Effect of HUVEC apoptosis inducing proteinase from Vipera lebetina venom (VLAIP) on viability of cancer cells and on platelet aggregation

Three cancer cell lines, the human androgen independent prostate cancer PC-3, androgen dependent LNCaP prostate cancer and human chronic myeloid leukaemia cell line K-562, were treated with Sephadex G-100 sf fractions of Vipera lebetina venom and with HUVEC apoptosis inducing heterodimeric metalloproteinase (VLAIP) from the same venom. The venom was separated into nine fractions using size-exclusion chromatography on Sephadex G-100 sf. The effect of V. lebetina venom gel filtration fractions on the viability of studied cancer cells was different: high molecular mass fractions were the most effective on PC-3 cells. The viability of LNCaP cells was inhibited most strongly by the third fraction. The first and the second fractions contain different metalloproteinases including VLAIP that also most effectively reduced the viability of PC-3 cells. VLAIP decreased PC-3 cell viability in a concentration- and time-dependent manner but did not induce apoptosis as shown by DNA fragmentation assay. VLAIP induced changes in cell shape, rounding up and detachment. VLAIP inhibited the PC-3 cell adhesion to extracellular matrix proteins collagen I, fibronectin and vitronectin but not to fibrinogen. VLAIP had no significant effect on the viability of LNCaP and K-562 cells.VLAIP was also capable to inhibit ADP- and collagen-induced platelet aggregation dose-dependently. IC₅₀ was determined to be 1.89 μM and 0.94 μM, respectively.     

Isolation and Biochemical Characterization of Rubelase, a Non-Hemorrhagic Elastase from Crotalus ruber ruber (Red Rattlesnake) Venom

A novel non-hemorrhagic basic metalloprotease, rubelase, was isolated from the venom of Crotalus ruber ruber. Rubelase hydrolyzes succinyl-L-alanyl-L-alanyl-L-alanyl p-nitroanilide (STANA), a specific substrate for elastase, and the hydrolytic activity was inhibited by chelating agents. It also hydrolyzes collagen and fibrinogen. However, hemorrhagic activity was not observed. By ESI/Q-TOF and MALDI/TOF mass spectrometry combined with Edman sequencing procedure, the molecular mass of rubelase was determined to be 23,266 Da. Although its primary structure was similar to rubelysin (HT-2), a hemorrhagic metalloprotease isolated from the same snake venom, the circumstances surrounding putative zinc binding domain HEXXHXXGXXH were found to be different when the three-dimensional computer models of both metalloproteases were compared. The cytotoxic effects of rubelase and rubelysin on cultured endothelial and smooth muscle cells were also different, indicating that the substitution of several amino acid residues causes the changes of active-site conformation and cell preference.     

2种海参胶原蛋白多肽对血管内皮细胞保护作用的比较研究

目的 观察革皮氏海参和北极刺参胶原蛋白多肽对氧化型低密度脂蛋白(ox-LDL)损伤的血管内皮细胞的保护作用,探讨其保护内皮细胞的作用机制。方法 采用ox-LDL处理血管内皮细胞(ECV304)建立氧化应激损伤模型,以MTT法测定ECV304的增殖活性,硫代巴比妥酸法测定细胞内的丙二醛(MDA)含量,比色法测定一氧化氮合酶(NOS)活力和一氧化氮(NO)释放量,Hoechst33258染色法检测细胞的凋亡,Western blotting法检测caspase-3蛋白的表达。结果 经2种海参胶原蛋白多肽预处理后,ECV304细胞的增殖率显著升高 (P<0.05,P<0.01),细胞凋亡比率和MDA含量显著降低(P<0.05,P<0.01),细胞NOS活力和NO释放量均显著提高 (P<0.05,P<0.01),凋亡蛋白caspase-3的表达量显著降低。结论 2种海参胶原蛋白多肽均能有效保护脂质过氧化物损伤的血管内皮细胞,其中北极刺参胶原蛋白多肽在抑制细胞凋亡和提高NOS活性方面效果更突出,可能与其氨基酸组成有关。     

Brown spider (Loxosceles intermedia) venom triggers endothelial cells death by anoikis

Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the α₅β₁ integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.     

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.     

