cytochrome-c-t and Bone-Neoplasms

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

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

2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) is a cyclohexanedione found in the roots of Averrhoa carambola L., commonly known as starfruit. Researchers have shown that DMDD has significant therapeutic potential for the treatment of diabetes; however, the effects of DMDD on human cancers have never been reported. We investigated the cytotoxic effects of DMDD against human breast, lung and bone cancer cells in vitro and further examined the molecular mechanisms of DMDD-induced apoptosis in human breast cancer cells. DMDD suppressed the growth of breast carcinoma cells, but not normal mammary epithelial cells, via induction of G1 phase cell cycle arrest, oxidative stress and apoptosis. DMDD increased the level of intracellular reactive oxygen species (ROS) and DMDD-induced ROS generation was found to be associated with the mitochondrial activity. The cytotoxicity that was induced by DMDD was attenuated by co-treatment with the antioxidant N-acetyl-L-cysteine (NAC). DMDD-induced cell apoptosis involved the activation of both the intrinsic mitochondrial pathway and the extrinsic receptor pathway. In addition, DMDD inhibited the canonical NF-κB signaling pathway at all steps, including TNF-α production, phosphorylation of NF-κB p65 and IκBα, as well as TNF-α activated NF-κB p65 nuclear translocation.Collectively, our studies indicate that DMDD has significant potential as a safe and efficient therapeutic agent for the treatment of breast cancer.

Topics: Acetylcysteine; Antineoplastic Agents; Antioxidants; Apoptosis; Averrhoa; Bone Neoplasms; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclohexenes; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hypoglycemic Agents; Inhibitory Concentration 50; Lung Neoplasms; MCF-7 Cells; NF-kappa B; Oxidative Stress; Phosphorylation; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

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

Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system.

Topics: Antacids; Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Breast Neoplasms; Calcium Carbonate; Cell Line, Tumor; Cytochromes c; Doxorubicin; Drug Delivery Systems; Female; Humans; Mitochondrial Proteins; Nanoparticles; Neoplasm Metastasis; Neoplasm Proteins

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

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

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

Clinical management of chondrosarcoma remains a challenging problem, largely due to the toxicity and resistance of this tumor to conventional chemotherapy. Programmed Cell Death 5 (PDCD5) is a protein that accelerates apoptosis in different cell types in response to various stimuli, and has been shown to be down-regulated in many cancer tissues. In this study, mRNA and protein levels of PDCD5 were found to be up-regulated in cisplatin-treated SW1353 chondrosarcoma cells compared with untreated cells. Recombinant human PDCD5 (rhPDCD5) was also shown to sensitize chondrosarcoma cells to cisplatin-based chemotherapy, with inhibition of cell growth and apoptosis detected both in vitro and in vivo. Increased expression of Bax and decreased expression of Bcl-2 were also observed, along with release of cytochrome c from mitochondria into the cytosol. Additionally, cleavage of caspase-9 and caspase-3, as well as the cleavage of poly (ADP-ribose) polymerase (PARP), were detected, suggesting that sensitization of chondrosarcoma cells involves the intrinsic mitochondrial apoptosis pathway. In vivo, the treatment of a xenograft model of chondrosarcoma with rhPDCD5 and cisplatin significantly inhibited tumor cell proliferation and induced apoptosis compared to treatment with cisplatin alone. Overall, these data provide a theoretical basis for the administration of rhPDCD5 and cisplatin for the treatment of patients with chondrosarcoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bone Neoplasms; Cell Line, Tumor; Chondrosarcoma; Cisplatin; Cytochromes c; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Recombinant Proteins; Xenograft Model Antitumor Assays

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