S-allyl-cysteine triggers cytotoxic events in rat glioblastoma RG2 and C6 cells and improves the effect of temozolomide through the regulation of oxidative responses.

Glioblastoma (GBM) is an aggressive form of cancer affecting the Central Nervous System (CNS) of thousands of people every year. Redox alterations have been shown to play a key role in the development and progression of these tumors as Reactive Oxygen Species (ROS) formation is involved in the modulation of several signaling pathways, transcription factors, and cytokine formation. The second-generation oral alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic drug used to treat of GBM, though patients often develop primary and secondary resistance, reducing its efficacy. Antioxidants represent promising and potential coadjutant agents as they can reduce excessive ROS formation derived from chemo- and radiotherapy, while decreasing pharmacological resistance. S-allyl-cysteine (SAC) has been shown to inhibit the proliferation of several types of cancer cells, though its precise antiproliferative mechanisms remain poorly investigated. To date, SAC effects have been poorly explored in GBM cells. Here, we investigated the effects of SAC in vitro, either alone or in combination with TMZ, on several toxic and modulatory endpoints-including oxidative stress markers and transcriptional regulation-in two glioblastoma cell lines from rats, RG2 and C6, to elucidate some of the biochemical and cellular mechanisms underlying its antiproliferative properties. SAC (1-750 µM) decreased cell viability in both cell lines in a concentration-dependent manner, although C6 cells were more resistant to SAC at several of the tested concentrations. TMZ also produced a concentration-dependent effect, decreasing cell viability of both cell lines. In combination, SAC (1 µM or 100 µM) and TMZ (500 µM) enhanced the effects of each other. SAC also augmented the lipoperoxidative effect of TMZ and reduced cell antioxidant resistance in both cell lines by decreasing the TMZ-induced increase in the GSH/GSSG ratio. In RG2 and C6 cells, SAC per se had no effect on Nrf2/ARE binding activity, while in RG2 cells TMZ and the combination of SAC + TMZ decreased this activity. Our results demonstrate that SAC, alone or in combination with TMZ, exerts antitumor effects mediated by regulatory mechanisms of redox activity responses. SAC is also a safe drug for testing in other models as it produces non-toxic effects in primary astrocytes. Combined, these effects suggest that SAC affords antioxidant properties and potential antitumor efficacy against GBM.

Anandamide and WIN 55212-2 Afford Protection in Rat Brain Mitochondria in a Toxic Model Induced by 3-Nitropropionic Acid: an In Vitro Study.

Mitochondrial dysfunction plays a key role in the development of neurodegenerative disorders. In contrast, the regulation of the endocannabinoid system has been shown to promote neuroprotection in different neurotoxic paradigms. The existence of an active form of the cannabinoid receptor 1 (CB1R) in mitochondrial membranes (mitCB1R), which might exert its effects through the same signaling mechanisms as the cell membrane CB1R, has been shown to regulate mitochondrial activity. Although there is evidence suggesting that some cannabinoids may induce protective effects on isolated mitochondria, substantial evidence on the role of cannabinoids in mitochondria remains to be explored. In this work, we developed a toxic model of mitochondrial dysfunction induced by exposure of brain mitochondria to the succinate dehydrogenase inhibitor 3-nitropropionic acid (3-NP). Mitochondria were also pre-incubated with the endogenous agonist anandamide (AEA) and the synthetic CB1R agonist WIN 55212-2 to evaluate their protective effects. Mitochondrial reduction capacity, reactive oxygen species (ROS) formation, and mitochondrial swelling were assessed as toxic markers. While 3-NP decreased the mitochondrial reduction capacity and augmented mitochondrial ROS formation and swelling, both AEA and WIN 55212-2 ameliorated these toxic effects. To explore the possible involvement of mitCB1R activation on the protective effects of AEA and WIN 55212-2, mitochondria were also pre-incubated in the presence of the selective CB1R antagonist AM281, which completely reverted the protective effects of the cannabinoids to levels similar to those evoked by 3-NP. These results show partial protective effects of cannabinoids, suggesting that mitCB1R activation may be involved in the recovery of compromised mitochondrial activity, related to reduction of ROS formation and further prevention of mitochondrial swelling.

Interactions Between the Ubiquitin-Proteasome System, Nrf2, and the Cannabinoidome as Protective Strategies to Combat Neurodegeneration: Review on Experimental Evidence.

