cytochrome-c-t and Osteosarcoma

In this study, we identified the multifaceted effects of atezolizumab, a specific monoclonal antibody against PD-L1, in tumor suppression except for restoring antitumor immunity, and investigated the promising ways to improve its efficacy. Atezolizumab could inhibit the proliferation and induce immune-independent apoptosis of osteosarcoma cells. With further exploration, we found that atezolizumab could impair mitochondria of osteosarcoma cells, resulting in increased release of reactive oxygen species and cytochrome-c, eventually leading to mitochondrial-related apoptosis via activating JNK pathway. Nevertheless, the excessive release of reactive oxygen species also activated the protective autophagy of osteosarcoma cells. Therefore, when we combined atezolizumab with autophagy inhibitors, the cytotoxic effect of atezolizumab on osteosarcoma cells was significantly enhanced in vitro. Further in vivo experiments also confirmed that atezolizumab combined with chloroquine achieved the most significant antitumor effect. Taken together, our study indicates that atezolizumab can induce mitochondrial-related apoptosis and protective autophagy independently of the immune system, and targeting autophagy is a promising combinatorial approach to amplify its cytotoxicity.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; B7-H1 Antigen; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Chloroquine; Cytochromes c; Humans; Immune Checkpoint Inhibitors; JNK Mitogen-Activated Protein Kinases; Male; Mice, SCID; Mitochondria; Osteosarcoma; Reactive Oxygen Species; Signal Transduction; Tumor Microenvironment; Xenograft Model Antitumor Assays

Cancer stem cells (CSCs) are mainly contributed to malignancy metastatic potential and resistant therapy of osteosarcoma (OS). The mitochondria-related apoptosis was generally accepted as the target of tumor therapy. However, the effect of N-myc downstream-regulated gene 1 (NDRG1) on CSCs and mitochondrial health in OS is still unknown.. In OS cells, MG63 and U2OS, the siRNA of NDRG1 were conducted. Transwell, western blot, RT-qPCR, and mitochondria isolation were used to identify the effect of NDRG on OS cells and mitochondria. Moreover, the differentiation-related factors of CSCs were determined.. After downregulation of NDRG1, the cell viability, invasion ability decreased whereas cell apoptosis increased. The expressions profiles of fibronectin, vimentin, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP) 2, MMP9, and MMP13 were downregulated, but E-cadherin expression level was upregulated by NDRG1 siRNA. At the same time, cytochrome (Cyt) C levels were increased in cytosol with the decreasing in mitochondria after siRNA treatment. The mitochondrial membrane potential (MMPs) was declined, and the function of mitochondria was impeded. The expressions of uncoupling proteins (UCP) 2, voltage dependent anion channel (VDAC), peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, and cyclooxygenase (COX) 2 were downregulated by NDRG1 silencing. Moreover, NDRG performed its function primarily through the Wnt pathway and could regulate the differentiation of osteosarcoma stem cells.. Silencing of NDRG1 could damage the function of mitochondria, promote the CSCs differentiation, alleviating OS progression.

Topics: Apoptosis; Cadherins; Cell Cycle Proteins; Cell Differentiation; Cell Survival; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Mitochondria; Neoplasm Invasiveness; Neoplastic Stem Cells; Osteosarcoma; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Tumor Cells, Cultured; Uncoupling Protein 2; Voltage-Dependent Anion Channels

The present study aimed to investigate the effects of cold atmospheric plasma (CAP)‑activated Ringer's solution on osteosarcoma cell lines MG63 and U2OS, and to identify the molecular mechanism underlying these effects. CAP‑activated Ringer's solution was used to treat osteosarcoma cell lines MG63 and U2OS for 30 min. Cell viability was measured using the MTT method. The apoptosis rate was detected using Annexin‑V and propidium iodide. The expression levels of cytochrome c, caspase‑3 and polyADP ribose polymerase (PARP) in MG63 cells were analyzed via western blotting. The change in mitochondrial membrane potential was detected via the JC‑1 dye method and verified by the level of reactive oxygen species (ROS). CAP‑activated Ringer's solution inhibited the proliferation of MG63 and U2OS cells in a dose‑ and time‑dependent manner. Furthermore, CAP‑activated Ringer's solution induced the apoptosis of MG63 cells, increased the intracellular ROS level, decreased the mitochondrial membrane potential level, and induced the release of cytochrome c. CAP‑activated Ringer's solution inhibits osteosarcoma cell proliferation through intracellular ROS‑mediated mitochondrial apoptosis.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Plasma Gases; Reactive Oxygen Species; Ringer's Solution

In the present study, a polysaccharide (ECP) from Enterobacter cloacae dose and time-dependently inhibited cell growth of human osteosarcoma U-2 OS cells via induction of apoptosis. ECP treatment was selectively toxic to U-2 OS cells whereas had no cytotoxic effect on normal human osteoblast cell line NHOst. ECP-induced apoptotic cell death was associated with collapse of mitochondrial membrane, cytochrome c release into the cytosol, activation of caspase-9 and-3, degradation of poly (ADP-ribose) polymerase (PARP), elevated the ratio of Bax/Bcl-2 protein and overexpression of p53, suggesting the involvement of the activation of p53 and mitochondrial intrinsic pathway in ECP-induced apoptosis. Likewise, ECP oral administration significantly inhibited the U-2 OS cancer growth in xenograft tumor model. All these first evidence indicated that ECP was a potential antitumor supplement for the treatment of human osteosarcoma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Cytosol; Enterobacter cloacae; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mitochondria; Mitochondrial Membranes; Osteosarcoma; Polysaccharides, Bacterial; Proto-Oncogene Proteins c-bcl-2; Tumor Burden; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

An accumulating body of evidence has shown that capsaicin induces apoptosis in various tumor cells as a mechanism of its anti-tumor activity. However, the effects of capsaicin on osteosarcoma have not been studied extensively. In the current study, we explore the molecular mechanism of capsaicin-mediated tumor suppressive function in osteosarcoma. We found that capsaicin-induced apoptosis and the activation of transient receptor potential receptor vanilloid 1 (TRPV1) in a dose- and time-dependent manner in human osteosarcoma MG63 cells

Topics: Adenylate Kinase; Apoptosis; Capsaicin; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Ion Channel Gating; JNK Mitogen-Activated Protein Kinases; Mitochondria; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase; TRPV Cation Channels; Tumor Suppressor Protein p53

Osteosarcoma is the most common type of solid bone cancer, which is the second leading cause of cancer-related death. Hypoxia is an ordinary phenomenon in solid tumor tissues and can induce cell apoptosis but the specific molecular mechanism remains unclear. In this study, we explored the effect and the molecular mechanism of Transglutaminase 2 (TG2) on cell apoptosis in osteosarcoma U2OS cells under hypoxia. We found the enzymatic activity of TG2 is significantly increased and the expression of TG2 is remarkably up-regulated under hypoxia condition. Cell apoptotic rate is markedly increased upon knockdown of TG2 by siRNA under hypoxia. We further investigated the mechanism of cell apoptosis and found Bax protein is significantly increased after depletion of TG2 under hypoxia. Moreover, our data also show that cytochrome C (Cyt C) is significantly increased in cytoplasm and markedly decreased in mitochondria of U2OS cells after depletion of TG2 under hypoxia. Our results suggest that TG2 can inhibit tumor cell apoptosis through down-regulation of Bax and prevention of release Cyt C from mitochondria into cytoplasm.

Topics: Apoptosis; bcl-2-Associated X Protein; Bone Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cytochromes c; Cytoplasm; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Silencing; GTP-Binding Proteins; Humans; Mitochondria; Osteosarcoma; Protein Glutamine gamma Glutamyltransferase 2; RNA, Small Interfering; Transglutaminases

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

Topics: Adaptor Proteins, Signal Transducing; Anemone; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Chromosomal Proteins, Non-Histone; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Gene Expression; HSP70 Heat-Shock Proteins; Humans; Osteosarcoma; Phytotherapy; Plant Extracts; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Sequestosome-1 Protein; Transcription Factors; Up-Regulation

The antitumor effects of curcumin have attracted widespread attention worldwide. One of its major functions is to induce the apoptosis of tumor cells, but the antitumor mechanism is currently unclear. In the present study, we found that cell mortality and curcumin concentration were dose dependent. Curcumin of low concentrations (10 μΜ) could reduce the level of reactive oxygen species (ROS) in tumor cells, while curcumin of high concentrations (80 μΜ) was able to significantly increase the content of ROS. In addition, Western blotting detection suggested that curcumin of high concentrations can induce the release of Cyto-C and the activation of Caspase-3, and that ROS scavenger NAC apparently inhibits apoptosis protein release and activation, consequently slowing the curcumin-induced apoptosis. Taken together, curcumin further activates the mitochondrial apoptotic pathway by inducing cells to generate ROS and ultimately promotes the apoptosis of tumor cells.

