SCCA-IC serum levels are predictive of clinical response in HCV chronic hepatitis to antiviral therapy: a multicentric prospective study.

The combination of pegylated interferon (Peg-IFN) and ribavirin is currently the gold standard therapy in patients with HCV chronic infection. The duration of therapy, as well as the therapeutic dosage, depend on the genotype. Identification of the genotype and rapid virological response (RVR) are widely accepted as the most important predictors of clinical outcome during antiviral therapy but to optimize cost-benefits and to reduce possible side effects, further prognostic factors are needed. Squamous cell carcinoma antigens immunocomplex (SCCA-IC) has been reported to be increased in the serum of patients with liver cancer. In this multicentric prospective study, we investigated the serum levels of SCCA-IC in 103 patients with HCV chronic infection. Serum HCV-RNA was detected before the beginning of treatment, after 4, 12, 24 or 48 weeks, and at week 24 during follow-up. RVR, early virological response and sustained virological response (SVR) were assessed following the international guidelines. SCCA-IC levels were higher in responders (238 AU, interquartile difference 130-556 AU) and decreased significantly to 125 AU (70-290 AU). The mean baseline value in nonresponders was 149 AU (86.5-306.5 AU), but after 4 weeks of treatment the serum levels decreased to 115 AU (80-280 AU): the profile of reduction was different between patients with or without a positive SVR. Logistic regression with SVR as dependent variable identified as significant independent variables: the reduction in SCCA-IC after 1 month (OR = 4.82; 95% CI 1.39-16.67; P = 0.131) and a genotype other than 1 (OR = 0.094; 95% CI 0.21-0.42; P = 0.002); sex and age were also significant factors influencing SVR. SCCA-IC seems to be a reliable independent prognostic marker of therapeutic effectiveness in anti-HCV positive patients undergoing antiviral therapy.

MMP-2, MMP-9, VEGF and CA 15.3 in breast cancer.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of extracellular matrix degrading proteinases. Owing to their matrix-degrading abilities and high expression in advanced tumours, MMPs were originally implicated in cancer progression, invasion and metastasis. PATIENTS AND METHODS: In this study, the correlation was determined between the expression of gelatinases (MMP-2 and MMP-9) in the sera of breast cancer patients from zymographic analysis and serum concentrations of VEGF and CA 15.3, before surgery and after 1 and 6 months; the association of both markers with clinicopathological features including histological type, stage of disease and estrogen (ER) and progesterone (PgR) receptors status were also analysed. In all, 88 breast cancer patients and 20 healthy women were involved in this study. RESULTS: No statistically significant correlation between pro MMP-2, pro MMP-9, VEGF and CA 15.3 serum levels was found (p>0.05). In breast cancer patients, a significant decrease of the pro MMP-2 serum expression 1 month after surgery with respect to serum levels before surgery (p=0.0008) was evident, as well as of CA 15.3 serum levels at baseline and after 1 month (p=0.017). Moreover a strong decrease of pro MMP-9 serum levels was found in 88 breast cancer patients after 1 month (p=0.028) and after 6 months (p =0.009) from surgery. On the other hand, no significant differences in the serum levels of VEGF, CA 15.3, pro MMP-2 or pro MMP-9 between 88 breast cancer patients preoperatively and 20 healthy women as controls were found. Our findings did indicate a significant positive association between higher preoperative levels of CA 15.3 and progression of disease (p=0.03), as well as a longer disease-free survival in patients who exhibited a decrease of serum pro MMP-9 expression compared to other biomarkers. No relationship between these four markers and the main clinical and pathological parameters was found. CONCLUSION: The present study failed to demonstrate any association between serum levels of MMPs, VEGF and CA 15.3 and well-known clinicopathological characteristics of breast carcinoma, while demonstrating the prognostic value of CA 15.3 and pro MMP-9 in the follow-up of breast cancer patients.

Laminin-5 with transforming growth factor-beta1 induces epithelial to mesenchymal transition in hepatocellular carcinoma.