Neurodegenerative disorders are chronic brain diseases that affect humans worldwide. Although many different factors are thought to be involved in the pathogenesis of these disorders, alterations in several key elements such as the ubiquitin-proteasome system (UPS), the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, and the endocannabinoid system (ECS or endocannabinoidome) have been implicated in their etiology. Impairment of these elements has been linked to the origin and progression of neurodegenerative disorders, while their potentiation is thought to promote neuronal survival and overall neuroprotection, as proved with several experimental models. These key neuroprotective pathways can interact and indirectly activate each other. In this review, we summarize the neuroprotective potential of the UPS, ECS, and Nrf2 signaling, both separately and combined, pinpointing their role as a potential therapeutic approach against several hallmarks of neurodegeneration.

Monoacylglycerol Lipase Inhibition Prevents Short-Term Mitochondrial Dysfunction and Oxidative Damage in Rat Brain Synaptosomal/Mitochondrial Fractions and Cortical Slices: Role of Cannabinoid Receptors.

Inhibition of enzymes responsible for endocannabinoid hydrolysis represents an invaluable emerging tool for the potential treatment of neurodegenerative disorders. Monoacylglycerol lipase (MAGL) is the enzyme responsible for degrading 2-arachydonoylglycerol (2-AG), the most abundant endocannabinoid in the central nervous system (CNS). Here, we tested the effects of the selective MAGL inhibitor JZL184 on the 3-nitropropinic acid (3-NP)-induced short-term loss of mitochondrial reductive capacity/viability and oxidative damage in rat brain synaptosomal/mitochondrial fractions and cortical slices. In synaptosomes, while 3-NP decreased mitochondrial function and increased lipid peroxidation, JZL184 attenuated both markers. The protective effects evoked by JZL184 on the 3-NP-induced mitochondrial dysfunction were primarily mediated by activation of cannabinoid receptor 2 (CB2R), as evidenced by their inhibition by the selective CB2R inverse agonist JTE907. The cannabinoid receptor 1 (CB1R) also participated in this effect in a lesser extent, as evidenced by the CB1R antagonist/inverse agonist AM281. In contrast, activation of CB1R, but not CB2R, was responsible for the protective effects of JZL184 on the 3-NP-iduced lipid peroxidation. Protective effects of JZL184 were confirmed in other toxic models involving excitotoxicity and oxidative damage as internal controls. In cortical slices, JZL184 ameliorated the 3-NP-induced loss of mitochondrial function, the increase in lipid peroxidation, and the inhibition of succinate dehydrogenase (mitochondrial complex II) activity, and these effects were independent on CB1R and CB2R, as evidenced by the lack of effects of AM281 and JTE907, respectively. Our novel results provide experimental evidence that the differential protective effects exerted by JZL184 on the early toxic effects induced by 3-NP in brain synaptosomes and cortical slices involve MAGL inhibition, and possibly the subsequent accumulation of 2-AG. These effects involve pro-energetic and redox modulatory mechanisms that may be either dependent or independent of cannabinoid receptors' activation.

Thallium-induced DNA damage, genetic, and epigenetic alterations.

Thallium (Tl) is a toxic heavy metal responsible for noxious effects in living organisms. As a pollutant, Tl can be found in the environment at high concentrations, especially in industrial areas. Systemic toxicity induced by this toxic metal can affect cell metabolism, including redox alterations, mitochondrial dysfunction, and activation of apoptotic signaling pathways. Recent focus on Tl toxicity has been devoted to the characterization of its effects at the nuclear level, with emphasis on DNA, which, in turn, may be responsible for cytogenetic damage, mutations, and epigenetic changes. In this work, we review and discuss past and recent evidence on the toxic effects of Tl at the systemic level and its effects on DNA. We also address Tl's role in cancer and its control.

Examining the Role of Histaminergic, Orexinergic, and Cannabinergic Systems in Redox Regulation in Gastric Adenocarcinoma.

