CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes

Background: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. Methods: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. Results: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. Conclusion: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development

Modificazione delle membrane in corso di invecchiamento

Tesi di dottorato in biologia e fisiopatologia dell'invecchiamento. 7. ciclo. Coordinatore G. Savioli; tutore G. SavioliConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - Piazza Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Methionine intolerance and plasma homocysteine after insulin infusion in type II diabetic patients

Background-To establish whether transsulfuration impairment (methionine intolerance) may influence total plasma homocysteine (Hcy) in response to insulin infusion in type II diabetic patients (DB).Materials and Methods- DB with normal (group A, n=13) and abnormal (hyperhomocysteinemia, HHcy)) (Group B, n=17) response to oral methionine load underwent a glucose/clamp study. At time 0, and 30, 60 and 120 minutes after hyperinsulinemia, Hcy and methionine (Met) plasma levels were assessed. All patients were assayed for fasting Homocysteine-Cysteine ratio (as marker of suspected heterozygosis for cystathionine--synthase deficit) and vitamin status [serum vitamin B6 (PLP), vitamin B12 and folate]. Results- After hyperinsulinemia, plasma methionine reduced (about –30% at 120 minutes vs. basal values) within both groups, whereas Hcy decreased in group A (up to –15.2 8.9 % at 120 minutes) and increased in group B (up to +30.6 10.9 % at 120 minutes). (see figure) PLP was higher in group A than in group B (94.2 42.6 vs. 54.6 32.4 nmol/L; p<0.01); group A showed a lower prevalence of suspected heterozygosis for cystathionine--synthase deficit (1/13 (7,7%) vs.13/17 (76.5%), p=0.000). Conclusions-Methionine intolerance may influence insulin effect on plasma Hcy. To prevent a possible acute HHcy due to insulin administration in case of transsulfuration impairment, it may be suggested to assess transsulfuration capacity in all patients due to be subjected to insulin therapy

Correlation between plasma malonyldialdeide levels and paroxonase activity in healthy subjects and in patients with atherosclerotic disease

Paraoxonase (PON) is a calcium-dependent HDL-associated ester hydrolase catalizying hydrolysis of organophosphates. Its biological role is still unknown: experimental studies suggest a hydroperoxidasic activity on lipoperoxides formed during LDL-oxidation, thus acting as a protective agent against atherogenesis. We evaluated PON activity in healthy subjects (HS) and in patients with cardiovascular (CHD) and cerebrovascular (CVD) disease ; PON activity was correlated with plasma malonyldialdeide (MDA) levels, as markers of lipid peroxidation Materials and Methods: 33 HS (age 7012 yr), 45 CHD (age 818 yr) and 38 CVD (age 8011 yr) patients were studied. Serum PON and plasma MDA levels were measured spectrophotometrically. Statististical analysis was performed by ANOVA univariate for groups comparation and by Pearson r test for values correlation Results: PON activity (mol min-1 ml-1) is higher in HS (0.0750.03) than in CHD (0.0450.03) and CVD (0.0560.03) patients (p<0.05 HS vs. CHD and CVD; ns CHD vs. CVD). MDA (mol/ml) levels are lower in HS (0.0280.023) than in CHD (0.0630.012) and in CVD (0.0560.036) patients (p<0.05 in HS vs. CHD and CVD, ns in CHD vs CVD). Table shows correlation between PON activity and MDA plasma levels in the three studied groups are shown below. rsignificanceHS-.43 p<0.05CHD-.39 p<0.01CVD-.33 p<0.05Conclusions: PON activity is significatively higher in HS than in CHD and CVD patients; whereas there are no significant difference between CHD e CVD patients. In all groups there is statistically significant negative correlation between PON activity and lipid peroxides content (MDA levels) in plasma. Our results suggest a protective role of PON activity against atherogenesis occurring with higher levels of lipoperoxides

Effect of N-acetylcysteine long term oral administration on plasma and urinary homocysteine and cysteine levels

Background: A decrease of plasma homocysteine (Hcy) may represent a therapeutic promise for reducing the impact of atherosclerosis. N-acetyl-cysteine (NAC) at high dosage seems able to interfere with endogenous thiols [cysteine (Cys) and Hcy] by forming mixed disulphides undergoing a more efficient renal clearance. Aim : to assess the effect of NAC oral chronic administration (different doses) on plasma and urine levels of different forms of plasma Cys and Hcy. Methods: In 40 healthy (44±15 years, 22 F) we assessed fasting total and free (= not protein bound) forms of plasma and urinary levels of Hcy and Cys. After informed consent subjects were randomly assigned to group A (n= 13, no therapy), group B (n= 14, NAC 600 mg once daily per os) and group C (n= 14, NAC once daily 1800 mg per os) for a month. Results: Group C showed a significant reduction of total Hcy (10.68 2.8 vs. 13.64 3.56 mmol/L, p= .001) a not significant reduction of free Hcy (3.48 0.72 vs. 3.55 0.74 mol/L, p=.702) a significant reduction of total Cys (320 93 vs. 377 122 mol/L, p=.013) a not significant reduction of free Cys (147 26.5 vs. 162 40.6 mol/L, p= .294), a significant increase of free/bound Hcy and Cys ratio (33.2 3.21 vs. 26.5 3.7 %, p= .000 and 48 7.6 vs. 42 6.0 %, p=.045, respectively).Group B showed a significant reduction of total plasma Hcy (11.6 3.7 vs. 12.88 3.9 mol/L, p=0.031) but not significant modification of other thiols forms levels.After NAC therapy Group C showed also significant higher daily Hcy and Cys urinary levels (11.73.5 vs. 9.13.1 mmol/ mg urinary creatinine , p<0.01, 24773 vs. 22074 mmol/ mg urinary creatinine, p=0.011).Also group B showed higher, but not significant, Hcy and Cys urinary levels (11.24.2 vs 10.513.3 mol/ mg urinary creatinine, p=0.056 and 23949 vs 22955 mol/ mg urinary creatinine, p=0.055). No significant variations in plasma and urinary thiols were noticed in group A.Conclusions: A chronic (one month) oral NAC administration induces a decrease of the main circulating plasma thiols (Cys and Hcy) by increasing their renal excretion. This effect seems dose-dependent, being significant only at higher NAC dosage; the modification of plasma thiols forms (reduction of total and free forms but increase of free/total ratio) support the hypothesis of displacement by NAC of thiols from their plasma protein binding sites to form mixed disulphides, which in turns may undergo a higher renal clearance, resulting in an increase of urinary excretion

Problemi dell'iperomocisteinemia nell'anziano

Il lavoro riguarda i problemi dell'iperomocisteinemia nell'anziano affrontando gli aspetti fisiopatologici e le possibili conseguenzze clinich

Affinit\ue0 dell'omocisteina per le lipoproteine

Il lavoro affronta in modo approfondito il legame dell'omocisteina alle lipoprpotein

Effetti dell’insulina sui livelli di omocisteina plasmatica in pazienti con diabete mellito tipo ii con e senza intolleranza alla metionina

Premessa- Una elevata prevalenza di iperomocisteinemia (HHcy) è stata osservata in pazienti con diabete mellito tipo II e malattia vascolare documentata; da qui l’ipotesi che la iperomocisteinemia possa contribuire alla mortalità generale nei pazienti diabetici. Il legame fra insulina e metabolismo dell'omocisteina (Hcy) non è stato ancora chiarito; in particolare, sono disponibili soltanto pochi dati circa gli effetti in vivo dell’insulina sul metabolismo dell' Hcy in presenza di quelle alterazioni (congenite e/o acquisite) del metabolismo degli aminoacidi solforati in grado di condizionare una condizione di HHcy (intolleranza alla metionina). Scopo- valutare in che modo la presenza di una condizione di intolleranza della metionina possa influenzare i livelli plasmatici di Hcy in risposta all'infusione dell'insulina in vivo in pazienti con diabete tipo II.Materiali e metodi - Abbiamo sottoposto 18 pazienti (gruppo A) con normale e 18 pazienti con anormale (iperomocisteinemia dopo carico, intolleranza alla metionina) risposta (gruppo B) al carico orale con metionina ad uno studio di clamp euglicemico. A tempo 0 ed a 30, 60 e 120 minuti dall’induzione di iperinsulinemia, abbiamo valutato i livelli circolanti di omocisteina (tHcy) e metionina (Met) (HPLC). Per valutare la causa di intolleranza della metionina, tutti i pazienti sono stati sottoposti a valutazione del rapporto omocisteina-cisteina a digiuno (indicatore di sospetta eterozigosi per deficit di CS), a valutazione genetica della mutazione C677T della MTHFR e al dosaggio sierico delle vitamine necessarie al corretto metabolismo dell’omocisteina [ vitamina B 6 ( PLP), vitamina B 12 e folati ]. Risultati - Dopo iperinsulinemia, i livelli di Met sono risultati ridotti (circa –30% a 120 minuti rispetto ai valori basali) in entrambi i gruppi, mentre la tHcy è risultata ridursi nel gruppo A (fino a - 6.6 3.6 % rispetto ai valori basali), ed aumentare nel gruppo B (fino a +29.05 8.3 % rispetto ai valori basali) . I livelli sierici di folati (7.45 2.8 contro. 4.82 2.5 nmol/L, p<0.05), Vit. B 12 (348 78 contro. 242 65 pmol/L, p< 0.05) e PLP (84.1 23.6 contro 50.6 32.4 nmol/L; p<0.01) sono risultati più alti nel gruppo A che nel gruppo B; i livelli di PLP sono risultati correlare significativamente con quelli della tHcy dopo carico (n=36; r=-.327, p<0.05); il gruppo A presentava inoltre una prevalenza inferiore di sospetta eterozigosi per CS [ 1/18 (11.1 %) contro 5/18(33.3%), p<0.05 ] e una ridotta prevalenza dell'allele mutato T della MTHFR [ 4/18 (22.2%) contro 11/18 (61.1%), p<0.01 ]. L’analisi di regressione logistica multipla con la variazione di Hcy sotto stimolo insulinico (evento A = riduzione; evento B = aumento) come variabile dipendente, ha indicato la carenza di folati e di PLP e la presenza dell'allele di MTHFR T come le variabili più significativamente predittive del tipo di risposta dell’Hcy all’iperinsulinemia.Conclusioni - La presenza di intolleranza alla metionina può influenzare l'effetto dell'insulina sull' omocisteina plasmatica in pazienti con diabete tipo 2. Per prevenire la possibilità di episodi acuti, ripetuti e potenzialmente dannosi, di iperomocisteinemia in questi pazienti in seguito alla assunzione di insulina, potrebbe essere utile la valutazione della presenza di una condizione di intolleranza della metionina (test orale alla metionina) e uno studio dello stato vitaminico Hcy-relato in tutti i pazienti diabetici da sottoporre alla terapia insulinica

Increase of protein and lipid oxidation during hyperhomocysteinemia induced by methionine oral loading

Introduction: Hyperhomocysteinemia (HHcy) is a well-defined risk factor for vascular disease by a not still well clear molecular mechanism. It is known a pro-oxidant effect of Hcy “in vitro” in presence of metal ions (Fe e Cu). To assay a similar effect in vivo, we studied plasma markers of lipid and protein oxidation during hyperhomocysteinemia induced by methionine oral load.Materials and methods: 16 subjects (aged 79 14 years; 16f), 14 of which underwent a methionine (100 mg/Kg) oral load were studied; in all patients we assayed total plasma HCY, malonaldehyde (MDA) and conjugates dienes (DIE), oxidized protein (PTOX) (carbonilic groups) in basal conditions and after 4, 6, 8 and 24 hours from the oral load. In the two subjects who did not take the methionine load (controls), were made the same assays with the same timing of the probands. In all subjects we assayed basal and after 8 hours from the methionine load total plasma antioxidant (ANTOX) capacity.Results: table shows values (mean DS) of considered parameters in subjects who underwent the methionine loadParameterBasal4 h6 h8 h24 hHcy (nmol/ml)20.7 11.550.6 19.157.2 25.561.6 28.3 45.3 30.7PTOX (nmol/mg prot.)0.38 0.210.49 0.270.68 0.390.68 0.270.58 0.40DIE(nmol/ml)493 163562 181526 233590 202545 182MDA(nmol/ml)1.66 0.801.91 0.942.19 1.321.96 0.931.95 0.99ANTOX(nmol/ml)1.76 +/- 0.511.38 +/- 0.86 Conclusions: HHCY induces a correspondent increase of plasma oxidation makers. In absence of HHCY, no significant modifications were observed. This data, together with the reduction of ANTOX in correspondence of maximum plasma HCY increase, are suggestive of pro-oxidant effect of HHCY in vivo