Juliprosopine and juliprosine from prosopis juliflora leaves induce mitochondrial damage and cytoplasmic vacuolation on cocultured glial cells and neurons.

Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 µg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 µg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 µg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in ß-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.

KM-34, a Novel Antioxidant Compound, Protects against 6-Hydroxydopamine-Induced Mitochondrial Damage and Neurotoxicity.

The etiology of Parkinson's disease is not completely understood and is believed to be multifactorial. Neuronal disorders associated to oxidative stress and mitochondrial dysfunction are widely considered major consequences. The aim of this study was to investigate the effect of the synthetic arylidenmalonate derivative 5-(3,4-dihydroxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (KM-34), in oxidative stress and mitochondrial dysfunction induced by 6-hydroxydopamine (6-OHDA). Pretreatment (2 h) with KM-34 (1 and 10 µM) markedly attenuated 6-OHDA-induced PC12 cell death in a concentration-dependent manner. KM-34 also inhibited H2O2 generation, mitochondrial swelling, and membrane potential dissipation after 6-OHDA-induced mitochondrial damage. In vivo, KM-34 treatment (1 and 2 mg/Kg) reduced percentage of asymmetry (cylinder test) and increased the vertical exploration (open field) with respect to untreated injured animals; KM-34 also reduced glial fibrillary acidic protein overexpression and increased tyrosine hydroxylase-positive cell number, both in substantia nigra pars compacta. These results demonstrate that KM-34 present biological effects associated to mitoprotection and neuroprotection in vitro, moreover, glial response and neuroprotection in SNpc in vivo. We suggest that KM-34 could be a putative neuroprotective agent for inhibiting the progressive neurodegenerative disease associated to oxidative stress and mitochondrial dysfunction.

Specific Cytostatic and Cytotoxic Effect of Dihydrochelerythrine in Glioblastoma Cells: Role of NF-κB/ß-catenin and STAT3/IL-6 Pathways.

BACKGROUND: A glioblastoma is a primary CNS tumor that is more aggressive and lethal than other brain tumors. Its location, rapid proliferation, invasive growth, angiogenesis and immunosuppression are the main factors that limit its treatment, making it a major challenge to neuro-oncology. OBJECTIVE: This study investigated the in vitro effects of the alkaloid dihydrochelerythrine (DHC), which is extracted from Zanthoxylum stelligerum, on the viability, proliferation, cell death and ß-catenin, NFκB, STAT3/pSTAT3 and interleukins roles. METHOD: In vitro experimental models of human (U251 and GL-15) and murine (C6) glioblastoma cells were cultured in the presence of DHC at increasing concentrations for MTT assay and exclusion trypan blue dye to determine EC50. Afterward, C6 and U251 cells were treated with 100 µM DHC or DMSO 0.1% for cell cycle, annexin and expression of ß-catenin/NFκB/STAT3/pSTAT3 by flow cytometry or immunofluorescence. Interleukin quantification was made by Cytometric Bead Array. RESULTS: A significant decrease was observed in C6 and U251 cell viability in a time and dose-dependent manner. GL-15 cell viability decreased only when treated with 200 µM DHC. This maximum concentration affected neither astrocytes nor microglia viability. A cytostatic effect of DHC was observed in C6 and U251 cells after 48 h of 100 µM DHC treatment. After 72 h of DHC treatment, C6 presented 80% of annexin-V+ cells compared to 10% of annexin-V+ U251 cells. C6 cells demonstrated significant high levels of NFκ B and ß-catenin cytoplasmic fraction. Additionally, DHC treatment resulted in higher significant levels of IL-6 than did other interleukins and STAT3 up-regulation in U251 cells. CONCLUSION: These results demonstrate that DHC acts as a chemosensitizing agent selective for glioma cells not affecting non-tumor cells. Considering tumor heterogeneity, DHC demonstrated an anti-cancer potential to activate different cell death pathways. DHC demonstrated could be used for chemotherapy and immunotherapy applications in glioblastomas in the future.

Cytotoxicity of the diterpene 14-O-methyl-ryanodanol from Erythroxylum passerinum in an astrocytic cells model.

Plant secondary metabolites, such as, specifically, alkaloids and terpenes, may present psychoactive properties that modify the function of the central nervous system (CNS) and induce neurotoxicity. Neurotoxicity involves the response of glial cells, mainly astrocytes, which play a fundamental role in the control of homeostasis of the CNS. Some Erythroxylum species are indigenous to the state of Bahia in Brazil. This study investigated the cytotoxic activity of the diterpene AEP-1, extracted from the fruit of E. passerinum in a GL-15 cell line, astrocytic, glial cells model. The effects on cell viability, analyzed by the MTT assay, demonstrated a dose-dependent cytotoxic effect, with maximum effect at 500 µg/mL of AEP-1, and with a reduction of about 40 and 47% on cellular viability after 24 h and 72 h treatment, respectively. Evidence for induction of apoptosis by AEP-1 was first obtained when GL-15 glial cells were incubated with 250 µg/mL AEP-1 causing reniform and/or pyknotic nuclei and apoptotic bodies revealed by chromatin staining with Hoechst 33258. Increase in DNA fragmentation was also observed by comet assays in cells incubated with 500 µg/mL of AEP-1. Moreover, cells exposed to a sub toxic dose of AEP-1 (250 µg/mL) showed significant changes in morphology--contraction of the cytoplasm and expansion of cellular projections--signifying the presence of astrocytic cytoskeletal protein and glial fibrillary acidic protein (GFAP). These findings indicated astrocytic cells as the target for terpene AEP-1 and suggest the involvement of glial cells with psychoactive symptoms observed in humans and animals after consumption of fruits of plants of the genus Erythroxylum.