Cyclopalladated Compound 7a Induces Apoptosis- and Autophagy-Like Mechanisms in Paracoccidioides and Is a Candidate for Paracoccidioidomycosis Treatment.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 µg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.

Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo.

Natural monoterpenes were isolated from the essential oil of Piper cernuum Vell. (Piperaceae) leaves. The crude oil and the individual monoterpenes were tested for cytotoxicity in human tumor cell lineages and B16F10-Nex2 murine melanoma cells. In the present work we demonstrate the activity of camphene against different cancer cells, with its mechanism of action being investigated in vitro and in vivo in murine melanoma. Camphene induced apoptosis by the intrinsic pathway in melanoma cells mainly by causing endoplasmic reticulum (ER) stress, with release of Ca(2+) together with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity. Importantly, camphene exerted antitumor activity in vivo by inhibiting subcutaneous tumor growth of highly aggressive melanoma cells in a syngeneic model, suggesting a promising role of this compound in cancer therapy.

Neolignans from Nectandra megapotamica (Lauraceae) Display in vitro Cytotoxic Activity and Induce Apoptosis in Leukemia Cells.

Nectandra megapotamica (Spreng.) Mez. (Lauraceae) is a well-known Brazilian medicinal plant that has been used in folk medicine to treat several diseases. In continuation of our ongoing efforts to discover new bioactive natural products from the Brazilian flora, this study describes the identification of cytotoxic compounds from the MeOH extract of N. megapotamica (Lauraceae) leaves using bioactivity-guided fractionation. This approach resulted in the isolation and characterization of eight tetrahydrofuran neolignans: calopeptin (1), machilin-G (2), machilin-I (3), aristolignin (4), nectandrin A (5), veraguensin (6), ganschisandrin (7), and galgravin (8). Different assays were conducted to evaluate their cytotoxic activities and to determine the possible mechanism(s) related to the activity displayed against human leukemia cells. The most active compounds 4, 5 and 8 gave IC50 values of 14.2 ± 0.7, 16.9 ± 0.8 and 16.5 ± 0.8 µg/mL, respectively, against human leukemia (HL-60) tumor cells. Moreover, these compounds induced specific apoptotic hallmarks, such as plasma membrane bleb formation, nuclear DNA condensation, specific chromatin fragmentation, phosphatidyl-serine exposure on the external leaflet of the plasma membrane, cleavage of PARP as well as mitochondrial damage, which as a whole could be related to the intrinsic apoptotic pathway.

Chemical composition and in vitro cytotoxic effects of the essential oil from Nectandra leucantha leaves.

CONTEXT: Nectandra (Lauraceae) species have been used in folk medicine as an antidiarrheal, analgesic, antifungal, etc., and have many pharmacological proprieties. OBJECTIVE: Investigation of the chemical composition and cytotoxicity of essential oil from Nectandra leucantha Nees & Mart. leaves. This is the first study involving N. leucantha reported in the literature. MATERIAL AND METHODS: The essential oil of N. leucantha leaves was obtained by hydrodistillation. Its chemical composition was determined using a combination of GC/FID, GC/MS, and determination of Kovats index (KI). In vitro cytotoxic activity was evaluated against six cancer cell lines - murine melanoma (B16F10-Nex2), human glioblastome (U-87), human cervical carcinoma (HeLa), human colon carcinoma (HCT), human breast adenocarcinoma (MCF7), and human cervical tumor (Siha) as well as against one non-tumorigenic cell line - human foreskin fibroblast (HFF). RESULTS: Thirty-three compounds were identified primarily sesquiterpenes (81.41%), the main compounds being bicyclogermacrene (28.44%), germacrene A (7.34%), spathulenol (5.82%), and globulol (5.25%). Furthermore, monoterpenes were also found in the analyzed oil (12.84%), predominantly α- and ß-pinenes (6.59 and 4.57%, respectively). The crude essential oil displayed significant cytotoxic activity against B16F10-Nex2 (IC50 33 ± 1 µg/mL) and U87 (IC50 75.95 ± 0.03 µg/mL) and HeLa (IC50 60 ± 12 µg/mL) cell lines. The main identified compound, bicyclogermacrene, displayed IC50 ranging from 3.1 ± 0.2 to 21 ± 6 µg/mL. DISCUSSION AND CONCLUSION: The results indicate that the crude oils from leaves of N. leucantha displayed cytotoxic activity being bicyclogermacrene, the main compound identified in the crude oil responsible, at least in part, for this potential.

Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis.

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

ß-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma.

Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that ß-actin is the receptor of C7H2 in the tumor cells. C7H2 induces ß-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.

Chemical composition and cytotoxicity evaluation of essential oil from leaves of Casearia sylvestris, its main compound α-zingiberene and derivatives.