Topics: Apoptosis; Bone Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Curcumin; Cytochromes c; Humans; Osteosarcoma; Reactive Oxygen Species; Signal Transduction

Ginsenosides, extracted from the traditional Chinese herb ginseng, are a series of novel natural anticancer products known for their favorable safety and efficacy profiles. The present study aimed to investigate the cytotoxicity of ginsenoside Rf to human osteosarcoma cells and to explore the anticancer molecular mechanisms of ginsenoside Rf. Five human osteosarcoma cell lines (MG-63, OS732, U-2OS, HOS and SAOS-2) were employed to investigate the cytotoxicity of ginsenoside Rf by MTT and colony forming assays. After treatment with ginsenoside Rf, MG-63 cells which were the most sensitive to ginsenoside Rf, were subjected to flow cytometry to detect cell cycle distribution and apoptosis, and nuclear morphological changes were visualized by Hoechst 33258 staining. Caspase-3, -8 and -9 activities were also evaluated. The expression of cell cycle markers including cyclin B1 and Cdk1 was detected by RT-PCR and western blotting. The expression of apoptotic genes Bcl-2 and Bax and the release of cytochrome c were also examined by western blotting. Change in the mitochondrial membrane potential was observed by JC-1 staining in situ. Our results demonstrated that the cytotoxicity of ginsenoside Rf to these human osteosarcoma cell lines was dose-dependent, and the MG-63 cells were the most sensitive to exposure to ginsenoside Rf. Additionally, ginsenoside Rf induced G2/M phase cell cycle arrest and apoptosis in MG-63 cells. Furthermore, we observed upregulation of Bax and downregulation of Bcl-2, Cdk1 and cyclin B1, the activation of caspase-3 and -9 and the release of cytochrome c in MG-63 cells following treatment with ginsenoside Rf. Our findings demonstrated that ginsenoside Rf induces G2/M phase cell cycle arrest and apoptosis in human osteosarcoma MG-63 cells through the mitochondrial pathway, suggesting that ginsenoside Rf, as an effective natural product, may have a therapeutic effect on human osteosarcoma.

Topics: Apoptosis; bcl-2-Associated X Protein; Bone Neoplasms; Caspase 3; Caspase 8; Caspase 9; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cytochromes c; Drugs, Chinese Herbal; Ginsenosides; Humans; M Phase Cell Cycle Checkpoints; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Panax; Plant Preparations; Proto-Oncogene Proteins c-bcl-2; Tumor Stem Cell Assay

Flavanones demonstrate a propensity to antiproliferation and induce apoptosis of malignant cells. Among the 4 flavanones under study, 2'-hydroxyflavanone exhibited the greatest potency to reduce the cell viability of 143 B cells in 4 osteosarcoma cells. Flow cytometry analysis showed that 2'-hydroxyflavanone increased the hypodiploid cells in the sub-G1 phase but resulted in the reduced DNA content in the G0/G1 phase in 143 B cells. The 2'-hydroxyflavanone-induced apoptosis in 143 B cells was confirmed by 4'-6-diamidino-2-phenylindole staining and mitochondrial membrane potential (Δψm) assay. Increasing expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor 5 (DR5) were found in 2'-hydroxyflavanone-treated cells. Moreover, 2'-hydroxyflavanone increased the expressions of B-cell lymphoma-extra small, cytochrome c, and cleavage poly (ADP-ribose) polymerase but downregulated B-cell lymphoma/leukemia-2expressions in 143 B cells. Furthermore, in vivo experiments showed that 2'-hydroxyflavanone inhibited the tumor growth of 143 B cells. 2'-hydroxyflavanone induced the apoptosis of 143 B cells via the extrinsic TRAIL- and intrinsic mitochondrial-dependent pathways, indicating its potential for inducing cancer apoptosis in osteosarcoma.

