cytochrome-c-t and Cachexia

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

While exercise benefits have been well documented in patients with chronic diseases, the mechanistic understanding of cachectic muscle's response to contraction is essentially unknown. We previously demonstrated that treadmill exercise training attenuates the initiation of cancer cachexia and the development of metabolic syndrome symptoms (Puppa MJ, White JP, Velazquez KT, Baltgalvis KA, Sato S, Baynes JW, Carson JA. J Cachexia Sarcopenia Muscle 3: 117-137, 2012). However, cachectic muscle's metabolic signaling response to a novel, acute bout of low-frequency contraction has not been determined. The purpose of this study was to determine whether severe cancer cachexia disrupts the acute contraction-induced response to low-frequency muscle contraction [low-frequency stimulation (LoFS)]. Metabolic gene expression and signaling was examined 3 h after a novel 30-min bout of contraction (10 Hz) in cachectic Apc(Min/+) (Min) and C57BL/6 (BL-6) mice. Pyrrolidine dithiocarbamate, a STAT/NF-κB inhibitor and free radical scavenger, was administered systemically to a subset of mice to determine whether this altered the muscle contraction response. Although glucose transporter-4 mRNA was decreased by cachexia, LoFS increased muscle glucose transporter-4 mRNA in both BL-6 and Min mice. LoFS also induced muscle peroxisome proliferator-activated receptor-γ and peroxisome proliferator-activated receptor-α coactivator-1 mRNA. However, in Min mice, LoFS was not able to induce muscle proliferator-activated receptor-α coactivator-1 targets nuclear respiratory factor-1 and mitochondrial transcription factor A mRNA. LoFS induced phosphorylated-S6 in BL-6 mice, but this induction was blocked by cachexia. Administration of pyrrolidine dithiocarbamate for 24 h rescued LoFS-induced phosphorylated-S6 in cachectic muscle. LoFS increased muscle phosphorylated-AMP-activated protein kinase and p38 in BL-6 and Min mice. These data demonstrate that cachexia alters the muscle metabolic response to acute LoFS, and combination therapies in concert with muscle contraction may be beneficial for improving muscle mass and function during cachexia.

Topics: AMP-Activated Protein Kinases; Animals; Cachexia; Cytochromes c; Disease Models, Animal; DNA-Binding Proteins; Electric Stimulation Therapy; Gene Expression Regulation; Genes, APC; Hand Strength; High Mobility Group Proteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscle Contraction; Muscle, Skeletal; Nuclear Respiratory Factor 1; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR gamma; Pyrrolidines; RNA, Messenger; STAT3 Transcription Factor; Thiocarbamates; Transcription Factors

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.

Topics: Adipose Tissue; Aldehydes; Animals; Body Weight; Cachexia; Catalase; Colonic Neoplasms; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex IV; Gene Expression; Genes, APC; GTP Phosphohydrolases; Hindlimb; Interleukin-6; Ion Channels; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Muscle; Mitochondrial Proteins; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Oxidative Phosphorylation; Oxidative Stress; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Sirtuin 1; STAT3 Transcription Factor; Succinate Dehydrogenase; Superoxide Dismutase; Trans-Activators; Transcription Factors; Uncoupling Protein 3

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.

Topics: Animals; Apoptosis; Cachexia; Cell Line; Cells, Cultured; Cytochromes c; Down-Regulation; Female; Genes, Lethal; GTP Phosphohydrolases; Lymphocytes; Metalloproteases; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins

Treatment of C(2)C(12) myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2, -3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 microM eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA. Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the anti-apoptotic protein bcl-2, which were both attenuated by 50 microM EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C(2)C(12) myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blood Proteins; Blotting, Western; Cachexia; Cell Line; Cells, Cultured; Chymotrypsin; Cysteine Endopeptidases; Cytochromes c; Cytosol; DNA Fragmentation; Dose-Response Relationship, Drug; Eicosapentaenoic Acid; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Mice; Multienzyme Complexes; Muscle, Skeletal; Nucleosomes; Proteasome Endopeptidase Complex; Proteoglycans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2