Histaminergic, orexinergic, and cannabinoid systems play a role in both physiologic and oncogenic mechanisms in digestive tissues. These three systems are important mediators of tumor transformation, as they are associated with redox alterations, which are key aspects in oncological disorders. The three systems are known to promote alterations in the gastric epithelium through intracellular signaling pathways, such as oxidative phosphorylation, mitochondrial dysfunction, and increased Akt, which might promote tumorigenesis. Histamine promotes cell transformation through redox-mediated alterations in the cell cycle, DNA repair, and immunological response. The increase in histamine and oxidative stress generates angiogenic and metastatic signals through the VEGF receptor and H2R-cAMP-PKA pathway. Immunosuppression in the presence of histamine and ROS is linked to a decrease in dendritic and myeloid cells in gastric tissue. These effects are counteracted by histamine receptor antagonists, such as cimetidine. Regarding orexins, overexpression of the Orexin 1 Receptor (OX1R) induces tumor regression through the activation of MAPK-dependent caspases and src-tyrosine. OX1R agonists are candidates for the treatment of gastric cancer by stimulating apoptosis and adhesive interactions. Lastly, cannabinoid type 2 (CB2) receptor agonists increase ROS, leading to the activation of apoptotic pathways. In contrast, cannabinoid type 1 (CB1) receptor agonists decrease ROS formation and inflammation in gastric tumors exposed to cisplatin. Overall, the repercussion of ROS modulation through these three systems on tumor activity in gastric cancer depends on intracellular and/or nuclear signals associated with proliferation, metastasis, angiogenesis, and cell death. Here, we review the role of these modulatory systems and redox alterations in gastric cancer.

Amyloid Beta Peptide-Mediated Alterations in Mitochondrial Dynamics and its Implications for Alzheimer's Disease.

Alzheimer's disease (AD) is considered the most frequent neurodegenerative disorder worldwide, compromising cognitive function in patients, with an average incidence of 1-3% in the open population. Protein aggregation into amyloidogenic plaques and neurofibrillary tangles, as well as neurodegeneration in the hippocampal and cortical areas, represent the neuropathological hallmarks of this disorder. Mechanisms involved in neurodegeneration include protein misfolding, augmented apoptosis, disrupted molecular signaling pathways and axonal transport, oxidative stress, inflammation, and mitochondrial dysfunction, among others. It is precisely through a disrupted energy metabolism that neural cells trigger toxic mechanisms leading to cell death. In this regard, the study of mitochondrial dynamics constitutes a relevant topic to decipher the role of mitochondrial dysfunction in neurological disorders, especially when considering that amyloid-beta peptides can target mitochondria. Specifically, the amyloid beta (Aß) peptide, known to accumulate in the brain of AD patients, has been shown to disrupt overall mitochondrial metabolism by impairing energy production, mitochondrial redox activity, and calcium homeostasis, thus highlighting its key role in the AD pathogenesis. In this work, we review and discuss recent evidence supporting the concept that mitochondrial dysfunction mediated by amyloid peptides contributes to the development of AD.

The Endocannabinoid System in Caenorhabditis elegans.

The existence of a formal Endocannabinoid System in C. elegans has been questioned due to data showing the absence of typical cannabinoid receptors in the worm; however, the presence of a full metabolism for endocannabinoids, alternative ligands, and receptors for these agents and a considerable number of orthologous and homologous genes regulating physiological cannabinoid-like signals and responses - several of which are similar to those of mammals - demonstrates a well-structured and functional complex system in nematodes. In this review, we describe and compare similarities and differences between the Endocannabinoid System in mammals and nematodes, highlighting the basis for the integral study of this novel system in the worm.

Modulatory Activity of the Endocannabinoid System in the Development and Proliferation of Cells in the CNS.

The Endocannabinoid System (ECS, also known as Endocannabinoidome) plays a key role in the function of the Central Nervous System, though the participation of this system on the early development - specifically in neuroprotection and proliferation of nerve cells - has been poorly studied. Here, we collect and describe evidence regarding how cannabinoid receptors CB1R and CB2R regulate several cell markers related to proliferation. While CB1R participates in the modulation of neuronal and glial proliferation, CB2R is involved in the proliferation of glial cells. The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) exert significant effects on nerve cell proliferation. AEA generated during embryogenesis induces major effects on the differentiation of neuronal progenitor cells, whereas 2-AG participates in modulating cell migration events rather than affecting the neural proliferation rate. However, although the ECS has been demonstrated to participate in neuroprotection, more characterization on its role in neuronal and glial proliferation and differentiation is needed, especially in brain areas with recognized high neurogenesis rates. This has encouraged scientists to elucidate and propose specific mechanisms related with these cell proliferation mechanisms to better understand some neurodegenerative disorders such as Parkinson, Huntington and Alzheimer diseases, in which neuronal loss and poor neurogenesis are crucial factors for their onset and progression. In this review, we collect and present recent evidence published pointing to an active role of the ECS in the development and proliferation of nerve cells.

