cytochrome-c-t and etomoxir

Myostatin (Mstn) is postulated to be a key determinant of muscle loss and cachexia in cancer. However, no experimental evidence supports a role for Mstn in cancer, particularly in regulating the survival and growth of cancer cells. In this study, we showed that the expression of Mstn was significantly increased in different tumor tissues and human cancer cells. Mstn knockdown inhibited the proliferation of cancer cells. A knockout (KO) of Mstn created by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 (CRISPR/Cas9) induced mitochondria-dependent apoptosis in HeLa cells. Furthermore, KO of Mstn reduced the lipid content. Molecular analyses demonstrated that the expression levels of fatty acid oxidation-related genes were upregulated and then increased rate of fatty acid oxidation. Mstn deficiency-induced apoptosis took place along with generation of reactive oxygen species (ROS) and elevated fatty acid oxidation, which may play a role in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, apoptosis induced by Mstn KO was partially rescued by antioxidants and etomoxir, thereby suggesting that the increased level of ROS was functionally involved in mediating apoptosis. Overall, our findings demonstrate a novel function of Mstn in regulating mitochondrial metabolism and apoptosis within cancer cells. Hence, inhibiting the production and function of Mstn may be an effective therapeutic intervention during cancer progression and muscle loss in cachexia.

Topics: A549 Cells; Animals; Antioxidants; Apoptosis; Cachexia; Caspases; Cell Line, Tumor; Cell Proliferation; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems; Cytochromes c; Epoxy Compounds; Fatty Acids; Female; Gene Knockout Techniques; HEK293 Cells; HeLa Cells; Humans; Lipid Metabolism; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Myostatin; Oxidation-Reduction; Reactive Oxygen Species; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

The overall goal of the investigation was to examine the role of uncoupling proteins (UCPs) in regulating late stage events in the chondrocyte maturation pathway. We showed for the first time that epiphyseal chondrocytes expressed UCP3. In hypoxia, UCP3 mediated regulation of the mitochondrial transmembrane potential (DeltaPsi(m)) was dependent on HIF-1alpha. We also showed for the first time that UCP3 regulated the induction of autophagy. Thus, suppression of UCP3 enhanced the expression of the autophagic phenotype, even in serum-replete media. Predictably, the mature autophagic chondrocytes were susceptible to an apoptogen challenge. Susceptibility was probably associated with a lowered expression of the anti-apoptotic proteins Bcl2 and BCL(xL) and a raised baseline expression of cytochrome c in the cytosol. These changes would serve to promote sensitivity to apoptogens. We conclude that in concert with HIF-1alpha, UCP3 regulates the activity of the mitochondrion by modulating the transmembrane potential. In addition, it inhibits induction of the autophagic response. When this occurs, it suppresses sensitivity to agents that promote chondrocyte deletion from the growth plate.

Topics: Animals; Autophagy; Cattle; Cells, Cultured; Chondrocytes; Cytochromes c; Enzyme Inhibitors; Epoxy Compounds; Gene Expression Regulation; Growth Plate; Hypoxia; Ion Channels; Membrane Potentials; Mice; Mitochondria; Mitochondrial Proteins; RNA, Small Interfering; Staurosporine; Uncoupling Protein 3

In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.. Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.. The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial beta-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid beta-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-independent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.. Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.

Topics: Apoptosis; Blotting, Western; Caspase 9; Caspases; Cells, Cultured; Cytochromes c; Endodeoxyribonucleases; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epoxy Compounds; Humans; Linoleic Acid; Mesangial Cells; Mitochondria; Oleic Acid; Palmitates; Signal Transduction; Stearates