Topics: Apoptosis; B-Lymphocytes; Cell Line, Tumor; Cytochromes c; Down-Regulation; Flavanones; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Poly(ADP-ribose) Polymerases; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand

Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Calpain; Caspase 3; Cell Line, Tumor; Cytochromes c; Down-Regulation; Egtazic Acid; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Mitochondria; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Small Interfering; Temperature; Up-Regulation

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. Although paclitaxel (PCX) has been considered one of the most important cancer chemotherapeutic drugs, the current protocols for OS treatment do not incorporate this agent. Therefore, the purpose of this study was to evaluate the induction of cell death in OS cells after exposure to PCX, to identify the cell death mechanism(s) activated by PCX and to investigate whether autophagy is associated with PCX-induced apoptosis. The results of the present study confirmed that exposure to low PCX concentrations can induce apoptotic cell death in Saos-2 cells; furthermore, caspase-3 activation, PARP degradation and XIAP downregulation were observed in combination with PCX-induced apoptosis. The potential involvement of mitochondrial events (intrinsic apoptotic pathway) in PCX-induced apoptosis in OS cells was verified by the alteration (depolarization) of mitochondrial membrane potential. In addition, pretreatment with 3-methyladenine (3-MA), a specific inhibitor of autophagy, significantly increased PCX-induced apoptotic cell death in Saos-2 cells. The augmentation of PCX-induced apoptosis by 3-MA was accompanied by increase in the cytochrome c release from the mitochondria, caspase-3 activity and XIAP downregulation, which suggests that inhibiting autophagy further stimulates the PCX-induced mitochondrion-related (intrinsic) apoptotic pathway by provoking caspase-3 activation. Thus, autophagy observed during PCX-induced apoptosis in Saos-2 OS cells represents the role of cytoprotection in cellular homeostatic processes. In conclusion, the results of this study revealed that PCX exposure effectively induces OS cell death by apoptosis associated with the mitochondrial-mediated caspase-dependent pathway. PCX can increase autophagic activity and suppressing autophagy enhances PCX-induced apoptosis in OS cells. Therefore, it is suggested that combination treatment involving low-dose PCX therapy and autophagy inhibitor therapy could be an effective and potent strategy for improved chemotherapy for OS in the near future.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Bone Neoplasms; Caspase 3; Cell Death; Cell Line, Tumor; Cytochromes c; Cytoprotection; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Paclitaxel; X-Linked Inhibitor of Apoptosis Protein

In this study antiproliferation, cell cycle arrest and apoptosis induced by daphnoretin in human osteosarcoma (HOS) cells were investigated. Antiproliferative activity was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) value of daphnoretin was 3.89 μM after 72 h treatment. Induction of apoptosis was evidenced by apoptotic body appearance and Annexin V-FITC/PI apoptosis detection kit. Flow cytometric analysis indicated daphnoretin arrested the cell cycle in the G2/M phase. Western-blot assay showed that the G2/M phase arrest was accompanied by down-regulation of cdc2, cyclin A and cyclin B1. Moreover, daphnoretin inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade. Our results demonstrated that daphnoretin caused death of HOS cells by blocking cells successively in G2/M phases and activating the caspase-3 pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Cell Shape; Coumarins; Cyclin A; Cyclin B; Cyclin B1; Cyclin-Dependent Kinases; Cytochromes c; G2 Phase Cell Cycle Checkpoints; Gene Expression; Humans; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Osteosarcoma

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment.

Topics: Apoptosis; bcl-2-Associated X Protein; Bone Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Shape; Cell Survival; Cytochromes c; Dose-Response Relationship, Radiation; Electromagnetic Radiation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Time Factors

Bufalin is the primary component of the traditional Chinese herb "Chan Su". Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s) is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX) sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27), decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27) were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Bufanolides; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; HSP27 Heat-Shock Proteins; Humans; Mice; Osteosarcoma; Proteome; Proteomics; Proto-Oncogene Proteins c-akt; Transcription Factor RelA; Xenograft Model Antitumor Assays

Solamargine, a steroidal glycoalkaloid isolated from S. incanum, has been shown to induce apoptosis in several cancer cell lines. In this study, the involvement of p53 in the pro-apoptotic action of solamargine was investigated in human osteosarcoma U2OS cells.. The cytotoxicity of solamargine was evaluated by MTT assay. Solamargine-induced apoptosis was evidenced by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine on the extracellular surface as revealed by DAPI nuclear staining, transmission electron microscopy and flow cytometry, respectively. mRNA expressions of p53 and Bax were investigated using real time-PCR. Western blot was used to examine the changes in the expression levels of p53, Bax, Bcl-2, caspase-3, caspase-9 and cytochrome c. Subcellular localization of p53 was verified by immunofluorescence staining and cell fractionation.. Solamargine substantially reduced cell viability and induced apoptosis in osteosarcoma U2OS cells. In this connection, solamargine increased the mRNA and protein expressions of p53 and Bax (a pro-apoptotic protein downstream to p53). The expression of Bcl-2 (an anti-apoptotic protein) was also reduced. Furthermore, solamargine induced mitochondrial translocation of p53, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. p53-specific transcriptional inhibitor pifithrin-α or mitochondrial translocation inhibitor pifithrin-μ partially reversed solamargine-induced apoptosis.. Solamargine activates the mitochondria-mediated apoptotic pathway in U2OS cells via both p53 transcription-dependent and -independent mechanisms. This compound may merit further investigation as a potential therapeutic agent for the treatment of cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Bone Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2; Solanaceous Alkaloids; Transcription, Genetic; Tumor Suppressor Protein p53

The response of osteosarcoma MG-63 cells to corosolic acid treatment has been investigated. The results showed that corosolic acid significantly inhibited cell viability in both a dose and a time dependent manner. It was found that corosolic acid increased the Bax/Bcl-2 ratio by up-regulating Bax expression, disrupted mitochondrial membrane potential and triggered the release of cytochrome c from mitochondria into the cytoplasm. Corosolic acid treatment triggered the activation of caspase-8, 9 and 3. The apoptosis was obviously inhibited by pretreatment with a general caspase inhibitor, z-VAD-FMK. Moreover, pretreatment of CsA, a cyclophilin D ligand that inhibits mitochondria potential uncoupling, prevented the activation of caspase-9 and caspase-3, but not caspase-8, and the apoptosis of MG-63 cells, triggered by corosolic acid. All these results indicated that corosolic acid-induced apoptosis was associated with the activation of caspases via a mitochondrial pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Triterpenes

Although rare, osteosarcoma is an aggressive cancer that often metastasizes to the lungs. Toward the goal of developing new treatment options for osteosarcoma, we show that the cyclin-dependent kinase (CDK) inhibitor SCH 727965 (SCH) induces the apoptosis of several osteosarcoma cell lines including those resistant to doxorubicin and dasatinib. Cell lines prepared in our laboratory from patients who had received adjuvant chemotherapy and explants derived from a human osteosarcoma xenograft in mice were also responsive to SCH. Apoptosis occurred at low nanomolar concentrations of SCH, as did CDK inhibition, and was p53-independent. SCH activated the mitochondrial pathway of apoptosis as evidenced by caspase-9 cleavage and accumulation of cytoplasmic cytochrome c. Amounts of the apoptotic proteins Bax and Bim increased in mitochondria, whereas amounts of the antiapoptotic proteins Mcl-1 and Bcl-x(L) declined. Osteosarcoma cells apoptosed when codepleted of CDK1 and CDK2 but not when depleted of other CDK combinations. We suggest that SCH triggers the apoptosis of osteosarcoma cells by inactivating CDK1 and CDK2 and that SCH may be useful for treatment of drug-resistant osteosarcomas. SCH also induced the apoptosis of other sarcoma types but not of normal quiescent osteoblasts or fibroblasts.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Bone Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Caspase 9; CDC2 Protein Kinase; Cell Line, Tumor; Cyclic N-Oxides; Cyclin-Dependent Kinase 2; Cytochromes c; Drug Resistance, Neoplasm; Humans; Indolizines; Membrane Proteins; Mice; Mitochondria; Osteosarcoma; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridinium Compounds; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Quercetin is the most abundant polyphenolic flavonoid found in plants. Several studies suggest that it has potent anticancer effects. The present study examines the apoptosis-inducing activity and the underlying mechanism of action of quercetin in a methotrexate (MTX)-resistant osteosarcoma model. Our results showed that quercetin inhibited cell viability in a dose-dependent manner and there was no cross-resistance between MTX and quercetin in U2-OS/MTX300 cells. The induction of apoptosis was observed by flow cyto-metry and fluorescence staining experiments. Quercetin-induced apoptosis was accompanied by a significant reduction of mitochondrial membrane potential, release of mitochondrial cytochrome c to the cytosol, activation of caspase-3, down-regulation of Bcl-2, p-Bad and up-regulation of Bax. A remarkable dephospho-rylation of Akt was also detected after quercetin treatment. Furthermore, transduction with constitutively active Akt protected against the quercetin-induced dephosphorylation of Akt and Bad as well as poly(ADP-ribose)polymerase (PARP) degradation, while combined treatment with quercetin and LY294002 enhanced the dephosphorylation of Akt, Bad and PARP cleavage in U2-OS/MTX300 cells. Taken together, our results demonstrate that quercetin-induced apoptosis in the MTX-resistant osteosarcoma cells U2-OS/MTX300 was mediated via mitochondrial dysfunction and dephosphorylation of Akt.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Methotrexate; Mitochondria; Osteosarcoma; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Quercetin

beta-Ionone is a constituent of vegetables and fruits, and can induce apoptosis in some types of malignant cells. However, the mechanism of apoptosis in osteosarcoma (U2os) cells is currently unclear. In this study, we determined whether beta-ionone can induce apoptosis in U2os cells in vitro and which signal pathway(s) is involved. We found that beta-ionone inhibited cell proliferation in U2os cells in a concentration- and time-dependent manner and caused cell cycle arrest at the G1-S phase. TUNEL assay, DNA ladder and assessment of Caspase 3 activity showed that apoptosis was the determinant in the effects of beta-ionone. Furthermore, Expression of the p53 protein increased in a concentration-dependent and time-dependent manner according to immunocytochemistry and immunoblotting after beta-ionone treatment. In addition, beta-ionone upregulated Bax protein and downregulated Bcl2 protein which led to Bax translocation and cytochrome c release, subsequently activated Caspase 3, thus resulting in apoptosis. In summary, these data suggested that beta-ionone induced apoptosis in a concentration-dependent manner in U2os cells via a p53-dependent mitochondrial pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Enzyme Activation; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Mitochondria; Norisoprenoids; Osteosarcoma; Signal Transduction; Tumor Suppressor Protein p53

Grifolin, a natural biologically active substance isolated from the edible bodies of the mushroom Albatrellus confluens, has been shown to inhibit proliferation and induce apoptosis in several cancer cell lines. But the mechanisms remain poorly understood. In this study, we investigated the apoptosis-inducing effects and the mechanisms of grifolin on human osteosarcoma cells. Our results demonstrated that grifolin induced concentration- and time-dependent suppression of proliferation and induction of apoptosis in U2OS and MG63 osteosarcoma cell lines. Grifolin induced the release of cytochrome c accompanied by activation of caspase-9, caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, z-VAD-fmk, a universal inhibitor of caspases, prevented caspase-3 activation and PARP cleavage and inhibted grifolin-induced cell growth inhibition. Furthermore, grifolin treatment resulted in a reduction in level of phosphorylated AKT, FOXO transcription factor, and glycogen synthase kinase 3 (GSK3). Knockdown of GSK3 with siRNA inhibited the apoptotic effects of grifolin. On the other hand, grifolin treatment down-regulated the expression of the inhibitor of apoptosis protein(IAP) in both osteosarcoma cells. Taken together, our results suggested that grifolin is able to suppress the phosphorylation of Akt and its substrates FOXO transcription factor and GSK3 in osteosarcoma cells causing the suppression of proliferation and induction of mitochondria- and caspase-dependent apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Inhibitors; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Mitochondria; Oncogene Protein v-akt; Osteosarcoma; Phosphatidylinositol 3-Kinases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; RNA, Small Interfering; Signal Transduction; Terpenes

The prognostic significance of bax, cytochrome c, and caspase-8 in patients with primary osteosarcoma is unknown. We examined the immunohistochemical expression of these genes in 35 surgically treated patients with primary osteosarcoma. Clinicopathological and survival data were correlated with the staining result. Eighteen tissue specimens from non-malignant osseous lesions were used as controls. Bax, cytochrome c, and caspase-8 positive staining was observed in 29 (82.9%), 16 (45.7%), and 0 (0%) patients, respectively, but did not stain any of the 18 benign osseous lesions used as controls. None of the genes studied predicted overall or disease-free survival. Patients, however, bearing bax(+)/cytochrome c(+) or bax(+)/cytochrome c(+ +) tumors had a decreased 4-year disease-free survival rate compared to the rest of the group (p = 0.0489 and p = 0.0208, respectively), identifying two groups of patients where more intensive adjuvant treatment could possibly be applied to prevent high postoperative recurrence rates.

