cytochrome-c-t and Fibrosarcoma

Chronic stress is regarded as a significant factor in the etiology of the many diseases. Numerous methods have been developed through which the effect of chronic stress is examined. The aim of this study is to demonstrate the new experimental model for analysis of immuno-suppression induced by chronic restraint stress, through challenge with conditionally tumorigenic cell line BHK-21/C13. 20 male NMRI mice were randomly divided into 2 groups-control and experimental. Each mouse was subcutaneously inoculated with BHK-21/C13 cells. Stress in the experimental group was induced for 20 days. After the experiment, tumor masses were removed, and analyzed using histology and immunohistochemistry techniques. We found a statistically significant difference (p = 0.034) in tumor expression and tumor volumes (p = 0.0061) between groups, as well as in immunopositivity on Ki67, cytochrome C and matrix metalloproteinase 9. Absence of immune infiltrate was noticed in experimental, and the presence of inflammatory infiltrate at tumor invasion front in control group.

Topics: Animals; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Fibrosarcoma; Immunohistochemistry; Ki-67 Antigen; Male; Matrix Metalloproteinase 9; Mice; Stress, Physiological; Tumor Burden

A recently studied endoplasmic reticulum (ER) stress regulator, Bax inhibitor-1 (BI-1) plays a regulatory role in mitochondrial Ca(2+) levels. In this study, we identified ER-resident and mitochondria-associated ER membrane (MAM)-resident populations of BI-1. ER stress increased mitochondrial Ca(2+) to a lesser extent in BI-1-overexpressing cells (HT1080/BI-1) than in control cells, most likely as a result of impaired mitochondrial Ca(2+) intake ability and lower basal levels of intra-ER Ca(2+). Moreover, opening of the Ca(2+)-induced mitochondrial permeability transition pore (PTP) and cytochrome c release were regulated by BI-1. In HT1080/BI-1, the basal mitochondrial membrane potential was low and also resistant to Ca(2+) compared with control cells. The activity of the mitochondrial membrane potential-dependent mitochondrial Ca(2+) intake pore, the Ca(2+) uniporter, was reduced in the presence of BI-1. This study also showed that instead of Ca(2+), other cations including K(+) enter the mitochondria of HT1080/BI-1 through mitochondrial Ca(2+)-dependent ion channels, providing a possible mechanism by which mitochondrial Ca(2+) intake is reduced, leading to cell protection. We propose a model in which BI-1-mediated sequential regulation of the mitochondrial Ca(2+) uniporter and Ca(2+)-dependent K(+) channel opening inhibits mitochondrial Ca(2+) intake, thereby inhibiting PTP function and leading to cell protection.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Calcium; Cell Proliferation; Cytochromes c; Endoplasmic Reticulum; Fibrosarcoma; Fluorescent Antibody Technique; Humans; Membrane Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Tumor Cells, Cultured

The objective of this study is to investigate the effect of molecular size and terminal structure of poly(ethylene glycol) (PEG) on the antitumor activity of PEG-modified fullerene (C60). PEG samples with different terminal structures and molecular weights were covalently coupled to C60 and their superoxide anion generation, in vitro or in vivo antitumor activity, and body distribution were assessed for the effect of the photosensitizer, used in photodynamic therapy (PDT), on the tumor. Irrespective of the molecular weight and terminal structure of PEG used, the C60-PEG conjugates exhibited a similar ability of superoxide anion generation and in vitro antitumor activity. On the contrary, a strong suppression of the in vivo tumor growth was observed for the C60-PEG conjugates prepared with methyl-terminated PEG with the highest molecular weight, which showed the longest half-life period in the blood circulation and the highest tumor accumulation among all the conjugates used. It is concluded that the superior tumor targetability of C60-PEG conjugates is one of the keys to enhance the PDT activity.

Topics: Animals; Cell Line, Tumor; Cytochromes c; Fibrosarcoma; Fullerenes; Iodine Radioisotopes; Mice; Molecular Weight; Neoplasm Transplantation; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Radionuclide Imaging; Radiopharmaceuticals; Superoxides; Treatment Outcome; Water

In this study, we have investigated the chemotherapeutic potential of a purple violet pigment (PVP), which was isolated from a previously undescribed Antarctic Janthinobacterium sp. (Ant5-2), against murine UV-induced 2237 fibrosarcoma and B16F10 melanoma cells.. The 2237, B16F10, C50, and NIH3T3 cells were treated with PVP at different doses and for different times, and their proliferation and viability were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle arrest induced by PVP in 2237 fibrosarcoma cells was assessed by flow cytometry and expression analysis of cell cycle regulatory proteins were done by Western blot. Apoptosis induced by PVP in 2237 cells was observed by annexin-V/propidium iodide double staining flow cytometry assay and fluorescence microscopy. To further determine the molecular mechanism of apoptosis induced by PVP, the changes in expression of Bcl-2, Bax and cytochrome c were detected by Western blot. The loss of mitochondrial membrane potential in PVP treated 2237 cells was assessed by staining with JC-1 dye following flow cytometry. Caspase-3, Caspase-9 and PARP cleavage were analyzed by Western blot and Caspase-3 and -9 activities were measured by colorimetric assays.. In vitro treatment of murine 2237 cells with the PVP resulted in decreased cell viability (13-79%) in a time (24-72 h) and dose (0.1-1 μM)-dependent manner. The PVP-induced growth inhibition in 2237 cells was associated with both G0/G1 and G2/M phase arrest accompanied with decrease in the expression of cyclin dependent kinases (Cdks) and simultaneous increase in the expression of cyclin dependent kinase inhibitors (Cdki) - Cip1/p21 and Kip1/p27. Further, we observed a significant increase in the apoptosis of the 2237 fibrosarcoma cells which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2, disruption of mitochondrial membrane potential, cytochrome c release, activation of caspase-3, caspase-9 and poly-ADP-ribose-polymerase (PARP) cleavage.. We describe the anti-cancer mechanism of the PVP for the first time from an Antarctic bacterium and suggest that the PVP could be used as a potent chemotherapeutic agent against nonmelanoma skin cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Cytochromes c; Fibrosarcoma; Indoles; Melanoma; Melanoma, Experimental; Membrane Potential, Mitochondrial; Mice; Oxalobacteraceae; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

Apoptosis and autophagy are genetically regulated, evolutionarily conserved processes that can jointly seal the fate of cancer cells. However, substantial gaps remain in our understanding of the molecular mechanisms that mediate the two cellular processes. In the present study, the exposure of murine fibrosarcoma L929 cells to oridonin led to the generation of intracellular reactive oxygen species (ROS) and, subsequently, the ROS triggered apoptosis by Bax translocation, cytochrome c release and ERK activations. In addition, oridonin induced autophagy in L929 cells, and the inhibition of autophagy by 3-MA or siRNA against LC3 and beclin 1 promoted oridonin-induced apoptosis. Furthermore, p38 and NFkappaB were confirmed to have roles in inhibiting apoptosis but promoting autophagy. Moreover, the inhibition of autophagy could reduce oridonin-induced activation of p38. Finally, NFkappaB activation was inhibited by blocking the p38 pathway. In conclusion, these findings indicate that oridonin-induced apoptosis can be regulated by ROS-mediated signaling pathways, and oridonin-induced autophagy may block apoptosis by upregulating p38 and NFkappaB activation.

Topics: Animals; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Survival; Cytochromes c; Diterpenes, Kaurane; Extracellular Signal-Regulated MAP Kinases; Fibrosarcoma; Mice; Models, Biological; NF-kappa B; Proteins; Reactive Oxygen Species; Signal Transduction

Acquired resistance to classic caspase-mediated apoptosis is a common problem for the treatment of human cancer. Here, we show that siramesine, a novel sigma-2 receptor ligand, effectively induces caspase-independent programmed cell death in immortalized and transformed cells of various origins. Siramesine-treated tumor cells displayed increased levels of reactive oxygen species, lysosomal membrane permeabilization, chromatin condensation, and shrinkage and detachment of cells. Lipid antioxidants (alpha-tocopherol and gamma-tocopherol), but not other tested antioxidants (butylated hydroxyanisol or N-acetyl cysteine), effectively inhibited siramesine-induced morphologic changes and cell death. Cathepsin B inhibitors (CA-074-Me and R-2525) conferred similar, but less pronounced protection, whereas ectopic expression of antiapoptotic protein Bcl-2, lack of wild-type p53 as well as pharmacologic inhibitors of caspases (zVAD-fmk, DEVD-CHO, and LEHD-CHO), calpains (PD150606), and serine proteases (N-tosyl-L-phenylalanine chloromethyl ketone and pefabloc) failed to protect cells against siramesine-induced death. Importantly, transformation of murine embryonic fibroblasts with activated c-src or v-Ha-ras oncogenes greatly sensitized them to siramesine-induced cytotoxicity. Furthermore, p.o. administration of well-tolerated doses of siramesine had a significant antitumorigenic effect in orthotopic breast cancer and s.c. fibrosarcoma models in mice. These results present siramesine as a promising new drug for the treatment of tumors resistant to traditional therapies.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cathepsins; Cell Line, Transformed; Cell Line, Tumor; Cytochromes c; Female; Fibrosarcoma; Humans; Indoles; Ligands; Lysosomes; Mice; Mice, Inbred BALB C; NIH 3T3 Cells; Oxidative Stress; Receptors, sigma; Spiro Compounds; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays