumor angiogenesis, facilitating tumor growth and metastasis. Connective tissue growth factor (CTGF, also known as CCN2), is a cysteine-rich protein that has been reported to promote metastasis of osteosarcoma. However, the effect of CTGF on Angpt2 regulation and angiogenesis in human osteosarcoma remains largely unknown. We found that overexpression of CTGF in osteosarcoma cells increased Angpt2 production and induced angiogenesis, in vitro and in vivo. Our findings demonstrate that CTGF-enhanced Angpt2 expression and angiogenesis is mediated by the phospholipase C (PLC)/protein kinase C (PKCδ) signaling pathway. Moreover, endogenous microRNA-543 (miR-543) expression was negatively regulated by CTGF via the PLC/PKCδ pathway. We also provide evidence showing clinical significance between CTGF, Angpt2, and miR-543 as well as tumor staging in human osteosarcoma tissue. CTGF may serve as a therapeutic target in the process of osteosarcoma metastasis and angiogenesis., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Osteosarcoma is the most frequent bone tumor, characterized by a high metastatic potential. However, the crosstalk between chemokine (C-C motif) ligand 3 (CCL3), which facilitates tumor progression and metastasis. Vascular endothelial growth factor-A (VEGF-A), an angiogenesis inducer and a highly specific mitogen for endothelial cells, has not been well explored in human osteosarcoma. Here we demonstrate the correlation of CCL3 and VEGF-A expressions, quantified by immunohistochemistry, with the tumor stage of human osteosarcoma tissues. Furthermore, CCL3 promotes VEGF-A expression in human osteosarcoma cells that subsequently induces human endothelial progenitor cell (EPC) migration and tube formation. Phosphorylation of JNK, ERK, and p38 was found after CCL3 stimulation. In addition, JNK, ERK, and p38 inhibitors also abolished CCL3-induced VEGF-A expression and angiogenesis. We noted that CCL3 reduces the expression of miR-374b and miR-374b mimic by reversing CCL3-promoted VEGF-A expression and angiogenesis in vitro and in vivo. This study shows that CCL3 promotes VEGF-A expression and angiogenesis in human osteosarcoma cells by down-regulating miR-374b expression via JNK, ERK, and p38 signaling pathways. Thus, CCL3 may be a new molecular therapeutic target in osteosarcoma angiogenesis and metastasis.

Osteosarcoma, the most common primary malignant bone tumor, shows potent capacity for local invasion and distant metastasis. Connective tissue growth factor (CTGF/CCN2), a secreted protein, binds to integrins, modulates invasive behavior of certain human cancer cells. Effect of CTGF in metastasis of human osteosarcoma is unknown. We found overexpression of CTGF increasing matrix metalloproteinases (MMPs)-2 and MMP-3 expression as well as promoting cell migration. MicroRNA (miRNA) analysis of CTGF-overexpressed osteosarcoma versus control cells probed mechanisms of CTGF-mediated promotion of migration. Among miRNAs regulated by CTGF, miR-519d was most downregulated after CTGF treatment. Co-transfection with miR-519d mimic reversed CTGF-mediated MMPs expression and cell migration. Also, MEK and ERK inhibitors or mutants reduced CTGF-increased cell migration and miR-519d suppression. By contrast, knockdown of CTGF diminished lung metastasis in vivo. Clinical samples indicate CTGF expression as linked with clinical stage and tumor metastasis. Taken together, data show CTGF elevating MMPs expression and subsequently promoting tumor metastasis in human osteosarcoma, down-regulating miR-519d via MEK and ERK pathways, making CTGF a new molecular therapeutic target in osteosarcoma metastasis.

WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matrix cellular proteins. Osteosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effect of WISP-1 on migration activity in human osteosarcoma cells is mostly unknown. In this study, we first found that the expression of WISP-1 in osteosarcoma patients was significantly higher than that in normal bone and corrected with tumor stage. Exogenous treatment of osteosarcoma cells with WISP-1 promoted cell motility and matrix metalloproteinase (MMP)-2 and MMP-9 expression. In addition, the Ras and Raf-1 inhibitor or siRNA abolished WISP-1-induced cell migration and MMP expression. On the other hand, activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after WISP-1 treatment was demonstrated, and WISP-1-induced expression of MMPs and migration activity were inhibited by the specific inhibitor, and mutant of MEK, ERK, and NF-κB cascades. Taken together, our results indicated that WISP-1 enhances the migration of osteosarcoma cells by increasing MMP-2 and MMP-9 expression through the integrin receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway., (© 2013. Published by Elsevier Inc. All rights reserved.)

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF-1 and CXCR4 (SDF-1 receptor). Treatment of osteosarcoma cells with SDF-1alpha increased the migration and cell surface expression of alphavbeta3 integrin. CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1alpha-induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF-1alpha-mediated migration and integrin expression. Stimulation of cells with SDF-1alpha increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited SDF-1alpha-mediated cell migration and integrin up-regulation. Stimulation of cells with SDF-1alpha induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the SDF-1alpha-mediated increasing kappaB-luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKalpha and IKKbeta mutants. Taken together, these results suggest that the SDF-1alpha acts through CXCR4 to activate MEK and ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of alphavbeta3 integrins and contributing the migration of human osteosarcoma cells., (Copyright 2009 Wiley-Liss, Inc.)

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system and is characterized by an extremely aggressive clinical course that lacks an effective treatment. This study is the first to investigate the anti-cancer effects of a new isoflavone-derived 7-hydroxy-3',4'-benzoisoflavone (HBI) in human osteosarcoma cells. HBI-induced cell apoptosis in human osteosarcoma cell lines. The accumulation of reactive oxygen species (ROS) is a critical mediator in HBI induced cell death. HBI also induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation, p38, JNK and p53 phosphorylation. Transfection with ASK1, p38 and JNK small interfering RNA (siRNA) antagonized HBI-induced cell apoptosis. HBI also triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl2 ratio. Bax knockdown using a Bax siRNA strategy reduced Bax expression and subsequent cell death. In addition, ASK1, p38 and JNK siRNA reduced HBI-induced p53 phosphorylation and Bax expression. These results suggest that the ROS-ASK1-p38/JNK-p53 and Bax pathway plays a critical role in HBI's anti-cancer effects.

Osteosarcoma is the most common primary bone tumor associated with childhood and adolescence. In the present study, we investigated the anticancer effect of a new isoflavone derivative, 3',4'-dichloro-3-(3,4-dichlorophenylacetyl)-2,4,6-trihydroxydeoxybenzoin (DDTD) in human osteosarcoma cells. DDTD induced cell apoptosis in human osteosarcoma cell lines (including: U2OS, MG-63, Saos2 and ROS 17/2.8). We found that the accumulation of reactive oxygen species is a critical mediator in DDTD-induced cell death. DDTD induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of osteosarcoma cells with DDTD induced p38 and p53 phosphorylation. Transfection with ASK1, mitogen activated protein kinase (MAPK) kinase (MKK)3/6, and p38 small interfering RNA (siRNA) antagonized the DDTD-induced cell apoptosis. DDTD also triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl2 ratio and Caspase-9 activation. Bax knockdown using a Bax siRNA strategy reduced Bax expression and subsequent cell death. In addition, transfection of cells with ASK1, MKK3/6, and p38 siRNA reduced DDTD-induced p38 activation, p53 phosphorylation and Bax expression. These results suggest that DDTD generates reactive oxygen species and activates the ASK1-MKK3/6-p38-p53-Bax pathway to cause osteosarcoma cell death.