Amelioration of experimental colitis after short-term therapy with glucocorticoid and its relationship to the induction of different regulatory markers.

The clinical benefits of short-term therapy with glucocorticoids (GC) in patients with inflammatory bowel disease (IBD) are widely known. However, the effects of this treatment towards the re-establishment of the regulatory network in IBD are not fully explored. We have evaluated the immunological effects of the abbreviated GC therapy in experimental colitis induced by 3% dextran sulphate sodium in C57BL/6 mice. Treatment with GC improved disease outcome, constrained circulating leucocytes and ameliorated intestinal inflammation. The control of the local inflammatory responses involved a reduction in the expression of interferon-γ and interleukin-1ß, associated with augmented mRNA levels of peroxisome proliferator-activated receptors (α and γ) in intestine. Furthermore, there was a reduction of CD4+ T cells producing interferon-γ, together with an increased frequency of the putative regulatory population of T cells producing interleukin-10, in spleen. These systemic alterations were accompanied by a decrease in the proliferative potential of splenocytes of mice treated in vivo with GC. Notably, treatment with GC also led to an increase in the frequency of the regulatory markers GITR, CTLA-4, PD-1, CD73 and FoxP3, more prominently in spleen. Taken together, our results pointed to a role of GC in the control of leucocyte responsiveness and re-establishment of a regulatory system, which probably contributed to disease control and the restoration of immune balance. Finally, this is the first time that GC treatment was associated with the modulation of a broad number of regulatory markers in an experimental model of colitis.

Adrenal-Derived Hormones Differentially Modulate Intestinal Immunity in Experimental Colitis.

The adrenal glands are able to modulate immune responses through neuroimmunoendocrine interactions and cortisol secretion that could suppress exacerbated inflammation such as in inflammatory bowel disease (IBD). Therefore, here we evaluated the role of these glands in experimental colitis induced by 3% dextran sulfate sodium (DSS) in C57BL/6 mice subjected to adrenalectomy, with or without glucocorticoid (GC) replacement. Mice succumbed to colitis without adrenals with a higher clinical score and augmented systemic levels of IL-6 and lower LPS. Furthermore, adrenalectomy negatively modulated systemic regulatory markers. The absence of adrenals resulted in augmented tolerogenic lamina propria dendritic cells but no compensatory local production of corticosterone and decreased mucosal inflammation associated with increased IFN-γ and FasL in the intestine. To clarify the importance of GC in this scenario, GC replacement in adrenalectomized mice restored different markers to the same degree of that observed in DSS group. Finally, this is the first time that adrenal-derived hormones, especially GC, were associated with the differential local modulation of the gut infiltrate, also pointing to a relationship between adrenalectomy and the modulation of systemic regulatory markers. These findings may elucidate some neuroimmunoendocrine mechanisms that dictate colitis outcome.

The acute phase of Trypanosoma cruzi infection is attenuated in 5-lipoxygenase-deficient mice.

In the present work we examine the contribution of 5-lipoxygenase- (5-LO-) derived lipid mediators to immune responses during the acute phase of Trypanosoma cruzi infection in 5-LO gene knockout (5-LO(-/-)) mice and wild-type (WT) mice. Compared with WT mice, the 5-LO(-/-) mice developed less parasitemia/tissue parasitism, less inflammatory cell infiltrates, and a lower mortality. This resistance of 5-LO(-/-) mice correlated with several differences in the immune response to infection, including reduced PGE2 synthesis; sustained capacity of splenocytes to produce high levels of interleukin (IL)-12 early in the infection; enhanced splenocyte production of IL-1ß, IL-6, and IFN-γ; rapid T-cell polarization to secrete high quantities of IFN-γ and low quantities of IL-10; and greater numbers of CD8(+)CD44(high)CD62L(low) memory effector T cells at the end of the acute phase of infection. The high mortality in WT mice was associated with increased production of LTB4/LTC4, T cell bias to produce IFN-γ, high levels of serum nitrite, and marked protein extravasation into the peritoneal cavity, although survival was improved by treatment with a cys-LT receptor 1 antagonist. These data also provide evidence that 5-LO-derived mediators negatively affect host survival during the acute phase of T. cruzi infection.