Topics: Adolescent; Adult; Aged; bcl-2-Associated X Protein; Biomarkers, Tumor; Bone Neoplasms; Caspase 8; Cytochromes c; Disease-Free Survival; Female; Humans; Male; Middle Aged; Osteosarcoma; Prognosis

Dexamethasone (Dx) is often used to alleviate the acute emetic toxicity associated with high dose cisplatin (cDDP) in osteosarcoma patients. However, in other tumour cell types, Dx has been reported to induce partial resistance to anticancer drugs.. We examined the effect of Dx on cDDP-induced apoptosis in the HOS human osteosarcoma cell line.. Exposure to cDDP, induced apoptosis via the mitochondrial apoptotic pathway as evidenced by cytochrome c release and caspase activation. Pre and cotreatment of HOS cells with Dx reduced cDDP induced apoptosis by 10-25%. Investigation of the mechanisms of this protective effect indicated both the upregulation of the survival factor Akt and a possible direct receptor-mediated action of Dx to attenuate the activation of the mitochondrial apoptotic pathway components.. These data indicate the need to carefully address the timing of glucocorticoid use in the clinical management of cancer patients.

Topics: Antiemetics; Antineoplastic Agents; Apoptosis; Blotting, Western; Bone Neoplasms; Caspase Inhibitors; Caspases; Cisplatin; Cytochromes c; Dexamethasone; Enzyme Activation; Humans; Immunoenzyme Techniques; Mitochondria; Osteosarcoma; Proto-Oncogene Proteins c-akt; Receptors, Glucocorticoid; Tumor Cells, Cultured

Cisplatin is an anticancer drug that can induce apoptosis. In this study, we investigated the effect of mitochondrial DNA (mtDNA) depletion on cisplatin-induced cell death using a human osteosarcoma cell line (143B) and mtDNA-depleted 143B cells (143B-rho0). Results showed that cisplatin decreased cell survival in 143B-rho0 cells. Moreover, cisplatin induced a greater extent of apoptosis-associated DNA fragmentation and caspase 3 activation in 143B-rho0 cells. The release of mitochondrial cytochrome c into cytosol by cisplatin was enhanced more obviously in 143B cells than in 143B-rho0 cells; however, in the control group of 143B-rho0 cells, it was already dramatically greater. Depletion of mtDNA may increase sensitivity of cells to cisplatin-induced apoptosis by enhancing caspase 3 activation via both cytochrome c-dependent and -independent pathways.

Topics: Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA, Mitochondrial; Enzyme Activation; Gene Deletion; Humans; Mitochondria; Nucleosomes; Osteosarcoma

Cell motility, growth, and proliferation are regulated by adhesion to the extracellular matrix. Detachment of adherent cells from extracellular matrix results in induction of apoptosis ("anoikis"). Transformed cells often show an anchorage-independent growth that enables them to acquire a motile, invasive phenotype. This phenotype has been associated with the altered expression and function of the integrin family of transmembrane proteins that mediate cell adhesion to the extracellular matrix. Although alpha4 integrin is normally expressed on leukocyte subpopulations, a number of metastatic melanomas and sarcomas express it as well. In this study, we demonstrated the expression of alpha4 integrins on the human osteosarcoma cell line SAOS and on metastatic osteosarcoma lesions from the lung and pericardium. We further demonstrated that alpha4 integrin is coupled to the beta1 subunit by biochemical analysis and by using a mAb directed against a combinatorial epitope unique to the alpha4beta1 molecule. SAOS cells undergo anoikis when adherence is denied. Anoikis involved the activation of caspase 3 and the release of cytochrome c from mitochondria. Treatment of non-adherent SAOS with an anti-alpha4 mAb increased anoikis while anti-beta1 integrin mAbs did not alter anoikis, thus indicating a novel function for the alpha4 subunit in the control of cell death. Since integrins can control cell migration, proliferation, and apoptosis these results demonstrate a potential role for alpha4 integrin during multiple aspects of osteosarcoma metastasis.

Topics: Anoikis; Antibodies, Monoclonal; Apoptosis; Cell Line; Cytochromes c; Heart Neoplasms; Humans; Integrin alpha4; Lung Neoplasms; Mitochondria; Neoplasm Metastasis; Osteosarcoma; Tumor Cells, Cultured

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Leupeptins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multienzyme Complexes; Osteosarcoma; Protease Inhibitors; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Retinoblastoma Protein; Time Factors; Transfection; Tumor Suppressor Protein p53