Phoneutria Nigriventer Spider Venom Research Articles

Unveiling hidden toxin diversity: Discovery of novel venom components through manual curation of highly expressed sequences annotated as “no hits” in Phoneutria nigriventer spider venom gland transcriptome

Spider venoms have evolved over thousands of years, optimizing feeding and defense mechanisms. Venom components show pharmacological and biotechnological potential, rising interest in their study. However, the isolation of spider toxins for experimental evaluation poses significant challenges. To address this, transcriptomic analysis combined with computational tools has emerged as an appealing approach to characterizing spider venoms. However, many sequences remain unidentified after automatic annotation. In this study, we manually curated a subset of previously unannotated sequences from the Phoneutria nigriventer transcriptome and identified new putative venom components. Our manual analysis revealed 29 % of the analyzed sequences were potential venom components, 29 % hypothetical/uncharacterized proteins, and 17 % cellular function proteins. Only 25 % of the originally unannotated dataset remained without any identification. Most reclassified components were cysteine-rich peptides, including 23 novel putative toxins. We also found glycine-rich peptides (GRP), corroborating the previous description of GRPs in Phoneutria pertyi venom glands. Furthermore, to emphasize the recurrence of the lack of annotation in spider venom glands transcripts, we provide a survey of the percentage of unidentified sequences in several published spider venom transcriptomics studies. In conclusion, our study highlights the importance of manual curation in uncovering novel venom components and underscores the need for improved annotation strategies to fully exploit the medical and biotechnological potential of spider venoms.

PnPP-15, a Synthetic Peptide Derived from a Toxin from Phoneutria nigriventer Spider Venom, Alleviates Diabetic Neuropathic Pain and Acts Synergistically with Pregabalin in Mice.

Diabetic neuropathic pain is one of the complications that affect a wide variety of the diabetic population and is often difficult to treat. Only a small number of patients experience pain relief, which usually comes with onerous side effects and low levels of satisfaction. The search for new analgesic drugs is necessary, given the limitations that current drugs present. Combining drugs to treat neuropathic pain has been attracting interest to improve their efficacy compared to single-drug monotherapies while also reducing dose sizes to minimize side effects. The aim of our study was to verify the antinociceptive effect of a synthetic peptide, PnPP-15, alone and combined with pregabalin, in male Swiss diabetic mice using the von Frey method. PnPP-15 is a synthetic peptide derived from PnPP19, a peptide representing a discontinuous epitope of the primary structure of the toxin PnTx2-6 from the venom of the spider Phoneutria nigriventer. The antinociceptive activity of both compounds was dose-dependent and showed synergism, which was verified by isobolographic analysis. Treatment with PnPP-15 did not cause spontaneous or forced motor changes and did not cause any damage or signs of toxicity in the analyzed organs (pancreas, lung, heart, kidney, brain, or liver). In conclusion, PnPP-15 is a great candidate for an analgesic drug against neuropathic pain caused by diabetes and exerts a synergistic effect when combined with pregabalin, allowing for even more efficient treatment.

Isolated Peptide from Spider Venom Modulates Dendritic Cells In Vitro: A Possible Application in Oncoimmunotherapy for Glioblastoma.

Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1β, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.

DDDR-15. SPIDER VENOM MOLECULES IMPAIR HUMAN GLIOMA CELLS MIGRATION: POTENTIAL APPLICATION IN CANCER THERAPY

Abstract Gliomas correspond to approximately 80% of malignant neoplasms of the central nervous system. Due to its aggressiveness, there is a growing need to seek new strategies to control the disease. The components of spider venoms are stable peptides with high specificity against biological molecular targets of therapeutic relevance. It has already been demonstrated by our group that two subfractions (SFs) isolated from the Phoneutria nigriventer spider venom (PnV) (called SF1 and SF11) can delay the migration of glioblastoma (GB) well-established cell lineage in invasions assays. Therefore, the present study aimed to evaluate the responsiveness of samples collected from Brazilian patients with a presumptive diagnosis of glioma (high and low grades), to PnV SFs, in order to contribute to the development of new drugs. After patient consent, 13 samples were collected for cultivation and attempt to reach the tenth passage, to ensure the establishment of the lineage. Four lineages were established: J01 and N07 – glioblastoma (high grade); C03 and L09 – astrocytoma and oligodendroglioma (low grade) respectively. Cells were incubated for 48 hours with SF1 and SF11, during the scratch wound-healing assay. The results demonstrated that the SFs, mainly SF11 (which proved to be a pure peptide with 1603,88 Da, sequenced as PyrKKDKKDKFY–GLM–NH2), harmed the migration of all these samples when compared to the control group. In addition, initial data with J01 lineage demonstrated that migration was also affected during the transwell invasion assay; as well as ROCK protein, involved in control of cell migration, was decreased (demonstrated by western blot) by both SFs, suggesting that ROCK is involved in the mechanism of the components. Ongoing tests continue to establish other samples collected so that the tests described above will also be carried out. These results will contribute to the development of new therapies against these tumors. Support: FAPESP #2019/10003-3.

Components from spider venom activate macrophages against glioblastoma cells: new potential adjuvants for anticancer immunotherapy.

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

Spider venom components decrease glioblastoma cell migration and invasion through RhoA-ROCK and Na+/K+-ATPase \u03b22: potential molecular entities to treat invasive brain cancer

BackgroundGlioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells, also evaluating RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) involvement.MethodsHuman (NG97) GB cells were treated with twelve subfractions (SFs—obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound healing and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Values were analyzed by the GraphPad Prism software package and the level of significance was determinate using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test.ResultsTwo (SF1 and SF11) of twelve SFs, decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase β2.ConclusionAll components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway.

EXTH-28. MOLECULAR CHARACTERIZATION OF HUMAN GLIOMAS AND IDENTIFICATION OF NEOPLASMS RESPONSIBLE FOR NEW BIOPHARMACEUTICALS OBTAINED FROM ANIMAL VENOM: A TRANSLATIONAL APPROACH

Abstract Gliomas correspond to approximately 80% of primary malignant brain tumors in adults. Associate histopathological classification to the identification of the molecular profile of these tumors is a great strategy to predict the tumor’s behavior and its responsiveness to the treatments. Studies have shown the potential of using biopharmaceuticals against cancer. Our research group has recently found two main subfractions isolated from Phoneutria nigriventer spider venom (PnV) (called SF1 and SF11) that can act decreasing the number of migrating cells during transwell assay. Therefore, the present study aimed to characterize, as regards molecular identity, the responsiveness of glioma samples collected from patients to the molecules purified from the PnV, in order to improve diagnostic and prognostic predictions, as well as contribute to the development of new drugs. After consent of the patients, samples of the tumors clinically diagnosed as glioma were collected during the surgical procedure to cultivation, that were performed until 10 passages to establish a lineage before use. To analyze its responsiveness to PnV in a migration assay, cultured human glioblastoma (1-JA63) cells were treated with PnV-isolated (HPLC) subfractions (SF1 and SF11 at 1 µg/ml) for 12 and 48 h (control stayed in IMDM) in a transwell system. SFs (mainly SF1) treatment induced a significant reduction in the number of migrating cells after 48 hours of exposure, in comparison to the control (untreated). Ongoing trials continue the establishment of another tumor samples of gliomas and migration tests with PnV subfractions will be carried out, to compare their effects in relation to other degrees of the disease also correlating the tumor response to the drug with the molecular profile. The results will contribute to the development of a potential individualized therapy also adding information about the correlation between the molecular identity of the tumor and its prognosis.

