benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Bone-Neoplasms

Osteosarcoma is the predominant form of primary bone malignancy. Although the combinational application of neoadjuvant chemotherapy and surgical resection significantly increases the survival rate, the therapeutic outcome remains unsatisfactory. Deoxyelephantopin (DET), an active ingredient of Elephantopus scaber, has been reported to have an anti-tumor effect in recent publications. This study aimed to investigate whether DET has antineoplastic effects on osteosarcoma cells and its underlying mechanism.. Cell viability and morphological changes were assessed by MTT and Live/dead assays. Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were detected utilizing Annexin V-FITC/PI double staining, DCFH-DA and JC-1 probes, respectively. Autophagy was detected by mRFP-GFP-LC3 adenovirus transfection and western blot.. DET dose-dependently reduced the viability of osteosarcoma cells following the increase in intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) reversed this effect. Furthermore, DET induced mitochondrial apoptosis. Depolarized cells were increased, and apoptosis-related proteins, such as Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3 and cleaved ploy ADP-ribose polymerase, were activated. Additionally, we found that DET could induce autophagy in osteosarcoma cells, but autophagy inhibition did not affect the decrease in cell viability.. DET induced apoptosis in osteosarcoma cells through ROS generation, mitochondrial dysfunction and caspase activation; in addition, autophagy was involved in the effects of DET on osteosarcoma cells.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Bone Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Humans; Lactones; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Reactive Oxygen Species; Sesquiterpenes

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Caspase Inhibitors; Cell Line, Tumor; Cyclosporine; Dose-Response Relationship, Drug; Humans; Imidazoles; Indoles; Microscopy, Electron, Transmission; Necrosis; Oligopeptides; Osteosarcoma; Time Factors

Prostate cancer is one of the most common cancers in men, with more than 500,000 new worldwide cases reported annually, resulting in 200,000 deaths of mainly older men in developed countries. Existing treatments have not proved very effective in managing prostate cancer, and continuing efforts therefore are ongoing to explore novel targets and strategies for future therapies. LAPSER1 has been identified as a candidate tumor suppressor gene in prostate cancer, but its true functions remain unknown. We report here that LAPSER1 colocalizes to the centrosomes and midbodies in mitotic cells with gamma-tubulin, MKLP1, and p80 katanin, and is involved in cytokinesis. Moreover, RNAi-mediated disruption of LAPSER1, which is accompanied by the mislocalization of p80 katanin, results in malformation of the central spindle. Significantly, the enhanced expression of LAPSER1 induces binucleation and renders the cells resistant to oncogenic transformation. In cells transformed by the v-Fps oncogene, overexpressed LAPSER1 induces abortive cytokinesis, followed by mitotic catastrophe in a p80 katanin-dependent manner. Cells that are rescued from this apoptotic pathway with Z-VAD-fmk display karyokinesis. These results suggest that LAPSER1 participates in cytokinesis by interacting with p80 katanin, the disruption of which may potentially cause genetic instability and cancer.

Topics: Adenocarcinoma; Adenosine Triphosphatases; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bone Neoplasms; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Centrosome; CHO Cells; Cricetinae; Cricetulus; Cytokinesis; Fusion Proteins, gag-onc; Genes, Tumor Suppressor; Humans; Katanin; Leucine Zippers; Male; Membrane Proteins; Microtubule-Associated Proteins; Oncogene Protein p21(ras); Oncogene Proteins v-abl; Osteosarcoma; Polyploidy; Prostatic Neoplasms; Protein Subunits; Protein-Tyrosine Kinases; Rats; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering; Spindle Apparatus; Subcellular Fractions; Tubulin; Tumor Suppressor Proteins

The type of cell death occurring in the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as three-dimensional spheroids after exposure to 5 Gy of ionizing radiation was determined. Morphological analyses using the chromatin dye Hoechst 33258 demonstrated that spheroids showed the typical characteristics of apoptosis, while monolayer cells revealed those typical of mitotic catastrophe. In order to better characterize these two types of cell death, the role of caspases was examined in irradiated monolayer cells and spheroids using the broad spectrum caspase inhibitor zVAD-fmk. Death in monolayer cells was caspase-independent, whereas spheroid death was characterized by caspase dependence. Members of the Bcl-2 family of proteins and survivin involved in cell death processes were also studied by Western blot analysis. The pro-apoptotic protein Bax increased in spheroids, whereas this protein remained unchanged in monolayer cells after the same 5-Gy irradiation. The anti-apoptotic protein Bcl-2, on the other hand, remained unchanged in both monolayer cells and spheroids. Finally, survivin increased significantly after irradiation in both cells in monolayer and spheroids. The results presented suggest that three-dimensional cell organization leads to a different type of cell death after exposure to ionizing radiation. Thus, the use of spheroids, a cell model which mimics in vivo solid tumors more closely than cells grown in monolayer, is more appropriate when investigating the effects of antineoplastic treatments such as ionizing radiation.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Bisbenzimidazole; Blotting, Western; Bone Neoplasms; Caspase 3; Cell Line, Tumor; Cell Survival; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Mitosis; Neoplasm Proteins; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2; Response Elements; Spheroids, Cellular; Survivin