Casearia sylvestris (Salicaceae), popularly known as "guaçatonga", is a plant widely used in folk medicine to treat various diseases, including cancer. The present work deals with the chemical composition as well as the cytotoxic evaluation of its essential oil, its main constituent and derivatives. Thus, the crude essential oil from leaves of C. sylvestris was obtained using a Clevenger type apparatus and analyzed by GC/MS. This analysis afforded the identification of 23 substances, 13 of which corresponded to 98.73% of the total oil composition, with sesquiterpene a-zingiberene accounting for 50% of the oil. The essential oil was evaluated for cytotoxic activity against several tumor cell lines, giving IC50 values ranging from 12 to 153 mg/mL. Pure a-zingiberene, isolated from essential oil, was also evaluated against the tumor cell lines showing activity for HeLa, U-87, Siha and HL60 cell lines, but with IC50 values higher than those determined for the crude essential oil. Aiming to evaluate the effect of the double bonds of a-zingiberene on the cytotoxic activity, partially hydrogenated a-zingiberene (PHZ) and fully hydrogenated a-zingiberene (THZ) derivatives were obtained. For the partially hydrogenated derivative only cytotoxic activity to the B16F10-Nex2 cell line (IC50 65 mg/mL) was detected, while totally hydrogenated derivative showed cytotoxic activity for almost all cell lines, with B16F10-Nex2 and MCF-7 as exceptions and with IC50 values ranging from 34 to 65 mg/mL. These results indicate that cytotoxic activity is related with the state of oxidation of compound.

Vesicle and vesicle-free extracellular proteome of Paracoccidioides brasiliensis: comparative analysis with other pathogenic fungi.

Microorganisms release effector molecules that modulate the host machinery enabling survival, replication, and dissemination of a pathogen. Here we characterized the extracellular proteome of Paracoccidioides brasiliensis at its pathogenic yeast phase. Cell-free culture supernatants from the Pb18 isolate, cultivated in defined medium, were separated into vesicle and vesicle-free fractions, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. In vesicle and vesicle-free preparations we identified, respectively, 205 and 260 proteins with two or more peptides, including 120 overlapping identifications. Almost 70% of the sequences were predicted as secretory, mostly using nonconventional secretory pathways, and many have previously been localized to fungal cell walls. A total of 72 proteins were considered as commonly transported by extracellular vesicles, considering that orthologues have been reported in at least two other fungal species. These sequences were mostly related to translation, carbohydrate and protein metabolism, oxidation/reduction, transport, response to stress, and signaling. This unique proteomic analysis of extracellular vesicles and vesicle-free released proteins in a pathogenic fungus provides full comparison with other fungal extracellular vesicle proteomes and broadens the current view on fungal secretomes.

The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic α-Galactosyl epitopes.

Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after ß-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.

Essential oils from Schinus terebinthifolius leaves - chemical composition and in vitro cytotoxicity evaluation.

CONTEXT: In folk medicine, Schinus terebinthifolius Raddi (Anacardiaceae), has been used as a remedy for ulcers, respiratory problems, wounds, rheumatism, gout, diarrhea, skin ailments and arthritis, as well as to treat tumors and leprosy. OBJECTIVE: To investigate the chemical composition and cytotoxicity of essential oil from leaves of S. terebinthifolius as well as the identification of active compounds from this oil. MATERIAL AND METHODS: Essential oil from S. terebinthifolius leaves, obtained by hydrodistillation using a Clevenger-type apparatus, was characterized in terms of its chemical composition. Also, the crude oil was subjected to chromatographic separation procedures to afford an active fraction composed of α- and ß-pinenes. These compounds, including hydrogenation (pinane) and epoxydation (α-pinene oxide) derivatives from α-pinene, were tested in vitro against murine melanoma cell line (B16F10-Nex2) and human melanoma (A2058), breast adenocarcinoma (MCF7), leukemia (human leukemia (HL-60) and cervical carcinoma (HeLa) cell lines. RESULTS: Forty-nine constituents were identified in the oil (97.9% of the total), with germacrene D (23.7%), bicyclogermacrene (15.0%), ß-pinene (9.1%) and ß-longipinene (8.1%) as the main compounds. The crude essential oil showed cytotoxic effects in several cell lines, mainly on leukemia and human cervical carcinoma. Fractions composed mainly of α- and ß-pinenes as well as those composed of individually pinenes showed effective activities against all tested cell lines. Aiming to determinate preliminary structure/activity relationships, α-pinene was subjected to epoxydation and hydrogenation procedures whose obtained α-pinene oxide showed an expressive depression in its cytotoxicity effect, similar as observed to pinane derivative. DISCUSSION AND CONCLUSION: The obtained results indicated that the monoterpenes α- and ß-pinenes could be responsible to the cytotoxic activity detected in the crude oil from leaves of S. terebinthifolius. In addition, it was possibly inferred that the presence of double bond in their structures, mainly at endocyclic position, is crucial to cytotoxic potential detected in these derivatives.

α-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model.

Malignant melanoma is one the most aggressive types of cancer and its incidence has gradually increased in the last years, accounting for about 75% of skin cancer deaths. This poor prognosis results from the tumor resistance to conventional drugs mainly by deregulation of apoptotic pathways. The aim of this work was to investigate the cell death mechanism induced by α-pinene and its therapeutic application. Our results demonstrated that α-pinene was able to induce apoptosis evidenced by early disruption of the mitochondrial potential, production of reactive oxygen species, increase in caspase-3 activity, heterochromatin aggregation, DNA fragmentation and exposure of phosphatidyl serine on the cell surface. Most importantly, this molecule was very effective in the treatment of experimental metastatic melanoma reducing the number of lung tumor nodules. This is the first report on the apoptotic and antimetastatic activity of isolated α-pinene.

A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells.

BACKGROUND: Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd(2) [S((-))C(2), N-dmpa](2) (µ-dppe)Cl(2)} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. METHODS: B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. RESULTS: Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. CONCLUSIONS: The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.

In vitro and in vivo trypanocidal effects of the cyclopalladated compound 7a, a drug candidate for treatment of Chagas' disease.

Chagas' disease, a neglected tropical infection, affects about 18 million people, and 100 million are at risk. The only drug available, benznidazole, is effective in the acute form and in the early chronic form, but its efficacy and tolerance are inversely related to the age of the patients. Side effects are frequent in elderly patients. The search for new drugs is thus warranted. In the present study we evaluated the in vitro and in vivo effect of a cyclopalladated compound (7a) against Trypanosoma cruzi, the agent of Chagas' disease. The 7a compound inhibits trypomastigote cell invasion, decreases intracellular amastigote proliferation, and is very effective as a trypanocidal drug in vivo, even at very low dosages. It was 340-fold more cytotoxic to parasites than to mammalian cells and was more effective than benznidazole in all in vitro and in vivo experiments. The 7a cyclopalladate complex exerts an apoptosis-like death in T. cruzi trypomastigote forms and causes mitochondrion disruption seen by electron microscopy.

Modulation of the exocellular serine-thiol proteinase activity of Paracoccidioides brasiliensis by neutral polysaccharides.

Our group characterized an exocellular serine-thiol proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a dimorphic human pathogen. The fungal proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans (GAGs) and neutral polysaccharides upon the serine-thiol proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the proteinase, while sulfated GAGs had no effect. Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 microM increased 6.8-fold the enzyme affinity for the substrate. In the presence of 8 microM of purified baker's yeast mannan, the apparent KM of the enzyme increased about 5.5-fold, reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-thiol proteolytic activity, which is possibly involved in fungal dissemination.

The Ig VH complementarity-determining region 3-containing Rb9 peptide, inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor.

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VHCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca2+ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.

A novel microtubule de-stabilizing complementarity-determining region C36L1 peptide displays antitumor activity against melanoma in vitro and in vivo.

Short peptide sequences from complementarity-determining regions (CDRs) of different immunoglobulins may exert anti-infective, immunomodulatory and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). In this sense, they resemble early molecules of innate immunity. C36L1 was identified as a bioactive light-chain CDR1 peptide by screening 19 conserved CDR sequences targeting murine B16F10-Nex2 melanoma. The 17-amino acid peptide is readily taken up by melanoma cells and acts on microtubules causing depolymerization, stress of the endoplasmic reticulum and intrinsic apoptosis. At low concentrations, C36L1 inhibited migration, invasion and proliferation of B16F10-Nex2 cells with cell cycle arrest at G2/M phase, by regulating the PI3K/Akt signaling axis involving Rho-GTPase and PTEN mediation. Peritumor injection of the peptide delayed growth of subcutaneously grafted melanoma cells. Intraperitoneal administration of C36L1 induced a significant immune-response dependent anti-tumor protection in a syngeneic metastatic melanoma model. Dendritic cells stimulated ex-vivo by the peptide and transferred to animals challenged with tumor cells were equally effective. The C36 VL CDR1 peptide is a promising microtubule-interacting drug that induces tumor cell death by apoptosis and inhibits metastases of highly aggressive melanoma cells.

Chemical composition and in vitro cytotoxic and antileishmanial activities of extract and essential oil from leaves of Piper cernuum.

Fractionation of the MeOH extract from leaves of Piper cernuum Vell. (Piperaceae) afforded six phenylpropanoid derivatives: 3',4'-dimethoxydihydrocinnamic acid (1), piplaroxide (2), methyl 4'-hydroxy-3',5'-dimethoxy cinnamate (3), 3',4',5'-trimethoxydihydrocinnamic acid (3), dihydropiplartine (5), and piplartine (6). The structures of isolated metabolites were characterized by NMR and MS spectral data analysis. The chemical composition of essential oil from the leaves was determined using GC/LREIMS followed by the determination of Kovats indexes. This procedure allowed the identification of nineteen terpenoids, with ß-elemene (7), bicyclogermacrene (8), germacrene D (9), and (E)-caryophyllene (10) as the main compounds. Compounds 1 and 3-6 displayed no in vitro cytotoxicity against cancer cell lineages B16F10-Nex2, U87, HeLa, HL-60, HCT, and A2058 while 2 showed moderate activity against B16F10-Nex2 and HL-60 lines. Otherwise, compounds 7-10 displayed high cytotoxic activity. Evaluation against non-tumorigenic HFF cells indicated a reduced selectivity of compounds 7-10 to tumoral cells. No antileishmanial activity on macrophages infected with L. (L.) amnazonensis was found for the crude MeOH extract and compounds 1-6. The crude essential oil and compounds 7-10 reduced parasitism and eliminated the majority of infected and non-infected cells at 50 µg/mL.

Mastoparan induces apoptosis in B16F10-Nex2 melanoma cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo.

Mastoparan is an α-helical and amphipathic tetradecapeptide obtained from the venom of the wasp Vespula lewisii. This peptide exhibits a wide variety of biological effects, including antimicrobial activity, increased histamine release from mast cells, induction of a potent mitochondrial permeability transition and tumor cell cytotoxicity. Here, the effects of mastoparan in malignant melanoma were studied using the murine model of B16F10-Nex2 cells. In vitro, mastoparan caused melanoma cell death by the mitochondrial apoptosis pathway, as evidenced by the Annexin V-FITC/PI assay, loss of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species, DNA degradation and cell death signaling. Most importantly, mastoparan reduced the growth of subcutaneous melanoma in syngeneic mice and increased their survival. The present results show that mastoparan induced caspase-dependent apoptosis in melanoma cells through the intrinsic mitochondrial pathway protecting the mice against tumor development.

Anti-tumor activities of peptides corresponding to conserved complementary determining regions from different immunoglobulins.

Short synthetic peptides corresponding to sequences of complementarity-determining regions (CDRs) from different immunoglobulin families have been shown to induce antimicrobial, antiviral and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). Presently, we studied the in vitro and in vivo antitumor activity of synthetic peptides derived from conserved CDR sequences of different immunoglobulins against human tumor cell lines and murine B16F10-Nex2 melanoma aiming at the discovery of candidate molecules for cancer therapy. Four light- and heavy-chain CDR peptide sequences from different antibodies (C36-L1, HA9-H2, 1-H2 and Mg16-H2) showed cytotoxic activity against murine melanoma and a panel of human tumor cell lineages in vitro. Importantly, they also exerted anti-metastatic activity using a syngeneic melanoma model in mice. Other peptides (D07-H3, MN20v1, MS2-H3) were also protective against metastatic melanoma, without showing significant cytotoxicity against tumor cells in vitro. In this case, we suggest that these peptides may act as immune adjuvants in vivo. As observed, peptides induced nitric oxide production in bone-marrow macrophages showing that innate immune cells can also be modulated by these CDR peptides. The present screening supports the search in immunoglobulins of rather frequent CDR sequences that are endowed with specific antitumor properties and may be candidates to be developed as anti-cancer drugs.

A subtraction tolerization method of immunization allowed for Wilms' tumor protein-1 (WT1) identification in melanoma and discovery of an antitumor peptide sequence.

On searching for melanoma transcription factors in a project focusing on internal antitumor peptide sequences from transcription factors, we found that a highly immunogenic component emerged upon using a subtraction tolerization method of immunization. While several conventional immunization procedures using whole melanoma cells induced a plethora of low affinity antibodies of various specificities, the subtraction tolerization method efficiently elicited mono-specific antibodies that recognized Wilms' tumor protein 1 (WT1), which is known as an important marker in melanoma prognosis and treatment. For the tolerization step, pre-immunization of Balb/c mice with a membrane-rich preparation of glioblastoma U87 cells was used. The subsequent immunizations with SK-MEL-28 melanoma cells elicited antibodies strongly reacting with 50 and 55 kDa proteins, identified as WT1. Remarkably, this was the only component strongly reactive with these antibodies in a melanoma cell lysate. WT1 was then chosen as a target for selecting internally bioactive peptides. A hydrophilic Trojan peptide containing most of the zinc finger-2 domain of WT1 was synthesized and shown to inhibit SK-MEL-28 melanoma growth in vitro. The peptide WT1-pTj was also protective in vivo in a metastatic melanoma model and peptide-stimulated syngeneic dendritic cells reproduced the anti-melanoma effect of the unprotected peptide. Identification of antitumor peptides derived from major transcription factors represents a new tool to be explored in cancer research aiming at new therapeutic drugs.