Targeting TNFα in severe psoriasis-mass spectrometry reveals a time-dependent specific inhibition of Factor H in responding patients.

Drugs targeting TNFα (eg, Etanercept®) provide effective control of severe psoriasis. In absence of validated biological parameters of inflammation in psoriasis most decisions on therapeutics have relied mostly on clinical criteria, namely the "Psoriasis Area and Severity Index" (PASI). The purpose of this study was to assess by mass spectrometry alterations in concentrations of serum proteins that specifically correlated with effectiveness of Etanercept treatment. This prospective study enrolled 10 patients suffering from moderate to severe psoriasis (PASI score > 10 and < 17) and treated with Etanercept over a period of 24 weeks; 10 healthy, age-matched volunteers provided controls. Serum proteins sensitive to Etanercept treatment were identified using SELDI-TOF (surface-enhanced laser desorption and ionization - time of flight) coupled to nano LC-ESI/MS (nano liquid chromatography-electrospray ionization/tandem mass spectrometry) technologies. For comparisons between groups of individuals p-values (considered significant when < 0.01) were estimated with non-parametric tests, namely Mann-Whitney (for unpaired data) and Wilcoxon signed-rank (for paired data). In responding patients it could be shown using SELDI-TOF spectrometry that two proteins (134 kDa and 4.3 kDa) return to control levels by 24 weeks of treatment. Using nano LC-ESI/MS the 134 kDa species was identified as complement Factor H. These observations deserve further analyses utilizing larger cohorts of patients. Determination of Factor H levels may become a complementary tool to follow remission or predict the onset of relapse in the follow-up of patients under treatment with Etanercept.

Interplay Between Membrane Lipid Peroxidation and Photoproduct Formation in the Ultraviolet A-Induced Phototoxicity of Vemurafenib in Skin Keratinocytes.

According to some authors, the phototoxic response to ultraviolet A (UVA) of patients treated with vemurafenib (VB) may involve VB metabolites. However, the production of singlet oxygen and free radicals and photoproduct formation upon UVA light absorption by the lipophilic VB have been demonstrated. This work is aimed at determining the contribution of reactive oxygen species (ROS), lipid photoperoxidation, and VB photochemistry in the UVA-induced photocytotoxicity in NCTC 2544 keratinocytes. The potent membrane lipid peroxidation effectiveness of VB-photosensitization has been proved by the observation of an effective photohemolysis accompanied by thiobarbituric reactive substances (TBARS) formation in 2% red blood cell (RBC) suspensions. Photohemolysis is inhibited by human serum albumin (HSA) that binds VB and by the antioxidants 2,6-di-tert-butyl-4-methylphenol and Trolox. These data on RBC suggest that VB is readily incorporated in cell membranes and provide clues for understanding the UVA-induced VB-photosensitization of keratinocytes. In keratinocytes, ROS and TBARS formation with 10 µM VB is inhibited by approximately 40% and 50% by 30 µM Trolox and 50 µM vitamin E, respectively, but the light dose-dependent cell survival is unaffected. Whereas cell photokilling depends on the VB concentration, much smaller changes in the lethal doses (LD) than theoretically expected are observed for 25% or 50% cell photokilling when changing absorbed UVA doses and irradiation wavelengths. The lack of antioxidant effect on cell survival and the unexpectedly small LD dependence on absorbed UVA light doses and on irradiation wavelengths strongly suggest that, instead of metabolites, membrane photosensitization and photoproduct formation contribute to the cell photocytotoxicity.

[Ferroptosis, a new form of cell death relevant to the medical treatment of cancer]. / La ferroptose, une nouvelle forme de mort cellulaire applicable au traitement médical des cancers.

Ferroptosis is a form of cell death that has recently been reported during exposure to erastin, a chemical compound identified in a screen for molecules able to kill cancer cells carrying an active Ras oncogene. In cells exposed to inducers of ferroptosis, a catastrophic alteration of the cellular redox metabolism occurs, resulting in massive lipid peroxidation in the plasma membrane and loss of cell viability. We present our recent observations suggesting that sorafenib, the only medical treatment with proven efficacy against hepatocellular carcinoma, induces ferroptosis, a new anti-oncogenic mode of action of this drug. The discovery of ferroptosis sheds light on the critical adaptations of the redox metabolism in cancer cells. It might also foster the discovery of new biomarkers and innovative approaches for the treatment of cancer.