Paclitaxel has been found to exhibit cytotoxic and antitumor activity. There is little information regarding the mechanisms of apoptotic-inducing effect of paclitaxel on human osteogenic sarcoma U-2 OS cells. Several key regulatory proteins are involved in the initiation of apoptosis. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. We examined the effect of paclitaxel on the cell cycle arrest and apoptosis in U-2 OS cells using flow cytometric analysis and Western blotting. We also measured the inhibition of paclitaxel-induced apoptosis and the caspase-3 activity by the broad-spectrum caspase inhibitor z-VAD-fmk on U-2 OS cells. The increased levels of casapse-3 were also confirmed by cDNA microarray. Our observations were: (1) paclitaxel treatment resulted in G2/M-cycle arrest in U-2 OS cells; (2) time and dose dependent apoptosis of U-2 OS cells was induced by paclitaxel; (3) in U-2 OS cells, z-VAD-fmk blocked the paclitaxel-induced apoptosis and caspase-3 activation. These results suggest that paclitaxel-induced G2/M-cycle arrest of the G2/M phase and apoptosis via a caspase-3 pathway in U-2 OS cells.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Bone Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Osteosarcoma; Paclitaxel

deltaNp63 is overexpressed in squamous carcinomas where it is associated with proliferation and is believed to enhance cell growth by blocking p53-mediated transactivation. In normal epithelium, deltaNp63alpha protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of deltaNp63alpha overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of deltaNp63alpha in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca(2+)], abrogates Ca(2)(+)-induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that deltaNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. deltaNp63alpha blocks transactivation of p53 responsive reporter constructs mediated by endogenous or exogenous p53 at 17 h postinfection, as expected. However, at 41 h, when p53-mediated transactivation is diminished, deltaNp63alpha enhances transactivation of these reporter constructs by 2.2-12-fold over control. Maximal deltaNp63alpha-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of deltaNp63alpha appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support deltaNp63alpha as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Animals; Binding Sites; Bone Neoplasms; Calcium; Cell Differentiation; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Fibroblasts; Filaggrin Proteins; Genes, Reporter; Genes, Tumor Suppressor; Humans; Keratinocytes; Mice; Mice, Inbred C57BL; Organ Specificity; Osteosarcoma; Phosphoproteins; Protein Isoforms; Recombinant Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Cell death processes are progressively inactivated during malignant development, in part by loss of tumor suppressors that can promote cell death. The Bin1 gene encodes a nucleocytosolic adaptor protein with tumor suppressor properties, initially identified through its ability to interact with and inhibit malignant transformation by c-Myc and other oncogenes. Bin1 is frequently missing or functionally inactivated in breast and prostate cancers and in melanoma. In this study, we show that Bin1 engages a caspase-independent cell death process similar to type II apoptosis, characterized by cell shrinkage, substratum detachment, vacuolated cytoplasm, and DNA degradation. Cell death induction was relieved by mutation of the BAR domain, a putative effector domain, or by a missplicing event that occurs in melanoma and inactivates suppressor activity. Cells in all phases of the cell cycle were susceptible to death and p53 and Rb were dispensable. Notably, Bin1 did not activate caspases and the broad spectrum caspase inhibitor ZVAD.fmk did not block cell death. Consistent with the lack of caspase involvement, dying cells lacked nucleosomal DNA cleavage and nuclear lamina degradation. Moreover, neither Bcl-2 or dominant inhibition of the Fas pathway had any effect. In previous work, we showed that Bin1 could not suppress cell transformation by SV40 large T antigen. Consistent with this finding, we observed that T antigen suppressed the death program engaged by Bin1. This observation was interesting in light of emerging evidence that T antigen has roles in cell immortalization and human cell transformation beyond Rb and p53 inactivation. In support of a link to c-Myc-induced death processes, AEBSF, a serine protease inhibitor that inhibits apoptosis by c-Myc, potently suppressed DNA degradation by Bin1. Our findings suggest that the tumor suppressor activity of Bin1 reflects engagement of a unique cell death program. We propose that loss of Bin1 may promote malignancy by blunting death penalties associated with oncogene activation.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Antigens, Polyomavirus Transforming; Apoptosis; Bone Neoplasms; Carcinoma, Hepatocellular; Carrier Proteins; Caspases; Cell Adhesion; Cell Size; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; DNA Fragmentation; Enzyme Activation; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mitochondria; Nuclear Proteins; Osteosarcoma; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Recombinant Fusion Proteins; Retinoblastoma Protein; Serine Proteinase Inhibitors; Sulfones; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins