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.

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.

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.

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.

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 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.

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.

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.

Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo.

BACKGROUND: Pyrostegia venusta (Ker. Gawl.) Miers (Bignoniacea) is a medicinal plant from the Brazilian Cerrado used to treat leucoderma and common diseases of the respiratory system. OBJECTIVE: To investigate the antitumor activity of P.venusta extracts against melanoma. MATERIALS AND METHODS: The cytotoxic activity and tumor induced cell death of heptane extract (HE) from P. venusta flowers was evaluated against murine melanoma B16F10-Nex2 cells in vitro and in a syngeneic model in vivo. RESULTS: We found that HE induced apoptosis in melanoma cells by disruption of the mitochondrial membrane potential, induction of reactive oxygen species and late apoptosis evidenced by plasma membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation, exposure of phosphatidylserine on the cell surface and activation of caspase-2,-3,-8,-9. HE was also protective against singeneyc subcutaneous melanoma HE compounds were also able to induce cell cycle arrest at G2/M phases on tumor cells. On fractionation of HE in silica gel we isolated a cytotoxic fraction that contained a mixture of saturated hydrocarbons identified by (1)H NMR and GC-MS analyses. Predominant species were octacosane (C28H58-36%) and triacontane (C30H62-13%), which individually showed significant cytotoxic activity against murine melanoma B16F10-Nex2 cells in vitro and a very promising antitumor protection against subcutaneous melanoma in vivo. CONCLUSION: The results suggest that the components of the heptane extract, mainly octasane and triacontane, which showed antitumor properties in experimental melanoma upon regional administration, might also be therapeutic in human cancer, such as in the mostly epidermal and slowly invasive melanomas, such as acral lentiginous melanoma, as an adjuvant treatment to surgical excision.

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 natural bacterial-derived product, the metalloprotease arazyme, inhibits metastatic murine melanoma by inducing MMP-8 cross-reactive antibodies.

The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

Palladacycle (BPC) antitumour activity against resistant and metastatic cell lines: the relationship with cytosolic calcium mobilisation and cathepsin B activity.

The search for new compounds that induce p53-independent apoptosis is the focus of many studies in cancer biology because these compounds could be more specific and would overcome chemotherapy resistance. In this study, we evaluated the in vitro antitumour activity of a Biphosphinic Palladacycle Complex (BPC) and extended preclinical studies to an in vivo model. Saos-2 cells, a p53-null human osteosarcoma drug-resistant cell line, were treated with BPC in the presence or absence of a cathepsin B inhibitor and a calcium chelator (CA074 and BAPTA-AM, respectively), and several parameters related to apoptosis were evaluated. Preclinical studies were performed with mice that were intravenously inoculated with murine melanoma B16F10-Nex2 cells and treated intraperitoneally (i.p.) with BPC (8 mg/kg/day) for ten consecutive days, when lung metastatic nodules were counted. In vitro data show that BPC induces cell death in Saos-2 cells mainly by apoptosis, which was accompanied by the effector caspase-3 activation. These events are most likely related to Bax translocation and increased cytosolic calcium mobilisation, mainly from intracellular compartments. Lysosomal Membrane Permeabilisation (LMP) was also observed after 12 h of BPC exposure. Interestingly, BAPTA-AM and CA074 significantly decreased BPC cytotoxicity, suggesting that both calcium and cathepsin B are required for BPC antitumour activity. In vivo studies demonstrated that BPC protects mice against murine metastatic melanoma. In conclusion, BPC complex is an effective anticancer compound against metastatic murine melanoma. This complex is cytotoxic to the drug-resistant osteosarcoma Saos-2 human tumour cells by inducing apoptosis triggered by calcium signalling and a lysosomal-dependent pathway.

A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems.

The Wilms tumor protein 1 (WT1) transcription factor has been associated in malignant melanoma with cell survival and metastasis, thus emerging as a candidate for targeted therapy. A lysine-arginine rich peptide, WT1-pTj, derived from the ZF domain of WT1 was evaluated as an antitumor agent against A2058 human melanoma cells and B16F10-Nex2 syngeneic murine melanoma. Peptide WT1-pTj quickly penetrated human melanoma cells and induced senescence, recognized by increased SA-ß-galactosidase activity, enhanced transcriptional activity of p53, and induction of the cell cycle inhibitors p21 and p27. Moreover, the peptide bound to p53 and competed with WT1 protein for binding to p53. WT1-pTj treatment led to sustained cell growth suppression, abrogation of clonogenicity and G2/M cell cycle arrest. Notably, in vivo studies showed that WT1-pTj inhibited both the metastases and subcutaneous growth of murine melanoma in syngeneic mice, and prolonged the survival of nude mice challenged with human melanoma cells. The 27-amino acid cell-penetrating WT1-derived peptide, depends on C(3) and H(16) for effective antimelanoma activity, inhibits proliferation of WT1-expressing human tumor cell lines, and may have an effective role in the treatment of WT1-expressing malignancies.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system.