Oxidized low density lipoprotein increases RANKL level in human vascular cells. Involvement of oxidative stress.

Receptor Activator of NFκB Ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) have been shown to play a role not only in bone remodeling but also in inflammation, arterial calcification and atherosclerotic plaque rupture. In human smooth muscle cells, Cu(2+)-oxidized LDL (CuLDL) 10-50 µg/ml increased reactive oxygen species (ROS) and RANKL level in a dose-dependent manner, whereas OPG level was not affected. The lipid extract of CuLDL reproduced the effects of the whole particle. Vivit, an inhibitor of the transcription factor NFAT, reduced the CuLDL-induced increase in RANKL, whereas PKA and NFκB inhibitors were ineffective. LDL oxidized by myeloperoxidase (MPO-LDL), or other pro-oxidant conditions such as ultraviolet A (UVA) irradiation, incubation with H2O2 or with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, also induced an oxidative stress and enhanced RANKL level. The increase in RANKL in pro-oxidant conditions was also observed in fibroblasts and endothelial cells. Since RANKL is involved in myocardial inflammation, vascular calcification and plaque rupture, this study highlights a new mechanism whereby OxLDL might, by generation of an oxidative stress, exert a deleterious effect on different cell types of the arterial wall.

Oxidized low density lipoprotein enhanced RANKL expression in human osteoblast-like cells. Involvement of ERK, NFkappaB and NFAT.

BACKGROUND: Receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand/osteoprotegerin ratio is of crucial importance in osteoclast differentiation and thus in bone dysregulation diseases. METHODS: Receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand and osteoprotegerin were determined under oxidized low density lipoprotein treatment of human osteoblast-like cells. The involvement of oxidative stress, of the extracellular signal regulated kinase and of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells and nuclear factor of activated T cells was demonstrated. RESULTS: Cu(2+)-oxidized low density lipoprotein increased cell-associated and extracellular receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand levels whereas osteoprotegerin levels were not affected. The increase in receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand was parallel to the generation of reactive oxygen species provoked by Cu(2+)-oxidized low density lipoprotein. The lipid extract of Cu(2+)-oxidized low density lipoprotein, together with other forms of oxidized low density lipoproteins such as smooth muscle cell-oxidized low density lipoprotein and myeloperoxidase-oxidized low density lipoprotein, also induced an increase in reactive oxygen species and cell-associated receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand. The effect of Cu(2+)-oxidized low density lipoprotein was prevented by the antioxidant vitamin E, and mimicked by the prooxidant compounds hydrogen peroxide and buthionine sulfoximine. Inhibitors of mitogen activated protein kinase/extracellular signal regulated kinase (PD 98059), nuclear factor kappa-light-chain-enhancer of activated B cells (Ro 106-9920) and nuclear factor of activated T cells (Vivit) reduced the effect of Cu(2+)-oxidized low density lipoprotein on receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand expression. Cu(2+)-oxidized low density lipoprotein signaling was also reduced by vitamin E. GENERAL SIGNIFICANCE: This work describes a new molecular mechanism and elucidates the signaling pathway whereby oxidized low density lipoprotein, by means of its lipid moiety, can modulate the crosstalk between osteoblasts/osteoclasts and bone remodeling, leading to an eventual risk of osteoporosis.

Plasma lipoproteins as mediators of the oxidative stress induced by UV light in human skin: a review of biochemical and biophysical studies on mechanisms of apolipoprotein alteration, lipid peroxidation, and associated skin cell responses.

There are numerous studies concerning the effect of UVB light on skin cells but fewer on other skin components such as the interstitial fluid. This review highlights high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as important targets of UVB in interstitial fluid. Tryptophan residues are the sole apolipoprotein residues absorbing solar UVB. The UVB-induced one-electron oxidation of Trp produces (•)Trp and (•)O2 (-) radicals which trigger lipid peroxidation. Immunoblots from buffered solutions or suction blister fluid reveal that propagation of photooxidative damage to other residues such as Tyr or disulfide bonds produces intra- and intermolecular bonds in apolipoproteins A-I, A-II, and B100. Partial repair of phenoxyl tyrosyl radicals (TyrO(•)) by α -tocopherol is observed with LDL and HDL on millisecond or second time scales, whereas limited repair of α -tocopherol by carotenoids occurs in only HDL. More effective repair of Tyr and α -tocopherol is observed with the flavonoid, quercetin, bound to serum albumin, but quercetin is less potent than new synthetic polyphenols in inhibiting LDL lipid peroxidation or restoring α -tocopherol. The systemic consequences of HDL and LDL oxidation and the activation and/or inhibition of signalling pathways by oxidized LDL and their ability to enhance transcription factor DNA binding activity are also reviewed.

Heterogeneous sensitivity of hepatocellular carcinoma to sorafenib revealed by the short-term culture of tumor fragments.

BACKGROUND/AIM: Sorafenib is currently the only medical treatment with proven efficacy against hepatocellular carcinoma (HCC). HCC cell lines display heterogeneous sensitivity to sorafenib, but little is known about the sensitivity of clinical tumors. We aimed to examine this aspect. MATERIALS AND METHODS: Using experimental tumors generated in nude mice, we set up a technique for short-term culture of HCC fragments. We applied this technique to six human HCC samples obtained from surgical resection. RESULTS: HCC fragments in culture retain their morphology and viability for at least 48 h, permitting an in vitro analysis of the effect of sorafenib on the Extracellular signal-regulated kinase (ERK) cascade. HCC exhibit heterogeneous individual responses, ranging from potent inhibition to paradoxical activation of this oncogenic cascade. CONCLUSION: Our observations highlight the heterogeneous sensitivity of HCC to sorafenib, and point to the potential interest of short-term culture of tumor fragments for personalizing the medical treatment of HCC.

Iron-dependent cell death of hepatocellular carcinoma cells exposed to sorafenib.

The multikinase inhibitor sorafenib is currently the treatment of reference for advanced hepatocellular carcinoma (HCC). In our report, we examined the cytotoxic effects of sorafenib on HCC cells. We report that the depletion of the intracellular iron stores achieved by using the iron chelator deferoxamine (DFX) strikingly protects HCC cells from the cytotoxic effects of sorafenib. The protective effect of the depletion of intracellular iron stores could not be explained by an interference with conventional forms of programmed cell death, such as apoptosis or autophagic cell death. We also found that DFX did not prevent sorafenib from reaching its intracellular target kinases. Instead, the depletion of intracellular iron stores prevented sorafenib from inducing oxidative stress in HCC cells. We examined the possibility that sorafenib might exert a cytotoxic effect that resembles ferroptosis, a form of cell death in which iron-dependent oxidative mechanisms play a pivotal role. In agreement with this possibility, we found that pharmacological inhibitors (ferrostatin-1) and genetic procedures (RNA interference against IREB-2) previously reported to modulate ferroptosis, readily block the cytotoxic effects of sorafenib in HCC cells. Collectively, our findings identify ferroptosis as an effective mechanism for the induction of cell death in HCC. Ferroptosis could potentially become a goal for the medical treatment of HCC, thus opening new avenues for the optimization of the use of sorafenib in these tumors.

Effects of phosphate on vascular function under normal conditions and influence of the uraemic state.

AIMS: Increased serum phosphorus levels are associated with cardiovascular disease in patients with chronic kidney disease (CKD) and in the general population. High phosphate levels may play a direct role in vascular dysfunction. We investigated here the effects of phosphate loading and of the phosphate binder sevelamer-HCl on vascular function. METHODS AND RESULTS: CKD and non-CKD C57/BL6 mice were used to study the effects of CKD, phosphate, and sevelamer-HCl on vascular function and structure. In vitro, phosphate exhibited a direct vasoconstrictor effect on aortic rings. This effect was smaller in vessels from CKD than non-CKD mice and it was abolished by reactive oxygen species inhibitor dimethylthiourea. A high-phosphate diet (1.3%) increased phenylephrine-induced contraction and lowered acetylcholine-induced relaxation of aortic rings ex vivo, both in non-CKD and CKD mice. It also induced endothelial cell detachment. Sevelamer-HCl exposure in vitro normalized the endothelial dysfunction induced by 3.0 mM phosphate and restored endothelial integrity. Sevelamer-HCl treatment of CKD mice under normal diet (0.65% phosphate) improved the endothelial dysfunction, aortic systolic expansion rate, and pulse wave velocity, and it reduced the endothelial expression of adhesion molecules. CONCLUSION: Changes in extracellular phosphorus concentrations may directly modulate vascular function and thereby modulate the vascular smooth muscle response to physiological or pathological stimuli in normal and CKD mice. Whether serum phosphorus lowering and/or dietary phosphate restriction can improve arterial function in humans remains to be established.

EGFR activation is a potential determinant of primary resistance of hepatocellular carcinoma cells to sorafenib.

Sorafenib is currently the medical treatment of reference for hepatocellular carcinoma (HCC), but it is not known whether sorafenib is equally active in all HCC. Here, our aim was to explore intrinsic differences in the response of HCC cells to sorafenib, to identify potential mechanisms leading to primary resistance to this treatment. We analyzed a panel of six human HCC cell lines and compared the activity of the main oncogenic kinase cascades, their clonogenic potential, proliferation and apoptosis upon exposure to sorafenib. We report that HCC cells present important differences in their response to sorafenib, and that some cell lines are more resistant to the actions of sorafenib than others. We identify the activated epidermal growth factor receptor (EGFR) as a parameter that promotes the resistance of HCC cells to sorafenib. In resistant cells, the efficacy of sorafenib was increased when EGFR was inhibited, as was demonstrated using two chemical inhibitors (erlotinib or gefitinib), a monoclonal antibody directed against EGFR (cetuximab), and RNA interference directed against EGFR. A combination of EGFR inhibitors and sorafenib affords a better control over HCC proliferation, most likely through an improved blockade of the RAF kinases. Our findings therefore confirm the importance of RAF kinases as therapeutic targets in HCC, and identify EGFR as a determinant of the sensitivity of HCC cells to sorafenib. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC, and might be useful for the identification of predictive biomarkers in this context.

The dependence of α-tocopheroxyl radical reduction by hydroxy-2,3-diarylxanthones on structure and micro-environment.

The flavonoid quercetin is known to reduce the α-tocopheroxyl radical (˙TocO) and reconstitute α-tocopherol (TocOH). Structurally related polyphenolic compounds, hydroxy-2,3-diarylxanthones (XH), exhibit antioxidant activity which exceeds that of quercetin in biological systems. In the present study repair of ˙TocO by a series of these XH has been evaluated using pulse radiolysis. It has been shown that, among the studied XH, only 2,3-bis(3,4-dihydroxyphenyl)-9H-xanthen-9-one (XH9) reduces ˙TocO, though repair depends strongly on the micro-environment. In cationic cetyltrimethylammonium bromide (CTAB) micelles, 30% of ˙TocO radicals are repaired at a rate constant of ~7.4 × 10(6) M(-1) s(-1) by XH9 compared to 1.7 × 10(7) M(-1) s(-1) by ascorbate. Water-soluble Trolox (TrOH) radicals (˙TrO) are restored by XH9 in CTAB (rate constant ~3 × 10(4) M(-1) s(-1)) but not in neutral TX100 micelles where only 15% of ˙TocO are repaired (rate constant ~4.5 × 10(5) M(-1) s(-1)). In basic aqueous solutions ˙TrO is readily reduced by deprotonated XH9 species leading to ionized XH9 radical species (radical pK(a) ~10). An equilibrium is observed (K = 130) yielding an estimate of 130 mV for the reduction potential of the [˙X9,H(+)/XH9] couple at pH 11, lower than the 250 mV for the [˙TrO,H(+)/TrOH] couple. A comparable value (100 mV) has been determined by cyclic voltammetry measurements.

Upregulation of BAD, a pro-apoptotic protein of the BCL2 family, in vascular smooth muscle cells exposed to uremic conditions.

Chronic kidney disease (CKD) has recently emerged as a major risk factor for cardiovascular pathology. CKD patients display accelerated atherosclerotic process, leading to circulatory complications. However, it is currently not clear how uremic conditions accelerate atherosclerosis. Apoptosis is an important homeostatic regulator of vascular smooth cells under pathological conditions. In the present study, we explored the regulation of apoptosis in cells of the vascular wall in the uremic context. We analysed the expression and regulation of the proteins of the BCL2 family that play an essential role in apoptosis. Our results, obtained in mice and primary human smooth muscle cells exposed to two uremic toxins, point to the existence of an alteration in expression and function of one pro-apoptotic member of this family, the protein BAD. We explore the regulation of BAD by uremic toxins and report the sensitization of vascular smooth muscle cells to apoptosis upon BAD induction.

Evidence for a slow and oxygen-insensitive intra-molecular long range electron transfer from tyrosine residues to the semi-oxidized tryptophan 214 in human serum albumin: its inhibition by bound copper (II).

A slow, long range electron transfer (SLRET) in human serum albumin (HSA) is observed from an intact tyrosine (Tyr) residue to the neutral tryptophan (Trp) radical (Trp·) generated in pulse radiolysis. This radical is formed, at neutral pH, through oxidation with Br (2) (·-) radical anions of the single Trp 214 present. The SLRET rate constant of ~0.2 s(-1) determined is independent of HSA concentration and radiation dose, consistent with an intra-molecular process. This is the slowest rate constant so far reported for an intra-molecular LRET. In sharp contrast with the LRET reported for other proteins, the SLRET observed here is insensitive to oxygen, suggesting that the oxidized Trp is inaccessible to-or do not react with radiolytically generated O (2) (·-) . In N(2)O-saturated solutions, the SLRET is inhibited by Cu(2+) ions bound to the His 3 residue of the N-terminal group of HSA but it is partially restored in O(2)-saturated solutions.

Oxidized low density lipoprotein induces cyclin A synthesis. Involvement of ERK, JNK and NFkappaB.

The cytotoxicity of oxidized LDL (OxLDL) towards different cell types of the arterial wall results in atherosclerotic plaque fissuring or rupture. The effects of OxLDL on cyclins A, E and D1 levels were investigated in human fibroblasts. A 24h incubation with Cu(2+)-oxidized (CuLDL) or monocyte-oxidized LDL (M-LDL), within the range of 10-50µg ApoB/ml, increased the intracellular level of cyclin A, both in the nuclear and in the cytoplasmic fraction. This increase is due to a stimulation of cyclin A mRNA synthesis, and cycloheximide had a preventive effect. The CuLDL-induced rise in cyclin A was accompanied by an augmentation of reactive oxygen species ROS and of lipid peroxidation end products. Furthermore, this effect was reproduced by the lipid extract of CuLDL and prevented by the antioxidant vitamin E, demonstrating the role of oxidized lipids and the involvement of oxidative stress. In addition, the use of specific inhibitors indicated that the increase in cyclin A involved ERK/JNK kinases and NFkappaB transcription factor. The augmentation of cyclin A was observed not only in fibroblasts but also in other cell types such as macrophages, T lymphocytes, endothelial and smooth muscle cells. Since cyclin A has been shown to be involved in cell cycle arrest, the described phenomenon might be related to the harmful effect of OxLDL, leading to plaque rupture.

Reduced CSF turnover and decreased ventricular Aß42 levels are related.

BACKGROUND: The appearance of Aß42 peptide deposits is admitted to be a key event in the pathogenesis of Alzheimer's disease, although amyloid deposits also occur in aged non-demented subjects. Aß42 is a degradation product of the amyloid protein precursor (APP). It can be catabolized by several enzymes, reabsorbed by capillaries or cleared into cerebrospinal fluid (CSF). The possible involvement of a decrease in CSF turnover in A4ß2 deposit formation is up to now poorly known. We therefore investigated a possible relationship between a reduced CSF turnover and the CSF levels of the A4ß2 peptide.To this aim, CSF of 31 patients with decreased CSF turnover were studied. These patients presented chronic hydrocephalus communicating or obstructive, which required surgery (ventriculostomy or ventriculo-peritoneal shunt). Nine subjects had idiopathic normal pressure hydrocephalus (iNPH), and the other 22 chronic hydrocephalus from other origins (oCH).The Aß42 peptide concentration was measured by an ELISA test in 31 ventricular CSF samples and in 5 lumbar CSF samples from patients with communicating hydrocephalus. RESULTS: The 5 patients with lumbar CSF analysis had similar levels of lumbar and ventricular Aß42. A significant reduction in Aß42 ventricular levels was observed in 24 / 31 patients with hydrocephalus. The values were lower than 300 pg/ml in 5 out of 9 subjects with iNPH, and in 15 out of 22 subjects with oCH. CONCLUSION: The decrease of CSF Aß42 seems to occur independently of the surgical hydrocephalus aetiology. This suggests that a CSF reduced turnover may play an important role in the decrease of CSF Aß42 concentration.