Oleamide Reduces Mitochondrial Dysfunction and Toxicity in Rat Cortical Slices Through the Combined Action of Cannabinoid Receptors Activation and Induction of Antioxidant Activity.

The potential treatment of neurodegenerative disorders requires the development of novel pharmacological strategies at the experimental level, such as the endocannabinoid-based therapies. The effects of oleamide (OEA), a fatty acid primary amide with activity on cannabinoid receptors, was tested against mitochondrial toxicity induced by the electron transport chain complex II inhibitor, 3-nitropropionic acid (3-NP), in rat cortical slices. OEA prevented the 3-NP-induced loss of mitochondrial function/cell viability at a concentration range of 5 nM-25 µM, and this protective effect was observed only when the amide was administered as pretreatment, but not as post-treatment. The preservation of mitochondrial function/cell viability induced by OEA in the toxic model induced by 3-NP was lost when the slices were pre-incubated with the cannabinoid receptor 1 (CB1R) selective inhibitor, AM281, or the cannabinoid receptor 2 (CB2R) selective inhibitor, JTE-907. The 3-NP-induced inhibition of succinate dehydrogenase (mitochondrial Complex II) activity was recovered by 25 nM OEA. The amide also prevented the increased lipid peroxidation and the changes in reduced/oxidized glutathione (GSH/GSSG) ratio induced by 3-NP. The cell damage induced by 3-NP, assessed as incorporation of cellular propidium iodide, was mitigated by OEA. Our novel findings suggest that the neuroprotective properties displayed by OEA during the early stages of damage to cortical cells involve the converging activation of CB1R and CB2R and the increase in antioxidant activity, which combined may emerge from the preservation of the functional integrity of mitochondria.

Alpha-Mangostin Alleviates the Short-term 6-Hydroxydopamine-Induced Neurotoxicity and Oxidative Damage in Rat Cortical Slices and in Caenorhabditis elegans.

The development, at the experimental level, of therapeutic strategies based on natural products to attenuate neurological alterations in degenerative disorders has gained attention. Antioxidant molecules exhibit both anti-inflammatory and neuroprotective properties. Alpha-mangostin (α-Man) is a natural xanthonoid isolated from the mangosteen tree with demonstrated antioxidant and cytoprotective properties. In this study, we investigated the antioxidant and protective properties of α-Man, both ex vivo and in vivo. We assessed the mitochondrial reductant capacity and oxidative damage to lipids in rat cortical slices, and several endpoints characteristic of physiological stress in the nematode, Caenorhabditis elegans (C. elegans), upon exposure to the parkinsonian neurotoxin, 6-hydroxydopamine (6-OHDA). In rat cortical slices, α-Man (25 and 50 µM) reduced the 6-OHDA (100 µM)-induced oxidative damage to lipid levels, but failed to reverse loss in cell viability. In wild-type (N2) C. elegans, α-Man (5-100 µM) protected against 6-OHDA (25 mM)-induced decrease in survival when administered either as pre- or post-treatment. Protective effects of α-Man were also observed on survival in the VC1772 strain (skn-1 KO-) exposed to 6-OHDA, though the extent of the protection was lesser than in the wild-type N2 strain. However, α-Man (5-50 µM) failed to attenuate the 6-OHDA-induced motor alterations in the N2 strain. The loss of lifespan induced by 6-OHDA in the N2 strain was fully reversed by high concentrations of α-Man. In addition, while 6-OHDA decreased the expression of glutathione S-transferase in the CL2166 C. elegans strain, α-Man preserved and stimulated the expression of this protein. α-Man (25 µM) also prevented 6-OHDA-induced dopaminergic neurodegeneration in the BZ555 C. elegans strain. Altogether, our novel results suggest that α-Man affords partial protection against several, but not all, short-term toxic effects induced by 6-OHDA in cortical slices and in a skn-1-dependent manner in C. elegans.

Thallium Induces Antiproliferative and Cytotoxic Activity in Glioblastoma C6 and U373 Cell Cultures via Apoptosis and Changes in Cell Cycle.