A novel RGD antagonist that targets both alphavbeta3 and alpha5beta1 induces apoptosis of angiogenic endothelial cells on type I collagen

Integrin-mediated cell adhesion is necessary for endothelial cell proliferation and apoptosis, which is a major determinant in tumor-induced angiogenesis. In this study, we compared two novel, structurally similar, Arg-Gly-Asp (RGD) peptidomimetic compounds having different integrin selectivities, for their inhibition of endothelial cell proliferation and induction of apoptosis on functionally relevant extracellular matrices (ECM) for angiogenesis. BCH-14661 was specific for integrin alphavbeta3, whereas BCH-15046 nonselectively antagonized integrins alphavbeta3, alphavbeta5, and alpha5beta1. Both compounds were potent inducers of endothelial cell apoptosis when plated on RGD-dependent ECM (vitronectin, VN), which was dependent on the ability to induce cell detachment. However, with endothelial cells plated on RGD-independent ECM (type I collagen, COL), only BCH-15046 was able to significantly prevent growth and induce apoptosis. This effect was not dependent on the induction of detachment. Experiments using the matrix metalloproteinase (MMP) inhibitor GM 6001 revealed that cleavage of COL was not required for the ability of BCH-15046 to induce apoptosis. However, the inhibition of growth factor-stimulated endothelial cell proliferation, required MMPs, and correlated with BCH-15046s' potent inhibition of endothelial cell attachment to denatured collagen. Antibody inhibition experiments showed that adhesion to denatured collagen required integrins alphavbeta3 and beta1, but not alphavbeta5. In addition, BCH-15046 exerted a significant inhibition of VEGF-stimulated angiogenesis in the chick chorioallontoic membrane in vivo. These results suggest that integrin antagonism of both alphavbeta3 and alpha5beta1 are important for MMP-independent induction of apoptosis on COL and MMP-dependent inhibition of endothelial cell-denatured collagen interactions required for proliferation.     

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.     

A mechanism of apigenin-induced apoptosis is potentially related to anti-angiogenesis and anti-migration in human hepatocellular carcinoma cells

Apigenin (APG) has been shown to have a strong anti-cancer effect on various cancer models via a programmed cell death, apoptosis. However, the fundamental mechanisms of these effects are still unclear. In the present study, we examined the question of whether or not APG can inhibit proliferation of hepatocellular carcinoma (HCC), huh-7 cells, resulting in apoptosis. In APG-treated cells, we observed typical features of apoptosis. To identify the proteins related to APG-induced apoptosis, we performed two-dimensional electrophoresis analysis and identified differentially expressed proteins. Among these proteins, we focused on vimentin, which plays a physiological role, such as cell migration and adhesion. We validated expression of vimentin in both mRNA and protein levels, verifying its decrease. In addition, we observed that APG down-regulated the expression levels of type I collagen, which collaborated with vimentin in cell migration and decreased the releasing amounts of VEGF and MMP-8, which are closely relevant to angiogenic activity. Finally, we confirmed the decreased capacity of cell migration due to down-regulation of vimentin, type I collagen, VEGF, and MMP-8 induced by APG. Based on the overall results, we suggested that vimentin was potentially associated with APG-induced apoptosis, as a key regulator in angiogenesis and migration.     

A snake venom metalloproteinase that inhibited cell proliferation and induced morphological changes of ECV304 cells.

TSV-DM, a basic metalloproteinase with a molecular weight of 110kDa, was purified from Trimeresurus stejnegeri venom. TSV-DM degraded the Aalpha chain of fibrinogen more rapidly than the Bbeta chain in a dose dependent manner. The cDNA of TSV-DM encoded a polypeptide of 622 amino acid residues, which comprises a signal peptide, proprotein, metalloproteinase domain, spacer, disintegrin-like domain and cysteine-rich domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIIb snake venom metalloproteinase, which comprises vascular apoptosis-inducing proteins. TSV-DM inhibited cell proliferation and induced cell morphologic changes transiently of ECV304 cells. However, DNA fragmentation and DNA content analysis demonstrated that this metalloproteinase could not induce ECV304 cells apoptosis.     

Protective effects of curcumin on methylglyoxal-induced oxidative DNA damage and cell injury in human mononuclear cells

AIM: To examine the effect of curcumin on oxidative DNA damage and cell apoptosis and injury caused by the reaction of methylglyoxal(MG) with amino acids. METHODS: We used DNA strand breaks to examine the effect of curcumin on oxidative DNA damage. In addition, reactive oxygen species(ROS) formation occurs in MG-treated mononuclear cells, so the effect of curcumin on ROS generation was measured using 2',7'-dichlorofluorescin diacetate(DCF-DA) as the detection reagent. Moreover, the impact effects of curcumin on MG-induced cell apoptosis and ROS injury were analyzed by TUNEL and ELISA assay. The collagen I attachment ability of mononuclear cells was examined by trypan blue staining. RESULTS: Our results revealed that curcumin prevented MG/lysine-induced oxidative stress and DNA damage. Curcumin also inhibited MG-induced apoptosis and generation of ROS in mononuclear cells. MG-treated mononuclear cells displayed a lower degree of attachment to collagen (the major component of the vessel wall subendo-thelium), whereas cells pretreated with curcumin before MG treatment exhibited restored affinities for collagen. CONCLUSION: These results demonstrated that oxidative stress plays a role in MG-induced cell injury and alterations in attachment ability, and that curcumin blocks these effects by virtue of its antioxidant properties.     

