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Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.
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Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.
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Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.
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Background: Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.
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Background: Endogenous phospholipases A2 (PLA2 ) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM médium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.(AU)
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Venenos de Serpentes/toxicidade , Bioquímica , Glioblastoma , NeurotoxinasRESUMO
Background: Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of β-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The β-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: β-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on β-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. β-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that β-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.
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Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -ß/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.
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Transdiferenciação Celular/efeitos dos fármacos , Células Espumosas/citologia , Fosfolipases A2 do Grupo II/farmacologia , Músculo Liso Vascular/citologia , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fenótipo , Ratos , Receptores Depuradores Classe A/metabolismo , Receptores Depuradores Classe E/metabolismoRESUMO
Viper snake Crotalus durissus ruruima (Cdr) is a subspecies found in northern area of Brazil. Among the snakes of Crotalus genus subspecies, the venom of Cdr presents highest level of crotoxin, which is the major component of Crotalus snake venoms, formed by two subunits (crotapotin and a phospholipase A2 named CBr) and presents potent neurotoxic activity. Curiously, the venom of C. d. ruruima (CdrV) is better neutralized by antibothropic than by anticrotalic serum, strongly suggesting that this venom has similarities with venom of Bothrops genus snakes with regard to the ability to induce inflammation. Macrophages are cells with a central role in inflammatory and immunological responses. Upon inflammatory stimuli, these cells exhibit increased numbers of lipid droplets, which are key organelles in the synthesis and release of inflammatory mediators. However, the effects of CdrV and CBr in macrophage functions are unknown. We herein investigated the ability of CdrV and CBr to activate macrophages with focus on the formation of lipid droplets (LDs), synthesis of lipid mediators, and mechanisms involved in these effects. The involvement of LDs in PGE2 biosynthesis was also assessed. Stimulation of murine macrophages with CdrV and CBr induced an increased number of LDs and release of prostanoids (PGE2, PGD2, and TXB2). Neither CdrV nor CBr induced the expression of COX-1 and COX-2 by macrophages. LDs induced by both CdrV and CBr are associated to PLIN2 recruitment and expression and were shown to be dependent on COX-1, but not COX-2 activity. Moreover, PGE2 colocalized to CdrV- and CBr-induced LDs, revealing the role of these organelles as sites for the synthesis of prostanoids. These results evidence, for the first time, the ability of a whole snake venom to induce formation of LDs and the potential role of these organelles for the production of inflammatory mediators during envenomation by Crotalus snakes.
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Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-gama and -ß/d. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.
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MT-III, a snake venom GIIA sPLA2, which shares structural and functional features with mammalian GIIA sPLA2s, activates macrophage defense functions including lipid droplet (LDs) formation, organelle involved in both lipid metabolism and inflammatory processes. Macrophages (MΦs) loaded with LDs, termed foam cells, characterize early blood vessel fatty-streak lesions during atherosclerosis. However, the factors involved in foam cell formation induced by a GIIA sPLA2 are still unknown. Here, we investigated the participation of lipid homeostasis-related factors in LD formation induced by MT-III in macrophages. We found that MT-III activated PPAR-γ and PPAR-ß/δ and increased the protein levels of both transcription factors and CD36 in macrophages. Pharmacological interventions evidenced that PPAR-γ, PPAR-ß/δ, and CD36 as well as the endoplasmic reticulum enzymes ACAT and DGAT are essential for LD formation. Moreover, PPAR-ß/δ, but not PPAR-γ, is involved in MT-III-induced PLIN2 protein expression, and both PPAR-ß/δ and PPAR-γ upregulated CD36 protein expression, which contributes to MT-III-induced COX-2 expression. Furthermore, production of 15-d-PGJ2, an activator of PPARs, induced by MT-III, was dependent on COX-1 being LDs an important platform for generation of this mediator.
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Células Espumosas/efeitos dos fármacos , Homeostase , Lipídeos/química , Fosfolipases A2/farmacologia , Venenos de Serpentes/enzimologia , Animais , Antígenos CD36/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , PPAR alfa/metabolismo , PPAR delta/metabolismo , PPAR gama/metabolismo , PPAR beta/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
MT-III, a snake venom GIIA sPLA(2), which shares structural and functional features with mammalian GIIA sPLA(2)s, activates macrophage defense functions including lipid droplet (LDs) formation, organelle involved in both lipid metabolism and inflammatory processes. Macrophages (M Phi s) loaded with LDs, termed foam cells, characterize early blood vessel fatty-streak lesions during atherosclerosis. However, the factors involved in foam cell formation induced by a GIIA sPLA(2) are still unknown. Here, we investigated the participation of lipid homeostasis-related factors in LD formation induced by MT-III in macrophages. We found that MT-III activated PPAR-gamma and PPAR-beta/delta and increased the protein levels of both transcription factors and CD36 in macrophages. Pharmacological interventions evidenced that PPAR-gamma, PPAR-beta/delta, and CD36 as well as the endoplasmic reticulum enzymes ACAT and DGAT are essential for LD formation. Moreover, PPAR-beta/delta, but not PPAR-gamma, is involved in MT-III-induced PLIN2 protein expression, and both PPAR-beta/delta and PPAR-gamma upregulated CD36 protein expression, which contributes to MT-III-induced COX-2 expression. Furthermore, production of 15-d-PGJ2, an activator of PPARs, induced by MT-III, was dependent on COX-1 being LDs an important platform for generation of this mediator.
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Crotoxin B (CB) is a catalytically active group IIA sPLA2 from Crotalus durissus terrificus snake venom. In contrast to most GIIA sPLA2s, CB exhibits anti-inflammatory effects, including the ability to inhibit leukocyte functions. Lipid droplets (LDs) are lipid-rich organelles associated with inflammation and recognized as a site for the synthesis of inflammatory lipid mediators. Here, the ability of CB to induce formation of LDs and the mechanisms involved in this effect were investigated in isolated macrophages. The profile of CB-induced 15-d-PGJ2 (15-Deoxy-Delta-12,14-prostaglandin J2) production and involvement of LDs in 15-d-PGJ2 biosynthesis were also investigated. Stimulation of murine macrophages with CB induced increased number of LDs and release of 15-d-PGJ2. LDs induced by CB were associated to PLIN2 recruitment and expression and required activation of PKC, PI3K, MEK1/2, JNK, iPLA2 and PLD. Both 15-d-PGJ2 and COX-1 were found in CB-induced LDs indicating that LDs contribute to the inhibitory effects of CB by acting as platform for synthesis of 15-d-PGJ2, a pro-resolving lipid mediator. Together, our data indicate that an immunomodulatory GIIA sPLA2 can directly induce LD formation and production of a pro-resolving mediator in an inflammatory cell and afford new insights into the roles of LDs in resolution of inflammatory processes.
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Fosfolipases A2 do Grupo II/metabolismo , Fosfolipases A2 do Grupo II/farmacologia , Gotículas Lipídicas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Prostaglandina D2/análogos & derivados , Venenos de Serpentes/enzimologia , Animais , Células Cultivadas , Ciclo-Oxigenase 1/metabolismo , Citoplasma/metabolismo , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Camundongos , Perilipina-2/metabolismo , Prostaglandina D2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The snake venom MT-III is a group IIA secreted phospholipase A2 (sPLA2) enzyme with functional and structural similarities with mammalian pro-inflammatory sPLA2s of the same group. Previously, we demonstrated that MT-III directly activates the innate inflammatory response of macrophages, including release of inflammatory mediators and formation of lipid droplets (LDs). However, the mechanisms coordinating these processes remain unclear. In the present study, by using TLR2-/- or MyD88-/- or C57BL/6 (WT) male mice, we report that TLR2 and MyD88 signaling have a critical role in MT-III-induced inflammatory response in macrophages. MT-III caused a marked release of PGE2, PGD2, PGJ2, IL-1ß and IL-10 and increased the number of LDs in WT macrophages. In MT-III-stimulated TLR2-/- macrophages, formation of LDs and release of eicosanoids and cytokines were abrogated. In MyD88-/- macrophages, MT-III-induced release of PGE2, IL-1ß and IL-10 was abrogated, but release of PGD2 and PGJ2 was maintained. In addition, COX-2 protein expression seen in MT-III-stimulated WT macrophages was abolished in both TLR2-/- and MyD88-/- cells, while perilipin 2 expression was abolished only in MyD88-/- cells. We further demonstrated a reduction of saturated, monounsaturated and polyunsaturated fatty acids and a release of the TLR2 agonists palmitic and oleic acid from MT-III-stimulated WT macrophages compared with WT control cells, thus suggesting these fatty acids as major messengers for MT-III-induced engagement of TLR2/MyD88 signaling. Collectively, our findings identify for the first time a TLR2 and MyD88-dependent mechanism that underlies group IIA sPLA2-induced inflammatory response in macrophages.
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Fosfolipases A2 do Grupo II/farmacologia , Macrófagos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Venenos de Serpentes/enzimologia , Receptor 2 Toll-Like/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Ácidos Graxos/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/deficiência , Perilipina-2 , Prostaglandinas/biossíntese , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Receptor 2 Toll-Like/deficiência , Regulação para Cima/efeitos dos fármacosRESUMO
The venom of viperid snakes is collected monthly at Butantan Institute for research purposes and production of antivenoms. Here we describe histological and ultrastructural changes on Crotalus durissus terrificus and Bothrops sp. venom glands with defective venom production. Secretory tubules commonly showed partial or total obliteration of their lumina by masses of necrotic cells and cellular debris. Secretory cells showed varying degrees of degenerative and/or metaplastic alterations seriously affecting the structures responsible for the synthesis and secretion of venom. The intertubular connective tissue presented fibroblast hyperplasia, inflammatory cells infiltration, vacuolated cells and blood vessels alterations. In two venom glands out of nineteen snakes examined, virus-like particles were found. The alterations observed in most of the glands could have been caused by excessive manual pressure, during venom extraction routine, causing disruption of the secretory tubules and leakage of venom to the intertubular connective tissue.
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Bothrops/anatomia & histologia , Venenos de Crotalídeos/metabolismo , Crotalus/anatomia & histologia , Glândulas Exócrinas/patologia , Animais , Glândulas Exócrinas/ultraestruturaRESUMO
MT-II, a Lys49PLA2 homologue devoid of catalytic activity from B. asper venom, stimulates inflammatory events in macrophages. We investigated the ability of MT-II to induce formation of lipid droplets (LDs), key elements of inflammatory responses, in isolated macrophages and participation of protein kinases and intracellular PLA2s in this effect. Influence of MT-II on PLIN2 recruitment and expression was assessed, and the effects of some synthetic peptides on LD formation were further evaluated. At noncytotoxic concentrations, MT-II directly activated macrophages to form LDs. This effect was reproduced by a synthetic peptide corresponding to the C-terminal sequence 115-129 of MT-II, evidencing the critical role of C-terminus for MT-II-induced effect. Moreover, MT-II induced expression and recruitment of PLIN2. Pharmacological interventions with specific inhibitors showed that PKC, PI3K, ERK1/2, and iPLA2, but not P38(MAPK) or cPLA2, signaling pathways are involved in LD formation induced by MT-II. This sPLA2 homologue also induced synthesis of PGE2 that colocalized to LDs. In conclusion, MT-II is able to induce formation of LDs committed to PGE2 formation in a process dependent on C-terminal loop engagement and regulated by distinct protein kinases and iPLA2. LDs may constitute an important inflammatory mechanism triggered by MT-II in macrophages.