BACKGROUND & AIMS: How hepatocellular carcinoma (HCC) cells acquire the ability to invade surrounding tissue is unknown, but epithelial mesenchymal transition (EMT) likely plays a role. We investigate how transforming growth factor (TGF)-beta1 and extracellular matrix protein Laminin-5 (Ln-5) induce EMT and cancer invasion. METHODS: Snail, Slug, E-cadherin, beta-catenin and Ln-5 were investigated on HCC tissues and on HCC cell lines. RESULTS: We show that in HCC but not in peritumoral tissue of the same HCC patients, Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated and beta-catenin is translocated into the nuclei. In vitro, HCC "invasive" cells, partially EMT-transformed, show low levels of E-cadherin. In presence of Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated, beta-catenin is translocated into the nuclei, and cells undergo a dramatic morphological change, becoming scattered and undergoing a complete EMT. This effect is reversed by anti-alpha3 but not by anti-alpha6 integrin blocking antibody. HCC "noninvasive" cells are not EMT-transformed, and have constitutively high levels of E-cadherin. In presence of Ln-5, cells undergo partial EMT, Snail, and Slug are up-regulated, E-cadherin is down-regulated but cells do not scatter. However, the presence of both Ln-5 and TGF-beta1 completes the EMT process, beta-catenin is translocated into the nuclei, cells scatter and become invasive, recalling the "invasive" cells. In this case, too, the effect is reversed by anti-alpha3 integrin blocking antibody. CONCLUSIONS: Our study shows that Ln-5 and TGF-beta1 cooperatively induce EMT in HCC, suggesting the microenvironment as a potential target for new biological therapies.

Laminin-5 offsets the efficacy of gefitinib ('Iressa') in hepatocellular carcinoma cells.

Prognosis and survival of patients with hepatocellular carcinoma (HCC) is still very poor, and no therapies are currently available to inhibit tumour growth and metastases. Recently, we reported that the expression of an extracellular matrix component (ECM), namely Laminin-5 (Ln-5), is directly related to poor prognosis in HCC patients. The aim of our study is to investigate the preclinical effect of gefitinib in an in vitro HCC model. We found that the IC(50) of gefitinib in HCC cells ranged from 0.7 to 10.0 muM, whereas Ln-5 inhibited the activity of gefitinib in a dose-dependent manner. Complete inhibition of phosphorylated (p)-EGFR (epidermal growth factor receptor) was obtained within 6 h exposure to gefitinib and complete restoration of the receptor status was obtained after 24 h. A downstream effect yields a decrease in p-Akt and p-Erk 1/2. The addition of exogenous Ln-5 has no effect on p-EGFR, whereas it restores p-Erk 1/2 and p-Akt. Consistently, Ln-5 induces recovery of HCC cells from Gefitinib-induced apoptosis. In conclusion, gefitinib inhibits HCC cell growth and we report for the first time that Ln-5, but not other ECM molecules, reduces the ability of gefitinib to inhibit cell growth via Akt. As patients with HCC have different Ln-5 expression levels, these results may help to better understand which patients might benefit from gefitinib treatment.

Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion.

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor of the liver; prognosis depends on the tendency to metastasize. Cancer cell invasion is regulated by proteolytic remodeling of extracellular matrix components and by integrin expression. We have shown that matrix metalloproteinase-2 (MMP-2) and membrane-type-1 matrix metalloproteinase (MT1-MMP) cleave Laminin-5 (Ln-5), stimulating cell migration. Here we report that all HCC cells express MT1-MMP, migrate on Ln-1 and Collagen IV, whereas only HCC cells that express alpha3beta1 integrin secrete detectable levels of gelatinases, migrate on Ln-5, and invade through a reconstituted basement membrane (BM). Migration on Ln-5 is blocked by BB-94, an MMP inhibitor, and by MIG1, a monoclonal antibody that hinders migration on MMP-2-cleaved Ln-5. Invasion through a reconstituted BM is also inhibited by BB-94. HCC alpha3beta1-negative cells migrate on Ln-1 and Collagen IV, but not on Ln-5, and do not invade through a reconstituted BM, although they express MT1-MMP. Anti-alpha3beta1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through MATRIGEL: In vivo, alpha3beta1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both alpha3beta1 integrin and gelatinase activity are required for HCC migration and invasion.

Integrin activation controls metastasis in human breast cancer.

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin alpha v beta 3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin alpha v beta 3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated alpha v beta 3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant alpha v beta 3(D723R), but not alpha v beta 3(WT), in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin alpha v beta 3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Modulation of cytochrome c-mediated extramitochondrial NADH oxidation by contact site density.

Data 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 + 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 + ADP when TMPD and endogenous cytochrome c are utilized as electron carriers. The results obtained are consistent with the proposal that the bi-trans-membrane 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 apoptosis [Biochem. Biophys. Res. Commun. 246, 556-561, 1998].

Inhibition by butylmalonate of proton influx in nonphosphorylating mitochondria.

The 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.

Mitochondrial membrane potential supported by exogenous cytochrome c oxidation mimics the early stages of apoptosis.