日本刺参酸性粘多糖、皂苷和胶原蛋白多肽对血管内皮细胞的保护作用

目的研究日本刺参酸性粘多糖、皂苷和胶原多肽对血管内皮细胞的保护作用。方法采用ox-LDL建立体外培养的人脐静脉血管内皮细胞株(ECV304)损伤模型。MTT法测定血管内皮细胞的增殖活性;硝酸还原酶法测定血管内皮细胞一氧化氮合酶(NOS)活力和NO释放量;TBA法测定细胞内MDA含量;DNA-Ladder法检测血管内皮细胞的凋亡。结果不同浓度的酸性粘多糖、胶原蛋白多肽和低浓度的皂苷能明显抑制ox-LDL对血管内皮细胞的损伤,促进血管内皮细胞增殖(P<0.05,P<0.01);降低细胞内MDA含量(P<0.05,P<0.01);拮抗ox-LDL引起的血管内皮细胞凋亡。酸性粘多糖和胶原蛋白多肽还能明显促进血管内皮细胞NO释放量(P<0.05,P<0.01),提高细胞NOS活力(P<0.05,P<0.01)。结论日本刺参酸性粘多糖、皂苷和胶原蛋白多肽对血管内皮细胞的脂质过氧化物损伤均具有明显的保护作用。     

NF-κB抑制剂PDTC诱导人肝星状细胞LX-2凋亡的药理机制（英文）

本研究旨在探讨NF-κB抑制剂吡咯烷二硫代氨基甲酸酯(PDTC)是否可以诱导人肝星状细胞LX-2凋亡,并分析其潜在药理机制。通过体外培养人肝星状细胞系LX-2细胞,将实验分为空白对照组和PDTC干预组。将LX-2细胞暴露于PDTC后,使用CCK8检测法评估LX-2细胞活力;采用AO/EB双染试剂盒检测PDTC的抗凋亡作用。此外,分别采用Western blotting法和免疫荧光染色法测定NF-κB、Fas/Fas L、凋亡相关蛋白的活性和AIF的细胞定位。PDTC处理12和24小时后,AO/EB双染呈现典型的凋亡变化,比如细胞体积缩小、细胞核固缩、核碎裂等。PDTC干预浓度在60μmol/L时可显著提高细胞增殖抑制率,减少LX-2细胞中的胶原蛋白I、胶原蛋白III和α-SMA的分泌。Westernblotting和RT-PCR结果显示,对照组与PDTC干预组的AIF表达水平无统计学意义,且各组均未观察到Fas和Fas L的表达(P>0.05)。PDTC还可以促进AIF从线粒体向细胞核的移位,激活细胞核内的凋亡信号,并可能参与LX-2细胞的凋亡过程。综上所述, PDTC抗肝纤维化...     

Humic acid induces apoptosis in human endothelial cells

Humic acid (HA) has been implicated as an etiologic factor in the vasculopathy of Blackfoot disease. In this study, the ability of HA to induce apoptosis was studied in cultured human umbilical vein endothelial cells. Treatment of endothelial cells with a variety of concentrations of HA (50-200 microg/ml) resulted in dose- and time-dependent sequences of events marked by apoptosis as shown by loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. Antioxidants (superoxide dismutase, vitamin C, and vitamin E) and Ca(2+) chelator (BAPTA) effectively suppressed HA-induced DNA fragmentation (apoptosis). Further studies have shown that HA induced dramatic Ca(2+)-dependent caspase activation (2, 3, 6, 8, and 9). In contrast, negligible caspase-1 activation was observed. The increase in HA-induced apoptosis correlated with a reduction in Bcl-2, a potent cell death inhibitor, and an increase in Bax protein levels, which heterodimerizes with and thereby inhibits Bcl-2. Both of the antioxidants vitamin C and vitamin E prevented the dysregulation of Bcl-2 and Bax in HA-treated endothelial cells. Furthermore, the increase in p53 protein levels correlated with an increase in HA-induced apoptosis. We concluded that both Ca(2+) and oxidative stress were mediators of apoptosis caused by HA and the induction of apoptotic cell death on endothelial cells may be important to the etiology of HA-induced vascular disorder of Blackfoot disease.     

