Oleocanthal Protects C2C12 Myotubes against the Pro-Catabolic and Anti-Myogenic Action of Stimuli Able to Induce Muscle Wasting In Vivo.

Oleocanthal (OC) is a monophenol of extra-virgin olive oil (EVOO) endowed with antibiotic, cardioprotective and anticancer effects, among others, mainly in view of its antioxidant and anti-inflammatory properties. OC has been largely investigated in terms of its anticancer activity, in Alzheimer disease and in collagen-induced arthritis; however, the possibility that it can also affect muscle biology has been totally overlooked so far. This study is the first to describe that OC modulates alterations induced in C2C12 myotubes by stimuli known to induce muscle wasting in vivo, namely TNF-α, or in the medium conditioned by the C26 cachexia-inducing tumor (CM-C26). C2C12 myotubes were exposed to CM-C26 or TNF-α in the presence or absence of OC for 24 and 48 h and analyzed by immunofluorescence and Western blotting. In combination with TNF-α or CM-C26, OC was revealed to be able to restore both the myotube's original size and morphology and normal levels of both atrogin-1 and MuRF1. OC seems unable to impinge on the autophagic-lysosomal proteolytic system or protein synthesis. Modulations towards normal levels of the expression of molecules involved in myogenesis, such as Pax7, myogenin and MyHC, were also observed in the myotube cultures exposed to OC and TNF-α or CM-C26. In conclusion, the data presented here show that OC exerts a protective action in C2C12 myotubes exposed to TNF-α or CM-C26, with mechanisms likely involving the downregulation of ubiquitin-proteasome-dependent proteolysis and the partial relief of myogenic differentiation impairment.

Anti-Proliferative Effects of an Extra-Virgin Olive Oil Extract Enriched in Ligstroside Aglycone and Oleocanthal on Human Liver Cancer Cell Lines.

Oleocanthal and ligstroside aglycone are olive oil-derived polyphenols. The former interferes with tumor growth with minor or no cytotoxicity on non-tumorigenic primary cell lines. The information about the bioactivity of ligstroside aglycone are scanty, with the exception of a known antioxidant power. Hepatocellular carcinoma is a malignant tumor with high mortality rates. Systemic chemotherapy is only marginally effective and is frequently complicated by toxicity. Previous observations have shown that hepatocellular carcinoma cell lines become more sensitive to taxol when it is combined with Tumor Necrosis Factor α (TNFα). The present work aimed to assess the effects of a polyphenolic extract containing both oleocanthal and ligstroside aglycone on proliferation and/or death in three liver cancer cell lines (HepG2, Huh7 and Hep3B). The possibility to enhance such effect by the addition of TNFα was also investigated. Both cell proliferation and death were enhanced by the exposure to the polyphenolic extract. Such effect was associated with induction of autophagy and could be potentiated by TNFα. The presence of ligstroside aglycone in the extract lowered the oleocanthal concentration required for cytotoxicity. These results show for the first time that the effects of a polyphenol extract can be potentiated by TNFα and that modulation of autophagy likely account for these effects.

Effects of chronic sugar consumption on lipid accumulation and autophagy in the skeletal muscle.

PURPOSE: In recent years, the increasing consumption of soft drinks containing high-fructose corn syrup or sucrose has caused a rise in fructose intake, which has been related to the epidemic of metabolic diseases. As fructose and glucose intake varies in parallel, it is still unclear what the effects of the increased consumption of the two single sugars are. In the present study, the impact of chronic consumption of glucose or fructose on skeletal muscle of healthy mice was investigated. METHODS: C57BL/6J male mice received water (C), 15 % fructose (ChF) or 15 % glucose (ChG) to drink for up to 7 months. Lipid metabolism and markers of inflammation and autophagy were assessed in gastrocnemius muscle. RESULTS: Increased body weight and gastrocnemius muscle mass, as well as circulating glucose, insulin, and lipid plasma levels were observed in sugar-drinking mice. Although triglycerides increased in the gastrocnemius muscle of both ChF and ChG mice (+32 and +26 %, vs C, respectively), intramyocellular lipids accumulated to a significantly greater extent in ChF than in ChG animals (ChF +10 % vs ChG). Such perturbations were associated with increased muscle interleukin-6 levels (threefold of C) and with the activation of autophagy, as demonstrated by the overexpression of LC3B-II (ChF, threefold and ChG, twofold of C) and beclin-1 (ChF, sevenfold and ChG, tenfold of C). CONCLUSIONS: The present results suggest that intramyocellular lipids and the pro-inflammatory signaling could contribute to the onset of insulin resistance and lead to the induction of autophagy, which could be an adaptive response to lipotoxicity.

Increase in ceramide level alters the lysosomal targeting of cathepsin D prior to onset of apoptosis in HT-29 colon cancer cells.

Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous N-acetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DH-C2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS- and PDMP-induced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis.