A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Abstract Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.(AU)

Proteomic characterization of Naja mandalayensis venom

Background Naja mandalayensis is a spitting cobra from Myanmar. To the best of our knowledge, no studies on this venom composition have been conducted so far. On the other hand, few envenomation descriptions state that it elicits mainly local inflammation in the victims' eyes, the preferred target of this spiting cobra. Symptoms would typically include burning and painful sensation, conjunctivitis, edema and temporary loss of vision. Methods We have performed a liquid-chromatography (C18-RP-HPLC) mass spectrometry (ESI-IT-TOF/MS) based approach in order to biochemically characterize N. mandalayensis venom. Results A wide variety of three-finger toxins (cardiotoxins) and metallopeptidases were detected. Less abundant, but still representative, were cysteine-rich secretory proteins, L-amino-acid oxidases, phospholipases A2, venom 5'-nucleotidase and a serine peptidase inhibitor. Other proteins were present, but were detected in a relatively small concentration. Conclusion The present study set the basis for a better comprehension of the envenomation from a molecular perspective and, by increasing the interest and information available for this species, allows future venom comparisons among cobras and their diverse venom proteins.(AU)

The modular nature of bradykinin-potentiating peptides isolated from snake venoms

Bradykinin-potentiating peptides (BPPs) are molecules discovered by Sergio Ferreira - who found them in the venom of Bothrops jararaca in the 1960s - that literally potentiate the action of bradykinin in vivo by, allegedly, inhibiting the angiotensin-converting enzymes. After administration, the global physiological effect of BPP is the decrease of the blood pressure. Due to this interesting effect, one of these peptides was used by David Cushman and Miguel Ondetti to develop a hypotensive drug, the widely known captopril, vastly employed on hypertension treatment. From that time on, many studies on BPPs have been conducted, basically describing new peptides and assaying their pharmacological effects, mostly in comparison to captopryl. After compiling most of these data, we are proposing that snake BPPs are 'modular' peptidic molecules, in which the combination of given amino acid 'blocks' results in the different existing peptides (BPPs), commonly found in snake venom. We have observed that there would be mandatory modules (present in all snake BPPs), such as the N-terminal pyroglutamic acid and C-terminal QIPP, and optionalmodules (amino acid blocks present in some of them), such as AP or WAQ. Scattered between these modules, there might be other amino acids that would 'complete' the peptide, without disrupting the signature of the classical BPP. This modular arrangement would represent an important evolutionary advantage in terms of biological diversity that might have its origins either at the genomic or at the post-translational modification levels. Regardless of the modules' origin, the increase in the diversity of peptides has definitely been essential for snakes' success on nature.(AU)

Preliminary molecular characterization of a proinflammatory and nociceptive molecule from the Echinometra lucunter spines extracts

Background Sea urchins are animals commonly found on the Brazilian shoreline, being Echinometra lucunter the most abundant species. Accidents caused by E. lucunter have been reported as one of the most frequent in Brazil, and are characterized by intense pain and inflammation, consequence of spine puncture in the skin. In order to characterize such toxic effects, we isolated one molecule that caused inflammatory and nociceptive effects. Methods E. lucunter specimens were collected without gender distinction. Spines were removed and molecules were extracted, fractionated by RP-HPLC and assayed for inflammatory and nociceptive activity, in a biological-driven fractionation way, until the obtainment of one active molecule and its subsequent analysis by mass spectrometry (MS and MS/MS). For inflammation, intravital microscopy was performed on the mouse cremaster muscle, in order to evaluate rolled, adherent and migrating leukocytes. Paw edema was also evaluated. For the nociceptive activity, the paw pressure test was performed in rats. Results One molecule could be isolated and related to the inflammatory and nociceptive activity. Regarding inflammation, increase in adherent and migrating cells was observed in the cremaster muscle after the administration of the molecule. Corroborating the inflammatory response, paw edema was also observed, although only in 20% of controls and 20 min after injection. Additionally, this molecule was able to decrease significantly the pain threshold, characterizing hyperalgesia. This molecule was analyzed by mass spectrometry, and according to the exact molecular mass, isotopic distribution and fragmentation profile, it was possible to propose the molecular formula C29H48N3O10. Conclusions One isolated molecule from the spine extract of E. lucunter is able to elicit inflammation and hypernociception in animal models, which is in agreement with the effects observed in sea urchin accidents.(AU)