N-(methylamino)isobutyric acid inhibits proliferation of CFSC-2C hepatic stellate cells

Activation of hepatic stellate cells (HSCs) involves the induction of ECM protein synthesis and rapid cell proliferation. Thus, agents that interfere with either process could potentially mitigate the development of liver disease by reducing the synthesis of proteins associated with fibrosis or by reducing the number of activated HSC. Previously, we described that the non-metabolizable amino acid analog N-(methylamino)isobutyric acid (MeAIB) reduced hepatic collagen content of rats in a model of CCl(4)-induced liver injury, and in vitro studies using CFSC-2G cells indicated that MeAIB directly reduced collagen synthesis. However, the MeAIB-mediated reduction of hepatic collagen, in vivo, following liver injury was associated with a decrease in hepatic alpha-smooth muscle actin (alpha-SMA) which suggested that MeAIB also inhibited the activation of HSCs. Because HSC activation is inseparable from proliferation, the purpose of this study was to examine the effect of MeAIB treatment on the proliferation of HSCs in an in vitro model utilizing CFSC-2G cell cultures. In these studies, MeAIB effectively inhibited the proliferation of CFSC-2G cells by interfering with the progression of the cells through the G(1)-phase of the cell cycle which delayed entry into S-phase. MeAIB prevented the phosphorylation of p70S6 kinase (p70S6K) at Thr389 and reduced the phosphorylation at Thr421/Ser424. Because p70S6K is required for G(1)-cell cycle progression and is known to be regulated by nutrient availability, this correlates well with MeAIB interfering with the proliferation of CFSC-2G HSCs. In addition, the rate of protein synthesis was reduced by MeAIB treatment following mitogenic stimulation, which agrees with a p70S6K-mediated reduction in translation. These data are consistent with MeAIB inhibiting the proliferation of CFSC-2G cells by altering the mitogen activated pathway(s) leading to phosphorylation of p70S6K by a yet to be described mechanism.     

Involvement of specific integrins in apoptosis induced by vascular apoptosis-inducing protein 1.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of vascular apoptosis-inducing protein 1 (VAP1) that specifically induces apoptosis in VEC. VAP1 belongs to the metalloprotease/disintegrin family. Yet the mechanism of inducing apoptosis by VAP1 is still not known. Since other various metalloproteases and disintegrins in snake venoms are known to influence extracellular matrix and cell adhesion, we investigated here the involvement of these adhesion molecules in VAP1-induced apoptosis. Consequently, VAP1 induced apoptosis without degrading extracellular matrix or inhibiting adhesion of VEC. However, VAP1-induced apoptosis was inhibited by antibodies for integrin alpha3, alpha6, beta1. Additionally, apoptosis was inhibited by antibody for CD9, an integrin associated protein. These results suggest that integrins are involved in VAP1-induced apoptosis by some specific role rather than that of adhesion to extracellular matrix.     

Inhibition of alpha7-nicotinic acetylcholine receptor expression by arsenite in the vascular endothelial cells

The alpha7-nicotinic acetylcholine receptor (alpha7-nAChR), expressed in the neuronal and non-neuronal cells, has been shown to regulate cell proliferation. However, the expression and function of the alpha7-nAChR in the proliferation of the vascular endothelial cells remain unclear. In this study, we investigated the expression of the alpha7-nAChR in the arsenite-exposed vascular endothelial cells. The vascular endothelial cells SVEC4-10 and porcine aorta endothelial cells (PAEC) expressed the alpha7-nAChR proteins. Moreover, the location of the alpha7-nAChR proteins on cell membrane of the vascular endothelial cells was identified by the alpha7-nAChR binding to a tetramethylrhodamine-labeled alpha-bungarotoxin (alpha-BTX). Arsenite (20 microM, 24 h) significantly induced the cytotoxicity, cell growth inhibition, and apoptosis in the vascular endothelial cells. The level of alpha7-nAChR proteins was concentration dependently decreased in the arsenite-treated endothelial cells. Furthermore, a specific alpha7-nAChR antagonist, alpha-BTX, inhibited the cell viability in the vascular endothelial cells. Nevertheless, alpha-BTX, and a alpha7-nAChR agonist, nicotine, did not significantly alter the cytotoxicity in the arsenite-treated endothelial cells. In addition, arsenite decreased the level of endothelial nitric oxide synthase proteins but did not alter choline acetyltransferase proteins in the SVEC4-10 endothelial cells. Together, our results indicate that arsenite can inhibit the alpha7-nAChR protein expression and cause the cell injury in the vascular endothelial cells.     

