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Modulation of cytochrome c-mediated extramitochondrial NADH oxidation by contact site density
No full textData presented in previous reports suggest that in
rat liver mitochondria a “bi-trans-membrane” electron
transport pathway is present which promotes the
transfer of reducing equivalents directly from cytosolic
NADH to molecular oxygen inside the mitochondria.
Here we show that the oxidation of external
NADH is stimulated by atractylate 1 ADP and greatly
inhibited by glycerol. These two conditions have been
documented to promote the increase and the decrease
respectively of the frequency of “contact sites” between
the two mitochondrial membranes. NADH oxidation
is not affected at all by glycerol and atractylate
1 ADP when TMPD and endogenous cytochrome c
are utilized as electron carriers. The results obtained
are consistent with the proposal that the bi-transmembrane
electron transport chain might be localized
at the level of respiratory contact sites having the
function of promoting the oxidation of the surplus
amount of cytosolic NADH. This electron transport
pathway has been suggested to play a decisive role in
the early stages of apoptosi
Blocking transforming growth factor-beta up-regulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells RID A-8169-2012
No full textHepatocellular carcinoma (HCC) treatment is challenging because the mechanisms underlying tumor progression are still largely unknown. Transforming growth factor (TGF)-beta 1 is considered a crucial molecule in HCC tumorigenesis because increased levels of patients' serum and urine are associated with disease progression. The aim of the present study was to investigate the inhibition of TGF-beta signaling and its impact on HCC progression. Human HCC cell lines were treated with a TGF-beta receptor kinase inhibitor (LY2109761) whose selectivity was determined in a kinase assay. Exogenous TGF-beta 1 phosphorylates the TGF-beta receptor, consequently activating Smad-2, whereas the drug selectively blocks this effect and dephosphorylates autocrine p-Smad-2 at concentrations ranging from 0.001 to 0.1 mu M. A cytotoxic effect documented by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), trypan blue, and propidium iodide staining assays was observed at 10 mu M, whereas the drug inhibits (P < 0.001) the migration of HCC cells on fibronectin, laminin-5, and vitronectin and invasion through Matrigel (P < 0.001) at concentrations up to 0.1 mu M. LY2109761 up-regulates (P < 0.001) E-cadherin mRNA and protein levels. This increase was localized at the cellular membrane where E-cadherin mediates anchorage that is cell-cell dependent. Consistently, a functional monoclonal antibody that inhibits E-cadherin-dependent cell-cell contact restores the migratory and invasive activity. Finally, nonmetastatic HCC tissues from 7 patients were cultured with TGF-beta 1 in the presence or absence of LY2109761. E-cadherin expression was reduced by TGF-beta 1 and was significantly (P < 0.0001) increased by LY2109761 treatment, measured by quantitative real-time PCR on microdissected tissues and by immunohistochemistry on serial sections. In 72 patients, E-cadherin tissue expression was more weakly expressed in metastatic than in nonmetastatic HCC (P < 0.0001). Conclusion: LY2109761 blocks migration and invasion of HCC cells by upregulating E-cadherin, suggesting that there could be a mechanistic use for this molecule in clinical trials
Inhibition of transforming growth factor beta receptor I kinase blocks hepatocellular carcinoma growth through neo-angiogenesis regulation
No full text
Targeting TGF-βRI inhibits activation of β1 integrin and blocks vascular invasion in Hepatocellular Carcinoma (HCC)
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Laminin-5 chains are expressed differentially in metastatic and nonmetastatic hepatocellular carcinoma
No full textInhibition by butylmalonate of proton influx in nonphosphorylating mitochondria
No full textThe impermeability of the inner membrane to protons is one of the four postulates of the chemiosmotic theory on the coupling mechanism between respiration and phosphorylation in mitochondria. However, oxygen uptake in isolated nonphosphorylating mitochondria requires that protons translocated from inside to outside must be, at least in part, retaken up. The nonohmic relationship between the respiration rate and the protonmotive force has been mainly ascribed to an increase in the proton conductance of the inner membrane (proton leak). In liver mitochondria oxygen pulse experiments the rate of both the efflux and the reentry of protons, linked to the oxygen consumption supported by succinate oxidation, is greatly stimulated by low concentrations of butylmalonate. The steady-state level of protons exported outside in the acidification-alkalinization cycle of the medium, generated by an oxygen pulse, is also increased but the rate of oxygen uptake is unaffected. However, in valinomycin-stimulated respiration butylmalonate inhibits the ratio of proton influx/oxygen consumption by 50% and also stimulates the ratio of proton efflux/oxygen consumption by 50%. Titration of the butylmalonate effect gives a saturation curve with a half-maximal effect at 5 microM. Identical results are obtained inthe presence of oligomycin which excludes the involvement of the ATP-synthase complex. The data obtained are not in contrast with the existence in the inner membrane of a channel-like system inhibited by butylmalonate and involved, together with other systems, in promoting the backflow of protons in nonphosphorylating state 4 respiration. Such a system, similar to thermogenin, could be involved in tissues, other than adipose, in a more general thermogenesis program by promoting the dissipation as heat of the energy given by the electrochemical proton gradient. The possibility that butylmalonate might inhibit the proton movement associated with cation and anion transport in mitochondria has also been considered
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