Structure-activity relationships in hydroxy-2,3-diarylxanthone antioxidants. Fast kinetics spectroscopy as a tool to evaluate the potential for antioxidant activity in biological systems.

A structure-activity relationship has been established for eight hydroxy-2,3-diarylxanthones (XH) bearing hydroxy groups on the two aryl rings. One-electron oxidation by superoxide radical-anions (˙O(2)(-)) and ˙Trp radicals as well as reaction with ˙CCl(3)O(2) and ˙CHCl(2)O(2) radicals demonstrates that two OH groups are required for efficient antioxidant reactivity in cetyltrimethylammonium bromide micelles. Hydroxy groups at the meta and para positions on either of the two phenyl rings confer enhanced reactivity, but XH bearing an OH at the para position of either phenyl ring is unreactive. While oxidation is favoured by OH in both meta and para positions of 2-aryl xanthone substituents, addition of a third and/or fourth OH enhances electron-donating capacity. In Cu(2+)-induced lipid peroxidation of human LDL, the lag period preceding the commencement of lipid peroxidation in the presence of XH bearing OH at meta and para positions on the 3-phenyl ring is extended to twice that observed with a comparable concentration of quercetin, a reference antioxidant. These antioxidants are also superior to quercetin in protecting human skin keratinocytes against tert-butylhydroperoxide-induced oxidative stress. While XH antioxidant activity in model biological systems is consistent with the structure-activity relationship, their response is also modulated by the localization of XH and by structural factors.

The Bcl-2 homology domain 3 (BH3) mimetic ABT-737 reveals the dynamic regulation of bad, a proapoptotic protein of the Bcl-2 family, by Bcl-xL.

The proteins of the B-cell lymphoma 2 (Bcl-2) family are important regulators of apoptosis under normal and pathological conditions. Chemical compounds that block the antiapoptotic proteins of this family have been introduced, such as 4-[4-[(4'-Chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide (ABT-737), a BH3-mimetic that neutralizes Bcl-2 and Bcl-xL. In this study, we used ABT-737 to explore the dynamic regulation of Bcl-2 proteins in living cells of different origins. Using ABT-737 as well as RNA interference or the application of growth factors, we examined the impact of the functional availability of the antiapoptotic proteins Bcl-2 and Bcl-2-extra large (Bcl-xL) on the Bcl-2 network. We report that ABT-737 increases the expression of Bcl-2-associated death promoter (Bad), a proapoptotic partner of the proteins Bcl-2 and Bcl-xL. Our observations indicate that Bad overexpression induced by ABT-737 results from the control of its normally rapid protein turnover, leading to the stabilization of this protein. We demonstrate the relevance of Bad post-translational regulation by Bcl-xL to the physiological setting using RNA interference against Bcl-xL as well as the application of epidermal growth factor, a growth factor that promotes the dissociation of Bad from Bcl-xL. Our results highlight a new facet of the mode of action of the antiapoptotic proteins Bcl-2 and Bcl-xL consisting of the regulation of the stability of the protein Bad. Finally, our results shed light on the mode of action of ABT-737, currently the best characterized inhibitor of the antiapoptotic proteins of the Bcl-2 family, and bear important implications regarding its use as an anticancer drug.

Chain-dependent photocytotoxicity of tricationic porphyrin conjugates and related mechanisms of cell death in proliferating human skin keratinocytes.