The modular nature of bradykinin-potentiating peptides isolated from snake venoms

Abstract Bradykinin-potentiating peptides (BPPs) are molecules discovered by Sergio Ferreira who found them in the venom of Bothrops jararaca in the 1960s that literally potentiate the action of bradykinin in vivo by, allegedly, inhibiting the angiotensin-converting enzymes. After administration, the global physiological effect of BPP is the decrease of the blood pressure. Due to this interesting effect, one of these peptides was used by David Cushman and Miguel Ondetti to develop a hypotensive drug, the widely known captopril, vastly employed on hypertension treatment. From that time on, many studies on BPPs have been conducted, basically describing new peptides and assaying their pharmacological effects, mostly in comparison to captopryl. After compiling most of these data, we are proposing that snake BPPs are modular peptidic molecules, in which the combination of given amino acid blocks results in the different existing peptides (BPPs), commonly found in snake venom. We have observed that there would be mandatory modules (present in all snake BPPs), such as the N-terminal pyroglutamic acid and C-terminal QIPP, and optionalmodules (amino acid blocks present in some of them), such as AP or WAQ. Scattered between these modules, there might be other amino acids that would complete the peptide, without disrupting the signature of the classical BPP. This modular arrangement would represent an important evolutionary advantage in terms of biological diversity that might have its origins either at the genomic or at the post-translational modification levels. Regardless of the modules origin, the increase in the diversity of peptides has definitely been essential for snakes success on nature.

Preliminary molecular characterization of a proinflammatory and nociceptive molecule from the Echinometra lucunter spines extracts

Abstract Background Sea urchins are animals commonly found on the Brazilian shoreline, being Echinometra lucunter the most abundant species. Accidents caused by E. lucunter have been reported as one of the most frequent in Brazil, and are characterized by intense pain and inflammation, consequence of spine puncture in the skin. In order to characterize such toxic effects, we isolated one molecule that caused inflammatory and nociceptive effects. Methods E. lucunter specimens were collected without gender distinction. Spines were removed and molecules were extracted, fractionated by RP-HPLC and assayed for inflammatory and nociceptive activity, in a biological-driven fractionation way, until the obtainment of one active molecule and its subsequent analysis by mass spectrometry (MS and MS/MS). For inflammation, intravital microscopy was performed on the mouse cremaster muscle, in order to evaluate rolled, adherent and migrating leukocytes. Paw edema was also evaluated. For the nociceptive activity, the paw pressure test was performed in rats. Results One molecule could be isolated and related to the inflammatory and nociceptive activity. Regarding inflammation, increase in adherent and migrating cells was observed in the cremaster muscle after the administration of the molecule. Corroborating the inflammatory response, paw edema was also observed, although only in 20% of controls and 20 min after injection. Additionally, this molecule was able to decrease significantly the pain threshold, characterizing hyperalgesia. This molecule was analyzed by mass spectrometry, and according to the exact molecular mass, isotopic distribution and fragmentation profile, it was possible to propose the molecular formula C29H48N3O10. Conclusions One isolated molecule from the spine extract of E. lucunter is able to elicit inflammation and hypernociception in animal models, which is in agreement with the effects observed in sea urchin accidents.

Initial peptidomic profiling of Brazilian sea urchins: Arbacia lixula, Lytechinus variegatus and Echinometra lucunter

Background Sea urchins can be found throughout the Brazilian coast and are reported to be one of the major causes of marine accidents on the shoreline. Although not lethal, these accidents are reported to be extremely painful. In order to understand the toxinology of the Brazilian urchins, a peptidomic approach was performed aiming to characterize the naturally occurring peptides in both the coelomic fluid and the spine. Methods Animals were collected without gender distinction and samples of the coelomic fluid and spines extracted were analyzed by RP-HPLC and mass spectrometry for peptide de novo sequencing. Results Several peptides were identified either in the coelomic fluid or the spine extract (except for E. lucunter). The peptide sequences were aligned with public deposited sequences and possible functions were inferred. Moreover, some peptides can be cryptides, since their sequences were identified within functional proteins, for example thymosin from Strongylocentrotus purpuratus. Conclusions Although preliminary, the peptidomic approach presented here reports, for the first time, the abundance of novel biological molecules derived from these animals. The discovery of such molecules may be of potential biotechnological application, as described for other organisms; nevertheless, further studies are required.(AU)

Initial peptidomic profiling of Brazilian sea urchins: Arbacia lixula, Lytechinus variegatus and Echinometra lucunter

Sea urchins can be found throughout the Brazilian coast and are reported to be one of the major causes of marine accidents on the shoreline. Although not lethal, these accidents are reported to be extremely painful. In order to understand the toxinology of the Brazilian urchins, a peptidomic approach was performed aiming to characterize the naturally occurring peptides in both the coelomic fluid and the spine. Methods Animals were collected without gender distinction and samples of the coelomic fluid and spines extracted were analyzed by RP-HPLC and mass spectrometry for peptide de novo sequencing. Results Several peptides were identified either in the coelomic fluid or the spine extract (except for E. lucunter). The peptide sequences were aligned with public deposited sequences and possible functions were inferred. Moreover, some peptides can be cryptides, since their sequences were identified within functional proteins, for example thymosin from Strongylocentrotus purpuratus. Conclusions Although preliminary, the peptidomic approach presented here reports, for the first time, the abundance of novel biological molecules derived from these animals. The discovery of such molecules may be of potential biotechnological application, as described for other organisms; nevertheless, further studies are required.

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the viruss glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.Conclusions This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.(AU)

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action...

Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins

Background Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in order to identify an enzyme that could contribute not only to the toxicity, but also participate in the spine growth and regeneration. Results The spine aqueous extract was tested for peptidase activity, with synthetic substrates, in the presence and absence of inhibitors and activators. For proper enzyme classification, the FRET-substrate cleavage pattern, pH-dependency activity and Western-blot analyses were performed. The spine extract was able to cleave Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubation with DTT, and was inhibited by E-64. Furthermore, the double-peaked pH curve (5 and 7) and the cleavage site proportion (4:6, R-A:A-K) indicate the presence of both mono and dicarboxypeptidase activities. Moreover, in Western-blot analysis, the spine extract was positive for anti-cathepsin B antibody. Conclusions E. lucunter spines extracts presented a cysteine peptidase activity that was identified as cathepsin B/X that would participate in the remodeling and growth processes of the spine, as well as in the inflammatory response to the accident.(AU)

Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins

Background Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in order to identify an enzyme that could contribute not only to the toxicity, but also participate in the spine growth and regeneration. Results The spine aqueous extract was tested for peptidase activity, with synthetic substrates, in the presence and absence of inhibitors and activators. For proper enzyme classification, the FRET-substrate cleavage pattern, pH-dependency activity and Western-blot analyses were performed. The spine extract was able to cleave Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubation with DTT, and was inhibited by E-64. Furthermore, the double-peaked pH curve (5 and 7) and the cleavage site proportion (4:6, R,A:A,K) indicate the presence of both mono and dicarboxypeptidase activities. Moreover, in Western-blot analysis, the spine extract was positive for anti-cathepsin B antibody. Conclusions E. lucunter spines extracts presented a cysteine peptidase activity that was identified as cathepsin B/X that would participate in the remodeling and growth processes of the spine, as well as in the inflammatory response to the accident.