Peroxisome Proliferator-Activated Receptor Alpha Mediates the Beneficial Effects of Atorvastatin in Experimental Colitis.

The current therapeutic options for Inflammatory Bowel Diseases (IBD) are limited. Even using common anti-inflammatory, immunosuppressive or biological therapies, many patients become unresponsive to the treatments, immunosuppressed or unable to restrain secondary infections. Statins are cholesterol-lowering drugs with non-canonical anti-inflammatory properties, whose underlying mechanisms of action still remain poorly understood. Here, we described that in vitro atorvastatin (ATO) treatment was not toxic to splenocytes, constrained cell proliferation and modulated IL-6 and IL-10 production in a dose-dependent manner. Mice exposed to dextran sulfate sodium (DSS) for colitis induction and treated with ATO shifted their immune response from Th17 towards Th2, improved the clinical and histological aspects of intestinal inflammation and reduced the number of circulating leukocytes. Both experimental and in silico analyses revealed that PPAR-α expression is reduced in experimental colitis, which was reversed by ATO treatment. While IBD patients also downregulate PPAR-α expression, the responsiveness to biological therapy relied on the restoration of PPAR-α levels. Indeed, the in vitro and in vivo effects induced by ATO treatment were abrogated in Ppara-/- mice or leukocytes. In conclusion, the beneficial effects of ATO in colitis are dependent on PPAR-α, which could also be a potential predictive biomarker of therapy responsiveness in IBD.

In vitro antitumour activity of orsellinates.

Lichen phenolic compounds exhibit antioxidant, antimicrobial, antiproliferative, and cytotoxic activities. The purpose of this study was to evaluate the anticancer activity of lecanoric acid, a secondary metabolite of the lichen Parmotrema tinctorum, and its derivatives, orsellinates, obtained by structural modification. A cytotoxicity assay was carried out in vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma, MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity index of the compounds. n-Butyl orsellinate was the most active compound, with IC50 values (the concentration that inhibits 50% of growth) ranging from 7.2 to 14.0 microg/mL, against all the cell lines tested. The compound was more active (IC50 = 11.4 microg/mL) against B16-F10 cells than was cisplatin (12.5 microg/mL). Conversely, lecanoric acid and methyl orsellinate were less active against all cell lines, having an IC50 value higher than 50 microg/mL. Ethyl orsellinate was more active against HEp-2 than against MCF7, 786-0, or B16-F10 cells. The same pattern was observed for n-propyl and n-butyl orsellinates. n-Pentyl orsellinate was less active than n-propyl or n-butyl orsellinates against HEp-2 cells. The orsellinate activity increased with chain elongation (from methyl to n-butyl), a likely consequence of an increase in lipophilicity. The results revealed that the structural modification of lecanoric acid increases the cytotoxic activity of the derivatives tested.

Vaginal lactobacilli as potential probiotics against Candida SPP.

Urogenital infections affect millions of people every year worldwide. The treatment of these diseases usually requires the use of antimicrobial agents, and more recently, the use of probiotic lactic acid bacteria (LAB) cultures for the management of vaginal infections has been extensively studied. In this work, 11 vaginal lactobacilli isolates, previously obtained from healthy patients, were studied to screen microorganisms with probiotic properties against Candida spp. The LAB were tested for their ability of auto-aggregation, co-aggregation with C. albicans, C. glabrata, C. krusei, and C. tropicalis, adhesion to Caco-2 epithelial cells and production of lactic acid and hydrogen peroxide (H2O2). All lactobacilli isolates tested were able to auto-aggregate (ranging from 25.3% to 75.4% assessed at 4 hours of incubation) and to co-aggregate with the four Candida species into different degrees; among them L. crispatus showed the highest scores of co-aggregation. The highest amount of lactic acid was produced by L. salivarius (13.9 g/l), followed by L. johnsonii (6.5 g/l), L. acidophilus (5.5 g/l), and L. jensenii (5.4 g/l). All isolates produced H2O2, but the highest levels (3 - 10 mg/l) were observed for L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, and L. vaginalis. Only L. agilis, L. jensenii, L. johnsonii and L. ruminus were able to adhere to epithelial Caco-2 cells. Among the isolates evaluated, L agilis, L. jensenii, L. johnsonii, and L. ruminus exhibited simultaneously several desirable properties as potential probiotic strains justifying future studies to evaluate their technological properties in different pharmaceutical preparations for human use.

ATP-induced apoptosis involves a Ca2+-independent phospholipase A2 and 5-lipoxygenase in macrophages.

Macrophages express P2X(7) and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X(7) receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1beta secretion. We investigated signaling pathways involved in the induction of cell death by ATP(e) in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6h after an induction period of 20 min in the presence of ATP. Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect on apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca(2+) were chelated during the induction period. Mepacrine, a generic PLA(2) inhibitor and BEL, an inhibitor of Ca(2+)-independent PLA(2) (iPLA(2)) blocked apoptosis, while pBPB and AACOOPF(3), inhibitors of secretory and Ca(2+)-dependent PLA(2) respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX(-/-) macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA(2) and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca(2+)-independent step involving an iPLA(2) and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X(7) in macrophages.

Antitumor effects of snake venom chemically modified Lys49 phospholipase A2-like BthTX-I and a synthetic peptide derived from its C-terminal region.

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115-129 C-terminal region. BPB-BthTX-I presented cytotoxicity of 10-40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer.

Myotoxic phospholipases A(2) isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: cytotoxic effect on microorganism and tumor cells.

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A(2) (PLA(2)s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M(r) approximately 14,000 for the monomer and 28,000Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA(2)s from snake venoms, MTX-I belonging to Asp49 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca(2+) ions for the enzymatic catalysis. Both PLA(2)s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA(2) proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer.

Biological activities from extracts of endophytic fungi isolated from Viguiera arenaria and Tithonia diversifolia.

A total of 39 endophytic fungi have been isolated from Viguiera arenaria and Tithonia diversifolia, both collected in São Paulo State, Brazil. The isolates were identified based on their ribosomal DNA sequences. The ethyl acetate (EtOAc) extracts of all endophytic fungi were evaluated for their antimicrobial, antiparasitic and antitumoral activity. Antimicrobial screening was conducted using an agar diffusion assay against three pathogenic microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans. Antiparasitic activity was determined by enzymatic inhibition of gGAPDH of Trypanosoma cruzi and adenine phosphorybosiltransferase (APRT) of Leishmania tarentolae. Antitumoral activity was tested against human T leukemia cells by the Mosmann colorimetric method. All extracts showed activity in at least one assay: 79.5% of the extracts were cytotoxic against leukemia cells, 5.1% of the extracts were active against S. aureus, 25.6% against E. coli and 64.1% against Candida albicans. Only one extract showed promising results in the inhibition of parasitic enzymes gGAPDH (95.0%) and three were found to inhibit APRT activity. The cytotoxic extract produced by the strain VA1 (Glomerella cingulata) was fractionated and yielded nectriapyrone and tyrosol. Nectriapyrone showed relevant cytotoxic activity against both human T leukemia and melanoma tumor cell lines.

Effects of periodontal therapy on glycemic control and inflammatory markers.

BACKGROUND: Periodontitis, a complication of diabetes mellitus (DM), can induce or perpetuate systemic conditions. This double-masked, placebo-controlled study evaluated the effects of periodontal therapy (scaling and root planing [SRP]) on the serum levels of glycated hemoglobin (HbA1c) and on inflammatory biomarkers. METHODS: Thirty subjects with type 2 DM and periodontitis were treated with SRP + placebo (SRP; N = 15) or with SRP + doxycycline (SRP+Doxy; N = 15), 100 mg/day, for 14 days. Clinical and laboratory data were recorded at baseline and at 3 months after treatment. RESULTS: After 3 months, the reduction in probing depth was 0.8 mm for the SRP group (P <0.01) and 1.1 mm for the SRP+Doxy group (P <0.01) followed by a 0.9% (SRP; P = 0.17) and 1.5% (SRP+Doxy; P <0.01) reduction in HbA1c levels. A significant reduction in interleukin (IL)-6; interferon-inducible protein 10; soluble fas ligand; granulocyte colony-stimulating factor; RANTES; and IL-12 p70 serum levels were also verified (N = 30). To our knowledge, this is the first report on the effects of periodontal therapy on multiple systemic inflammatory markers in DM. CONCLUSIONS: Periodontal therapy may influence the systemic conditions of patients with type 2 DM, but no statistical difference was observed with the adjunctive systemic doxycycline therapy. Moreover, it is possible that the observed improvement in glycemic control and in the reduction of inflammatory markers could also be due to diet, which was not controlled in our study. Therefore, a confirmatory study with a larger sample size and controlled diet is necessary.

B cells modulate T cells so as to favour T helper type 1 and CD8+ T-cell responses in the acute phase of Trypanosoma cruzi infection.

In this study, we have evaluated the production of pro- and anti-inflammatory cytokines and the formation of central and effector memory T cells in mice lacking mature B cells (mu MT KO). The results show that Trypanosoma cruzi infection in C57Bl/6m mu MT KO mice is intensified in relation to control mice and this exacerbation is related to low levels of inflammatory cytokines produced during the acute infection and the lower numbers of central and effector memory CD4(+) and CD8(+) T cells generated during the acute phase of the infection. In addition, a marked reduction in the CD8(+) T-cell subpopulation was observed in mu MT KO infected mice. In agreement to this, the degree of tissue parasitism was increased in mu MT mice and the tissue inflammatory response was much less intense in the acute phase of the infection, consistent with a deficit in the generation of effector T cells. Flow cytometry analysis of the skeletal muscle inflammatory infiltrate showed a predominance of CD8(+) CD45Rb low in B-cell-sufficient C57Bl/6 mice, whereas the preponderant cell type in mu MT KO skeletal muscle inflammatory infiltrate was CD4(+) T cells. In addition, CD8(+) T cells found in skeletal muscle from mu MT KO infected mice were less activated than in control B-cell sufficient infected mice. These results suggest that B cells may participate in the generation of effector/memory T cells. In addition and more importantly, B cells were crucial in the maintenance of central and effector memory CD8(+) T cell, as well as the determination of the T cell cytokine functional pattern, and they may therefore account for critical aspects of the resistance to intracellular pathogens, such as T. cruzi.

Cytotoxic L-amino acid oxidase from Bothrops moojeni: biochemical and functional characterization.

An L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) was purified to a high degree using sequential CM-Sepharose ion-exchange and phenyl-Sepharose chromatography. When analyzed by mass spectrometry, the purified BmooLAAO-I presented a molecular weight of 64,889 and 130,779 under denaturing and nondenaturing conditions, respectively. BmooLAAO-I is a homodimeric acidic glycoprotein with a pI approximately 4.7, and the N-terminal sequence shows close structural similarity to other snake venom LAAOs. This enzyme was inactivated by freezing or low pH, and secondary structural analysis by circular dichroism revealed 48% alpha-helix, 20% beta-sheet, 12% beta-turn, and 20% random coil structures. BmooLAAO-I exhibited bactericidal, antitumoral, trypanocidal, edematogenic, and platelet-aggregating activities. All of these effects were inhibited by catalase, suggesting that these biological effects are mediated by the production of H(2)O(2). BmooLAAO-I induced typical apoptotic DNA fragmentation in HL-60 cells, which was also inhibited by catalase. These results point to the potential use of BmooLAAO-I as a therapeutic agent for treatment of diseases in which induction of H(2)O(2) production can be beneficial.

Participation of Leukotrienes in the Immune Modulation of Oral Tolerance.

Oral tolerance (OT) is characterized as a peripheral immune tolerance form, in which, mature lymphocytes in lymphoid tissues associated with mucosa, become non-functional or hypo responsive due to prior oral administration of antigen. OT is an important immunological phenomenon due to its therapeutic potential in inflammatory processes and others diseases. Here we evaluated leukotriene role in the induction of OT, as well as, the production of cytokines IL-5 and IFN-γ in leukotriene deficient animals (knock-out). Our results suggested that even in the presence of OT and leukotrienes absence, cytokine IFN-γ remains being secreted, which gives us an indication of immune system specificity and also that IFN-γ participates in various immune processes.

V gamma 1 gammadelta T cells regulate type-1/type-2 immune responses and participate in the resistance to infection and development of heart inflammation in Trypanosoma cruzi-infected BALB/c mice.

Many different cell populations or lineages participate in the resistance to Trypanosoma cruzi infection. gammadelta T cells may also take part in a network of interactions that lead to control of T. cruzi infection with minimal tissue damage by controlling alphabeta T cell activation, as was previously suggested. However, the gammadelta T cell population is not homogeneous and its functions might vary, depending on T cell receptor usage or distinct stimulatory conditions. In this study, we show that the in vivo depletion of V gamma 1-bearing gammadelta T cells, prior to the infection of BALB/c mice with the Y strain of T. cruzi, induces an increased susceptibility to the infection with lower amounts of IFN-gamma being produced by conventional CD4+ or CD8+ T cells. In addition, the production of IL-4 by spleen T cells in V gamma 1-depleted mice was increased and the production of IL-10 remained unchanged. Since V gamma 1(+) gammadelta T cell depletion diminished the conversion of naive to memory/activated CD4 T cells and the production of IFN-gamma during the acute infection, these cells appear to function as helper cells for conventional CD4+ Th1 cells. Depletion of V gamma 1(+) cells also reduced the infection-induced inflammatory infiltrate in the heart and skeletal muscle. More importantly, V gamma 1(+) cells were required for up-regulation of CD40L in CD4+ and CD8+ T cells during infection. These results show that a subset of gammadelta T cells (V gamma 1(+)), which is an important component of the innate immune response, up-regulates the type 1 arm of the adaptative immune response, during T. cruzi infection.

Mechanism of the cytotoxic effect of l-amino acid oxidase isolated from Bothrops alternatus snake venom.

BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI-5.3, 15% sugar and a related molecular mass of 66,000Da in its monomeric form, and 123,000Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy.

Cloning and identification of a complete cDNA coding for a bactericidal and antitumoral acidic phospholipase A2 from Bothrops jararacussu venom.

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.

Effect of iontophoresis on topical delivery of doxorubicin-loaded solid lipid nanoparticles.

The combination of iontophoresis with solid lipid nanoparticles (SLNs) for targeting drug delivery to the epidermis has not been explored. The goal of this paper was to study the influence of iontophoresis on the penetration of doxorubicin (DOX) delivered in SLNs (DOX-SLNs). We measured the contribution of electroosmotic flow to the transport of DOX, and the accumulation of DOX in the stratum corneum (SC) and in the viable epidermis was determined. In addition, we evaluated the cytotoxicity of DOX-SLNs against skin cancer cells. Iontophoresis of unloaded SLNs decreased the electroosmotic flow by a factor of 5 and increased the skin resistance. Nevertheless, iontophoresis of DOX-SLNs increased DOX delivery to the viable epidermis, with 56% of all DOX penetrating this skin layer. Only 26% of the drug was retained in the SC. In contrast, passive delivery retained 43% of DOX in the SC and 26% in the viable epidermis. DOX-SLNs increased DOX cytotoxicity against melanoma cells by 50%. These results suggest the use of DOX-SLN iontophoresis in the topical treatment of skin cancer.

Antitumoral activity of snake venom proteins: new trends in cancer therapy.

For more than half a century, cytotoxic agents have been investigated as a possible treatment for cancer. Research on animal venoms has revealed their high toxicity on tissues and cell cultures, both normal and tumoral. Snake venoms show the highest cytotoxic potential, since ophidian accidents cause a large amount of tissue damage, suggesting a promising utilization of these venoms or their components as antitumoral agents. Over the last few years, we have studied the effects of snake venoms and their isolated enzymes on tumor cell cultures. Some in vivo assays showed antineoplastic activity against induced tumors in mice. In human beings, both the crude venom and isolated enzymes revealed antitumor activities in preliminary assays, with measurable clinical responses in the advanced treatment phase. These enzymes include metalloproteases (MP), disintegrins, L-amino acid oxidases (LAAOs), C-type lectins, and phospholipases A2 (PLA2s). Their mechanisms of action include direct toxic action (PLA2s), free radical generation (LAAOs), apoptosis induction (PLA2s, MP, and LAAOs), and antiangiogenesis (disintegrins and lectins). Higher cytotoxic and cytostatic activities upon tumor cells than normal cells suggest the possibility for clinical applications. Further studies should be conducted to ensure the efficacy and safety of different snake venom compounds for cancer drug development.

Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.