Photodynamic therapy (PDT) is a poor treatment option for nodular basal cell carcinomas and squamous cell carcinomas. As a result, the search for new photosensitizers with better effectiveness is of current interest. The photocytotoxicity of conjugates (P-R) of a water-soluble tri-cationic porphyrin (P-H) having similar efficiency of production of singlet oxygen, the PDT cytotoxin, has been assessed in vitro. Links between uptake, intracellular localization, photooxidative stress, photocytotoxicity and ability to induce programmed cell death are established. Conjugates bearing methyl (P-Me), Di-O-isopropylidene-(-d-galactopyranosyl (P-OGal) or N,N'-dicyclohexylureidooxycarbonyl (P-DDC) chains are efficiently taken-up by proliferating NCTC 2544 keratinocytes. The relative order of photocytotoxicity is P-OGal >P-DDC=P-Me≫P-H. The photocytotoxic potential of P-Me, P-OGal and P-DDC equals that of endogenous protoporphyrin IX induced by δ-aminolevulinic acid or its esters, the pro-drugs currently employed for PDT of skin lesions. Microfluorometry shows that P-Me, P-OGal, and P-DDC localize in endocytotic or pinocytotic vesicles but not in mitochondria or nucleus. Absence of annexin V binding, caspase activation or chromatin condensation suggests that cell photosensitization by P-R does not induce apoptosis. On the other hand, P-OGal photocytotoxicity correlates with appearance of multiple vesicles that have hallmarks of autophagy compartments, being decorated with the marker LC3 in cells transfected with an expression vector encoding GFP-LC3. p38 and JNK phosphorylation and inhibition of ERK1/2 phosphorylation suggest close relationship between mortality of NCTC 2544 keratinocytes and MAPK pathway impairment. Given their potentially easy formulation, water-soluble P-R are promising powerful photosensitizers for PDT of skin lesions.

Oxidized low density lipoprotein inhibits phosphate signaling and phosphate-induced mineralization in osteoblasts. Involvement of oxidative stress.

BACKGROUND: It is well admitted that oxidized LDL (OxLDL) plays a major role in the generation and progression of atherosclerosis. Since atherosclerosis is often accompanied by osteoporosis, the effects of OxLDL on phosphate-induced osteoblast mineralization were investigated. METHODS: Calcium deposition, expression of osteoblast markers and inorganic phosphate (Pi) signaling were determined under OxLDL treatment. RESULTS: OxLDL, within the range of 10-50 µg protein/ml, inhibited Pi-induced UMR106 rat osteoblast mineralization. In parallel, the expression of Cbfa1/Runx2 transcription factor was decreased, and the intracellular level of the osteoblast marker osteopontin (OPN) was reduced. The extracellular level of another marker, receptor activator of nuclear factor kappa B ligand (RANKL), was also diminished. OxLDL inhibited Pi signaling via ERK/JNK kinases and AP1/CREB transcription factors. OxLDL triggered the generation of reactive oxygen species (ROS), either in the absence or presence of Pi. Furthermore, the effects of OxLDL on Pi-induced mineralization, generation of ROS and extracellular level OPN were reproduced by the lipid extract of the particle, whereas the antioxidant vitamin E prevented them. CONCLUSIONS: This work demonstrates that OxLDL, by generation of an oxidative stress, inhibits of Pi signaling and impairs Pi-induced osteoblast differentiation. GENERAL SIGNIFICANCE: This highlights the role of OxLDL in bone remodeling and in degenerative disorders other than atherosclerosis, especially in osteoporosis.

BAD, a proapoptotic member of the BCL2 family, is a potential therapeutic target in hepatocellular carcinoma.

Proteins of the BCL2 family are key regulators of apoptosis. Their expression levels are frequently altered in cancers, enabling tumor cells to survive. To gain insight into the pathogenesis of hepatocellular carcinoma (HCC), we performed a comprehensive survey of the expression of the members of the BCL2 family in samples obtained from surgically resected HCCs. Here, we report the occurrence of a new molecular anomaly, consisting of a strong reduction in the expression of the proapoptotic protein BAD in HCC compared with surrounding nontumoral tissue. We investigate the function of BAD in a panel of HCC cell lines. Using gene overexpression and RNA interference, we show that BAD is involved in the cytotoxic effects of sorafenib, a multikinase blocker, which is currently the sole therapeutic drug effective for the treatment of HCC. Finally, we report that ABT-737, a compound that interacts with proteins of the BCL2 family and exhibits a BAD-like reactivity, sensitizes HCC cells toward sorafenib-induced apoptosis. Collectively, our findings indicate that BAD is a key regulator of apoptosis in HCC and an important determinant of HCC cell response to sorafenib.