内皮抑素对胶质瘤病理特征影响的实验研究

目的探讨内皮抑素对胶质瘤病理特征的影响及分子药理学机制。方法利用经抗性筛选可表达内皮抑素的C6细胞裸鼠皮下注射制作胶质瘤模型,光镜和电镜观察其病理特征,ELISA法检测瘤组织中VEGF含量。结果转染有内皮抑素cDNA片段的C6胶质瘤组织中VEGF含量明显低于仅转染有空载体的C6胶质瘤组(P<0.01)。前者肿瘤边界清楚,瘤体未见明显的出血和囊性变,瘤体和瘤周均无明显的水肿;瘤组织内血管稀少,未见类微血管样结构;瘤体内可见大片不规则坏死,但坏死灶周和瘤周均无明显的血管反应,瘤周侵袭少见,可见凋亡瘤细胞;血管内皮细胞胞质内未见内VVO样结构,基板不连续,基底膜疏松。而仅转染有空载体的C6胶质瘤瘤周血管反应明显,瘤体和瘤周水肿严重,瘤细胞可侵袭瘤周组织,瘤内组织明显出血坏死,且可见瘤细胞形成类微血管样结构,血管内皮细胞胞质内VVO样结构较多,水肿越明显VVO样结构越多见,并与移植瘤组织中VEGF表达相一致,血管基板完整、连续,多数基质疏松,呈多层排列,外层可见少量胶原纤维。所观察的几种肿瘤中的微血管内皮细胞孔窗及血管周胶原纤维与转染的目的基因无明显的关系,且内皮细胞的凋亡均不明显。结论内皮抑素不会直接诱导胶质瘤组织内血管内皮细胞的凋亡,但可通过诱导VEGF的表达的下调而抑制瘤周血管反应和肿瘤新血管的生成。     

Effect of free iron on collagen synthesis,cell proliferation and MMP-2 expression in rat hepatic stellate cells

Various studies on hepatic fibrosis occurring in iron overload suggest that excess of tissue iron may be involved in the stimulation of collagen synthesis. Anyway, up to date, direct evidence on the role of iron in hepatic fibrosis is lacking. Moreover, it is not clear whether iron acts as direct initiator of fibrogenesis or as mediator of hepatocellular necrosis. In the present study, we investigated the effect of nontoxic doses of iron on collagen metabolism and proliferation, key features of liver fibrosis, by means of cultures of hepatic stellate cells, the liver cells responsible for collagen production. Iron treatment increased collagen synthesis without affecting noncollagen proteins. The maximum effect was observed at 5 microM iron (+132%). At this dose, no cell damage or proliferation was detected. Conversely, higher doses of iron (10 and 25 microM) induced cell proliferation and a lower increase in collagen synthesis, suggesting the prevalence of proliferative effect on the synthetic one. These effects occurred without the intervention of serum factors and were not mediated by lipid peroxidation. Our results strongly support the hypothesis that iron "per sé" may act as a profibrogenic agent. Finally, we provide evidence that iron plays a role also in matrix degradation, by stimulating some metalloprotease activities. Iron treatment increased metalloprotease-2 activity in hepatic stellate cells, while no changes were observed for interstitial collagenase activity suggesting that, in these conditions, a pathological accumulation of hepatic extracellular matrix may occur.     

Effect of BJcuL (a lectin from the venom of the snake Bothrops jararacussu) on adhesion and growth of tumor and endothelial cells.

Lectins are polyvalent carbohydrate-binding proteins of non-immune origin. Recently, we have isolated and characterized a lectin from the venom of the snake Bothrops jararacussu. This lectin (BJcuL) has been shown to bind to lactose moieties and induce agglutination of erythrocytes. In the present work, we observed that cells from human metastatic breast cancer (MDA-MB-435) and human ovarian carcinoma (OVCAR-5) cell lines adhere, although weakly, to BJcuL. However, BJcuL did not inhibit adhesion of these cells to the extracellular matrix proteins fibronectin, laminin and type I collagen. Importantly, viability of these tumor cells and cells from other human tumor cell lines and a bovine brain endothelial cell line was suppressed by BJcuL. These findings suggest that the lectin BJcuL may serve as an interesting tool for combating tumor progression by inhibiting tumor cell and endothelial cell growth.