EXTH-42. SPIDER VENOM COMPONENTS DECREASE GLIOBLASTOMA CELL MIGRATION AND INVASION THROUGH RHOA-ROCK AND NA+/K+-ATPASE β2: POTENTIAL MOLECULAR ENTITIES TO TREAT INVASIVE BRAIN CANCER

Abstract Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells. The involvement of RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) was also evaluated. Human (NG97) GB cells were treated with twelve subfractions (SFs - obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound heling and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Two (SF1 and SF11) of twelve SFs decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase β2. All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway. Support: FAPESP #2015/04194-0; CNPq #431465/2016–9.

VEGF/VEGFR-2 system exerts neuroprotection against Phoneutria nigriventer spider envenomation through PI3K-AKT-dependent pathway

This study was undertaken to elucidate why VEGF/VEGFR-2 is elevated in the hippocampus of rats injected with Phoneutria nigriventer spider venom (PNV). PNV delays Na+ channels inactivation; blocks Ca2+ and K+ channels, increases glutamate release, causes blood-brain breakdown (BBBb), brain edema and severe excitotoxicity. Analytical FT-IR spectroscopy showed profound alteration in molecular biochemical state, with evidences for VEGFR-2 (KDR/Flk-1) signaling mediation. By blocking VEGF/VEGFR-2 binding via pre-treatment with itraconazole we demonstrated that animals' condition was deteriorated soon at 1–2 h post-PNV exposure concurrently with decreased expression of VEGF, BBB-associated proteins, ZO-1, β-catenin, laminin, P-gp (P-glycoprotein), Neu-N (neuron's viability marker) and MAPKphosphorylated-p38, while phosphorylated-ERK and Src pathways were increased. At 5 h and coinciding with incipient signs of animals' recuperation, the proteins associated with protection (HIF-1α, VEGF, VEGFR-1, VEGFR-2, Neu-N, occludin, β-catenin, laminin, P-gp efflux protein, phosphorylated-p38) increased thus indicating p38 pathway activation together with paracellular route strengthening. However, the BBB transcellular trafficking and caspase-3 increased (pro-apoptotic pathway activation). At 24 h, the transcellular route reestablished physiological state but the pro-survival pathway PI3K/(p-Akt) dropped in animals underwent VEGF/VEGFR-2 binding inhibition, whereas it was significantly activated at matched interval in PNV group without prior itraconazole; these results demonstrate impaired VEGF′ survival effects at 24 h. The inhibition of VEGF/VEGFR-2 binding identified 5 h as turning point at which multi-level dynamic interplay was elicited to reverse hippocampal damage. Collectively, the data confirmed VEGFR-2 signaling via serine-threonine kinase Akt as neuroprotective pathway against PNV-induced damage. Further studies are needed to elucidate mechanisms underlying PNV effects.

PnAn13, an antinociceptive synthetic peptide inspired in the Phoneutria nigriventer toxin PnTx4(6-1) (δ-Ctenitoxin-Pn1a).

Animal venoms are an almost inexhaustible source for promising molecules with biological activity and the venom of Phoneutria nigriventer spider is a good example of this. Among several other toxins obtained from this venom, PnTx4(6–1), also called δ-Ctenitoxin-Pn1a, was isolated and initially described as an insect toxin that binds to the site 3 of sodium channels in cockroach nerve cord synaptosomes (Periplaneta americana) and slows down sodium current inactivation in isolated axons of this animal. This toxin did not cause any apparent toxicity to mice when intracerebroventricularly injected (30 μg). Subsequently, it was demonstrated that PnTx4(6–1) has an antinociceptive effect in three different pain models: inflammatory, induced by carrageenan; nociceptive, induced by prostaglandin E2 and neuropathic, induced by sciatic nerve constriction. Using diverse antagonists from receptors, it was shown that the cannabinoid system, via the CB1 receptor, and the opioid system, through the μ and δ receptors, are both involved in the antinociceptive effect of PnTx4(6–1). In the present work, it was synthesized a peptide, named PnAn13, based on the amino acid sequence of PnTx4(6–1) in order to try to reproduce or increase the analgesic effect of the toxin. As it was seen for the toxin, PnAn13 had antinociceptive activity, when intrathecally injected, and this effect involved the cannabinoid and opioid systems. In addition, when it was evaluated the peripheral effect of PnAn13, via intraplantar administration, this peptide was able to reverse the hyperalgesic threshold, evoked by prostaglandin E2. Therefore, using different pharmacological tools, it was shown the participation of cannabinoid and opioid systems in this effect.

Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model

Molecules from animal venoms are promising candidates for the development of new drugs. Previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (PnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG−/− mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. Tumors from PnV-treated animals were smaller and did not uptake detectable amounts of 18F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro- and anti-inflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from PnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. Natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of PnV on tumor development in vivo, considering the complex neoplastic microenvironment. The venom was effective at impairing tumor development in murine xenogeneic model, activating the innate immune response and increasing tumor infiltrating macrophages. In addition, PnV activated macrophages in vitro for a different profile of M1. These activated PnV-macrophages have potential to fight the tumor without promoting tumorigenesis. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. We aim to synthesize and carry out a formulation with these antineoplastic molecules for clinical trials. Spider venom biomolecules induced smaller and necrotic xenogeneic GB; spider venom activated the innate immune system; venom increased blood monocytes and the migration of macrophages to the tumor; activated PnV-macrophages have a profile different of M1 and have a potential to fight the tumor without promote tumorigenesis.

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature.

Background: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown.Methods:Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences.Results:The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences.Conclusions:Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.

Intravitreal injection of peptides PnPa11 and PnPa13, derivatives of Phoneutria nigriventer spider venom, prevents retinal damage.

Background: PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides.Methods: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified.Results: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation.Conclusions: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.

Transcriptome analysis of the spider Phoneutria pertyi venom glands reveals novel venom components for the genus Phoneutria

Phoneutria nigriventer spider venom has been studied for more than 40 years and several components with pharmacological potential have been described in it. However, studies on venoms from other species of the Phoneutria genus are scarce. In this work, a conventional cDNA library from the species Phoneutria pertyi venom glands was constructed, aiming to identify novel putative cysteine-rich peptide toxins for the genus Phoneutria. 296 unique sequences were identified and 51 sequences corresponded to putative cysteine-rich peptide toxins. Besides cysteine-rich peptide toxins, other putative venom components such as protease inhibitors, defensins and serine proteinases were identified. Furthermore, by manual curation of the sequences with no match at UniProt, we were able to identify glycine-rich proteins (GRP), a class of venom component never described in Phoneutria genus. This work describes the first complete sequences of toxins from the venom of P. pertyi and reveals that, despite most of the retrieved toxins show a high identity to toxins identified in Phoneutria genus, novel putative toxins remains to be described.

Antinociceptive effect of PnTx4(5-5), a peptide from Phoneutria nigriventer spider venom, in rat models and the involvement of glutamatergic system.

ABSTRACTBackground: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

Venom of the Phoneutria nigriventer spider alters the cell cycle, viability, and migration of cancer cells.

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 µg/ml) and high (280 µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules.

Inhibition of VEGF-Flk-1 binding induced profound biochemical alteration in the hippocampus of a rat model of BBB breakdown by spider venom. A preliminary assessment using FT-IR spectroscopy