Mitochondria isolated from rat liver, incubated with inhibitors of respiratory Complexes I and III but in the presence of added NADH as a source of reducing equivalents, generate a membrane potential, the extent of which is comparable to that supported by the oxidation of intramitochondrial respiratory substrates. A catalytic amount of exogenously added cytochrome c is required in order for this membrane potential to be observed. Such an experimental approach mimics the early stages of the apoptotic program of mammalian cells. This has been reported to be characterized by the extrusion of the bulk of intermembrane pool of cytochrome c while the mitochondria continue to preserve their membrane potential. The data obtained are consistent with the proposal that the bi-trans-membrane electron transport pathway, activated by the transfer of cytochrome c from mitochondria to the cytosolic compartment, may be directly involved in the generation of the mitochondrial membrane potential at the beginning of the cell death process.

Cycloheximide sensitivity of orotic acid biosynthesis induced by ammonia and glycine administration.

Administration of either ammonia or glycine to both rats and mice results in an increased synthesis in the liver and urinary excretion of orotic acid. The two most relevant observations obtained are that carbamoyl phosphate synthesized inside the mitochondria is involved in the increased synthesis of orotic acid and that this latter process is almost completely abolished by cycloheximide and actinomycin D, inhibitors of protein and RNA synthesis. Orotic acid synthesis could be controlled by an induction-suppression mechanism. Inhibition of synthesis of excess orotic acid brought about by N-(phosphonacetyl)-L-aspartic acid but not by acivicin, suggests that glutamine-dependent cytosolic synthesis of carbamoyl phosphate, is not involved. Administration of ornithine together with glycine completely suppressed the synthesis of orotic acid, but promoted a twofold increase of urea excretion. The concentration of ornithine rather than that of carbamoyl phosphate or the activity of the enzymes involved, may represent a limiting factor controlling both the flux of ammonia in the urea cycle and the availability of mitochondrial carbamoyl phosphate for orotic acid synthesis. Two enzymes have been found to be induced by glycine: ornithine decarboxylase and aspartate transcarbamoylase (aspartate carbamoyltransferase). Both enzymes may contribute to the increase in orotic acid synthesis, aspartate transcarbamoylase more directly and ornithine decarboxylase by lowering the ornithine concentration. Ornithine decarboxylase activity was completely suppressed but that of aspartate transcarbamoylase was further increased by cycloheximide treatment. Inhibition of orotic acid biosynthesis by cycloheximide appears to be the result of a decreased availability in the cytosol of carbamoyl phosphate synthesized inside the mitochondria.

Proton translocation linked to the activity of the bi-trans-membrane electron transport chain.

Recently we have proposed and presented evidence suggesting the existence of a "bi-trans-membrane" electron transport chain, located at the contact sites between outer and inner mitochondrial membranes, which can be utilized to promote either the oxidation of exogenous NADH in the presence of catalytic amounts of added cytochrome c or the reduction of exogenous cytochrome c supported by the oxidation of respiratory substrates present inside the mitochondria. Here we show that the oxidation of exogenous NADH is accompanied by a net alkalinization of the incubation medium preceded by a transient acidification phase. In oxygen-pulse experiments, the alcohol oxidation (induced by the addition of alcohol dehydrogenase) was used to mimic a cytosolic source of reducing equivalents. Oxygen pulses promote an acidification-alkalinization proton cycle which is insensitive to antimycin and myxothiazol inhibitory effect, is stimulated by valinomycin, inhibited by trypsin-aprotinin complex, abolished by the protonophore carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and is absent or at least inverted (alkalinization-acidification cycle) in broken mitochondria. The oxidation of cytosolic substrates, mediated by the bi-trans-membrane electron transport chain, does not involve endogenous cytochrome c and is associated with a vectorial proton translocation from the inside to the outside of the mitochondria. In the out-->in electron transport pathway the components involved appear to be cytosolic reduced substrates-->NADH produced by cytosolic dehydrogenases activity-->NADH-cytochrome b5 oxidoreductase complex leaning out the external side of the external membrane-->exogenous cytochrome c-->cytochrome oxidase of contact sites-->molecular oxygen. The possible components of the in-->out pathway are matrix respiratory substrates-->primary dehydrogenases of the matrix-->Complexes I, II, and III of the respiratory chain present in the inner membrane-->NADH-cytochrome b5 oxidoreductase system of the external membrane-->exogenous cytochrome c-->additional cytosolic electron acceptors or, alternatively, cytochrome oxidase of contact sites. The two pathways can be considered a bi-trans-membrane electron channeling system which, at the level of bridges set up by the contact points between the outer and the inner mitochondrial membrane, may represent a link between the redox processes occurring inside with those present outside the mitochondrion.