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased ß-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished.

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.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished. .

Characterization of cell wall lipids from the pathogenic phase of Paracoccidioides brasiliensis cultivated in the presence or absence of human plasma.

BACKGROUND: The fungal cell wall is a complex and dynamic outer structure. In pathogenic fungi its components interact with the host, determining the infection fate. The present work aimed to characterize cell wall lipids from P. brasiliensis grown in the presence and absence of human plasma. We compared the results from isolates Pb3 and Pb18, which represent different phylogenetic species that evoke distinct patterns of experimental paracoccidioidomycosis. METHODOLOGY/PRINCIPAL FINDINGS: We comparatively characterized cell wall phospholipids, fatty acids, sterols, and neutral glycolipids by using both electrospray ionization- and gas chromatography-mass spectrometry analyses of lipids extracted with organic solvents followed by fractionation in silica-gel-60. We detected 49 phospholipid species in Pb3 and 38 in Pb18, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. In both Pb3 and Pb18, PC and PE had the most numerous species. Among the fatty acids, C18:1 and C18:2 were the most abundant species in both isolates, although C18:2 was more abundant in Pb18. There was a different effect of plasma supplementation on fatty acids depending on the fungal isolate. The prevalent glycolipid species was Hex-C18:0-OH/d19:2-Cer, although other four minor species were also detected. The most abundant sterol in all samples was brassicasterol. Distinct profiles of cell wall and total yeast sterols suggested that the preparations were enriched for cell wall components. The presence of plasma in the culture medium specially increased cell wall brassicasterol abundance and also other lipids. CONCLUSIONS/SIGNIFICANCE: We here report an original comparative lipidomic analysis of P. brasiliensis cell wall. Our results open doors to understanding the role of cell wall lipids in fungal biology, and interaction with anti-fungal drugs and the host.

Chemical constituents and cytotoxic evaluation of essential oils from leaves of Porcelia macrocarpa (Annonaceae).

This work reports the chemical composition and cytotoxic evaluation of the essential oils from three different samples of the leaves of Porcelia macrocarpa R. E. Fries (Annonaceae). The crude oils, obtained by hydrodistillation procedures, were chemically analyzed by GC/MS. The obtained data indicated the predominance of sesquiterpenes (89.8 +/- 0.7%), the main compounds being germacrene D (47 +/-+/- 1%) and bicyclogermacrene (37 +/- 1%). These oils also contained the monoterpene verbanyl acetate (0.5 +/- 0.06%) and the diterpene phytol (1.2 +/- 0.3%). The crude oils obtained from leaves were pooled and tested in vitro against six cancer cell lineages--murine melanoma (B16F10-Nex2), human glioblastome (U87), human cervical carcinoma (HeLa), human leukemia (HL-60), human colon carcinoma (HCT), human breast adenocarcinoma (SKBr), and human melanoma (A2058), as well as against a non-tumorigenic human cell line (HFF). Since the essential oil reduced more than 50% of the viability of several tumor cells at 100 microg/mL, indicating the presence of active compounds, the crude material was subjected to fractionation over a SiO2/AgNO3 column. This procedure afforded different fractions composed of pure as well as different mixtures of bicyclogermacrene and germacrene D, which were tested against the same tumor cell lines, indicating a significant cytotoxic potential against HL-60 cells. These results suggested that the crudeoils and their components, mainly germacrene D, could be used as prototypes for the development of new anti-cancer agents for the treatment of human leukemia.

Identification of human plasma proteins associated with the cell wall of the pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermodimorphic species that cause paracoccidioidomycosis. The cell wall is the outermost fungal organelle to form an interface with the host. A number of host effector compounds, including immunologically active molecules, circulate in the plasma. In the present work, we extracted cell-wall-associated proteins from the yeast pathogenic phase of P. brasiliensis, isolate Pb3, grown in the presence of human plasma and analyzed bound plasma proteins by liquid chromatography-tandem mass spectrometry. Transport, complement activation/regulation, and coagulation pathway were the most abundant functional groups identified. Proteins related to iron/copper acquisition, immunoglobulins, and protease inhibitors were also detected. Several human plasma proteins described here have not been previously reported as interacting with fungal components, specifically, clusterin, hemopexin, transthyretin, ceruloplasmin, alpha-1-antitrypsin, apolipoprotein A-I, and apolipoprotein B-100. Additionally, we observed increased phagocytosis by J774.16 macrophages of Pb3 grown in plasma, suggesting that plasma proteins interacting with P. brasiliensis cell wall might be interfering in the fungal relationship with the host.