Phoneutria nigriventer spider venom (PNV) contains ion channels-acting neuropeptides that in rat induces transitory blood-brain barrier breakdown (BBBb) in hippocampus in parallel with VEGF upregulation. We investigated whether VEGF has a neuroprotective role by inhibiting its binding to receptor Flk-1 by itraconazole (ITZ). FT-IR spectroscopy examined the biochemical status of hippocampus and evaluated BBBb in rats administered PNV or ITZ/PNV at periods with greatest toxicity (1-2h), recovery (5h) and visual absence of symptoms (24h), and compared to saline and ITZ controls. The antifungal treatment before venom intoxication aggravated the venom effects and increased BBB damage. FT-IR spectra of venom, hippocampi of controls, PNV and ITZ-PNV showed a 1400 cm−1 band linked to symmetric stretch of carboxylate and 1467 cm−1 band (CH2 bending: mainly lipids) that were considered biomarker and reference bands, respectively. Inhibition of VEGF/Flk-1 binding produced marked changes in lipid/protein stability at 1-2h. The largest differences were observed in spectra regions assigned to lipids, both symmetric (2852 cm−1) and asymmetric (2924 and 2968 cm−1). Quantitative analyses showed greatest increases in the 1400 cm−1/1467 cm−1 ratio also at 1h. Such changes at period of rats' severe intoxication referred to wavenumber region from 3106 cm−1 to 687 cm−1 assigning for C-H and N-H stretching of protein, Amide I, C=N cytosine, N-H adenine, Amide II, CH2 bending: mainly lipids, C-O stretch: glycogen, polysaccharides, glycolipids, z-type DNA, C-C, C-O and CH out-of-plane bending vibrations. We conclude that VEGF has a neuroprotective role and can be a therapeutic target in PNV envenomation. FT-IR spectroscopy showed to be instrumental for monitoring biochemical changes in this model of P. nigriventer venom-induced BBB disruption.

PnTx2-6 (or δ-CNTX-Pn2a), a toxin from Phoneutria nigriventer spider venom, releases l-glutamate from rat brain synaptosomes involving Na+ and Ca2+ channels and changes protein expression at the blood-brain barrier

PhTx2 is the most toxic fraction from the venom of the spider Phoneutria nigriventer, being responsible to sodium entry into cortical synaptosomes, increasing the release of neurotransmitters, such as l-glutamate (L-Glu) and; acetylcholine. In this study, we investigated the action of a toxin purified from; PhTx2 fraction, called PnTx2-6 or δ-CNTX-Pn2a, on L-Glu release from rat; brain cortex synaptosomes, as well as its ability to induce blood-brain barrier permeability. PnTx2-6 increased L-Glu release from rat cortical brain synaptosomes in a time- and dose-dependent manner (EC50 = ∼20 nM; Tm = 16min), as measured by a fluorimetric method. The increase of L-Glu by PnTx2-6 was inhibited by tetrodotoxin. And partially inhibited by EGTA. Calcium channel blockers ω-conotoxin MVIIC (P/Q-types) and ω-conotoxin GVIA (N-type), were able to reduce the PnTx2-6-induced release of L-Glu, while nifedipine (L-type) did not show any inhibition. These findings suggest that thew release of L-Glu by PnTx2-6 is due its primary action on sodium channels, well-known to be target of this toxin. PnTx2-6 is able to potentiate penile erection and this effect may be related with the release of l-glutamate from the CNS, besides a local effect on corpus carvenosum, as previously shown by our group. If L-Glu release and penile erection potentiation are indeed correlated, then this toxin should be able to cross the blood brain barrier (BBB). Results by immunoblotting assays indicated a change in the expression of proteins associated with the paracellular and transcellular transport at the blood-brain barrier, suggesting a BBB dysfunction mediated by PnTx2-6. Therefore, PnTx2-6 may induce the release l-glutamate in the central nervous system, when injected peripherally.

PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system

Background and aimsCholinergic agents cause antinociception by mimicking the release of acetylcholine (ACh) from spinal cholinergic nerves. PhKv is a peptide isolated from the venom of the armed spider Phoneutria nigriventer. It has an antiarrythmogenic activity that involves the enhanced release of acetylcholine. The aim of this study was to investigate whether PhKv had an antinociceptive action in mice. MethodsMale albino Swiss mice (25–35g) were used in this study. The PhKv toxin was purified from a PhTx3 fraction of the Phoneutria nigriventer spider's venom. Because of its peptide nature, PhKv is not orally available and it was delivered directly into the central nervous system by an intrathecal (i.t.) route. PhKV on the thermal and mechanical sensitivity was evaluated using plantar test apparatus and the up-and-down method. The analgesic effects of PhKv were studied in neuropathic pain (CCI) and in the peripheral capsicin test. In order to test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (5mg/kg, i.p.) or mecamylamine (0.001mg/kg, i.p.) and the PhKv toxin (30pmol/site i.t.) or neostigmine (100pmol/site) were applied 15min before the intraplantar capsaicin (1nmol/paw) administrations. To investigate PhKv action on the AChE activities, was performed in vitro and ex vivo assay for AChE. For the in vitro experiments, mice spinal cord supernatants of tissue homogenates (1mg/ml) were used as source of AChE activity. The AChE assay was monitored at 37°C for 10min in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 405nm. ResultsPhKv (30 and 100pmol/site, i.t.) had no effect on the thermal or mechanical sensitivity thresholds. However, in a chronic constriction injury model of pain, PhKv (10pmol/site, i.t.) caused a robust reduction in mechanical withdrawal with an antinociceptive effect that lasted 4h. A pretreatment in mice with PhKv (30pmol/site, i.t.) or neostigmine (100pmol/site, i.t.) 15min before an intraplantar injection of capsaicin (1nmol/paw) caused a maximal antinociceptive effect of 69.5±4.9% and 85±2.5%, respectively. A pretreatment in mice with atropine; 5mg/kg, i.p. or mecamylamine 0.001mg/kg, i.p. inhibited a neostigimine and PhKv-induced antinociception, suggesting a cholinergic mechanism. Spinal acetylcholinesterase was inhibited by PhKv with ED50 of 7.6 (4.6–12.6pmol/site, i.t.). PhKv also inhibited the in vitro AChE activity of spinal cord homogenates with an EC50 of 20.8 (11.6–37.3nM), shifting the Km value from 0.06mM to 18.5mM, characterizing a competitive inhibition of AChE activity by PhKv. ConclusionsOur findings provide, to our knowledge, the first evidence that PhKv caused inhibition of AChE, it increased the ACh content at the neuronal synapses, leading to an activation of the cholinergic system and an antinociceptive response. ImplicationsStudies regarding the nociceptive mechanisms and the identification of potential targets for the treatment of pain have become top priorities. PhKv, by its action of stimulating the cholinergic receptors muscarinic and nicotinic system, reduces pain it may be an alternative for controlling the pain processes.

Lethality and histopathological alterations caused by Phoneutria nigriventer spider venom from Argentina: Neutralization of lethality by experimental and therapeutic antivenoms

Although the spiders of the genus Phoneutria cause envenomation and their presence has been described in several provinces of the north of Argentina, they are not as common as other spiders of sanitary importance. In the present work, we studied the toxicity of samples of venom of Phoneutria spiders from the provinces of Misiones (where severe envenomation and deaths by Phoneutria have been recorded) and Jujuy (where no deaths have been recorded and severe envenomations are not frequent). To this end, we assessed the lethal potency in mice and guinea pigs and the histopathological alterations caused by both venoms, as well as the neutralization by the commonly used therapeutic antivenom produced by the Butantan Institute in Brazil and by an experimental antivenom developed with venom of P. nigriventer from Misiones. There were no differences in the lethality of the venoms of spiders from both regions. Post mortem examination showed that the heart and lungs were the most affected organs, while important pulmonary edema was seen macroscopically. Histological analysis showed edema, atelectasis, emphysema and cardiac lesion in both experimental models. The antivenoms assayed showed good neutralization of the venoms in the two experimental models. Despite the different geographic origins, the venoms showed similar toxicity and both the experimental antivenom and therapeutic antivenmos were able to neutralize the venoms of Argentinean P. nigriventer.