Thallium (Tl+) is a heavy metal that causes toxicity in several organs, including the brain. Its cytotoxic profile, combined with its affinity for tumor cells when used as a radioligand for labeling these cells, suggests its potential use as antitumor therapy. In this study, glioblastoma cell lines C6 (from rat) and U373 (from human) were exposed to increased concentrations of thallium(I) acetate (5, 10, 50, 100, or 200 µM) and several toxic endpoints were evaluated, including loss of confluence and morphological changes, loss of cell viability, changes in cell cycle, and apoptosis. Tl+ was detected in cells exposed to thallium(I) acetate, demonstrating efficient uptake mechanism. Confluence in both cell lines decreased in a concentration-dependent manner (50-200 µM), while morphological changes (cell shrinkage and decreased cell volume) were more evident at exposures to higher Tl+ concentrations. For both parameters, the effects of Tl+ were more prominent in C6 cells compared to U373 cells. The same trend was observed for cell viability, with Tl+ affecting this parameter in C6 cells at low concentrations, whereas U373 cells showed greater resistance, with significant changes observed only at the higher concentrations. C6 and U373 cells treated with Tl+ also showed morphological characteristics corresponding to apoptosis. The cytotoxic effects of Tl+ were also assessed in neural and astrocytic primary cultures from the whole rat brain. Primary neural and astrocytic cultures were less sensitive than C6 and U373 cells, showing changes in cell viability at 50 and 100 µM concentrations, respectively. Cell cycle in both brain tumor cell lines was altered by Tl+ in G1/G2 and S phases. In addition, when combined with temozolamide (500 µM), Tl+ elicited cell cycle alterations, increasing SubG1 population. Combined, our novel results characterize and validate the cytotoxic and antiproliferative effects of Tl+ in glioblastoma cells.

Chemical structure of three basic Asp-49 phospholipases A2 isolated from Crotalus molossus nigrescens venom with cytotoxic activity against cancer cells.

Snake venoms are complex mixtures of molecules with several biological activities. Among these molecules, the enzymes with phospholipase A2 activity have been extensively studied in the venoms from snakes because of their importance in the envenomation process and symptoms. The Mexican rattlesnake Crotalus molossus nigrescens is widely distributed in the Mexican plateau. Unlike other crotalids, its venom components have been poorly studied. Here, we characterized the phospholipase activity of one fraction isolated from the venom of this snake and we determined the cytotoxic and neurotoxic effects on brain tumor cells and neuronal primary cultures, respectively. After reverse phase chromatography, we obtained a fraction which was analyzed by mass spectrometry showing higher activity than that from a PLA2 from bee venom used as control. This fraction was enriched with three basic Asp49 phospholipases with molecular masses of 12.5, 13.9 and 14.2 kDa. Their complete amino acid sequences were determined, and their predicted tertiary structures were generated using the model building softwares I-tasser and Chimera. Viability assays revealed that the fraction showed cytotoxic activity against brain tumor cells (C6, RG2 and Daoy) with IC50 values ranging between 10 and 100 ng/ml, whereas an IC50 > 100 ng/ml was exerted in rat primary astrocytes. These findings might be relevant in oncological medicine due to their potential as anticancer agents and low neurotoxic effects compared to conventional drugs.

An Update on the Critical Role of α-Synuclein in Parkinson's Disease and Other Synucleinopathies: from Tissue to Cellular and Molecular Levels.

The aggregation of alpha-synuclein (α-Syn) plays a critical role in the development of Parkinson's disease (PD) and other synucleinopathies. α-Syn, which is encoded by the SNCA gene, is a lysine-rich soluble amphipathic protein normally expressed in neurons. Located in the cytosolic domain, this protein has the ability to remodel itself in plasma membranes, where it assumes an alpha-helix conformation. However, the protein can also adopt another conformation rich in cross-beta sheets, undergoing mutations and post-translational modifications, then leading the protein to an unusual aggregation in the form of Lewy bodies (LB), which are cytoplasmic inclusions constituted predominantly by α-Syn. Pathogenic mechanisms affecting the structural and functional stability of α-Syn - such as endoplasmic reticulum stress, Golgi complex fragmentation, disfunctional protein degradation systems, aberrant interactions with mitochondrial membranes and nuclear DNA, altered cytoskeleton dynamics, disrupted neuronal plasmatic membrane, dysfunctional vesicular transport, and formation of extracellular toxic aggregates - contribute all to the pathogenic progression of PD and synucleinopathies. In this review, we describe the collective knowledge on this topic and provide an update on the critical role of α-Syn aggregates, both at the cellular and molecular levels, in the deregulation of organelles affecting the cellular homeostasis and leading to neuronal cell death in PD and other synucleinopathies.

On the Biomedical Properties of Endocannabinoid Degradation and Reuptake Inhibitors: Pre-clinical and Clinical Evidence.

The endocannabinoid system (ECS) is composed of endogenous cannabinoids; components involved in their synthesis, transport, and degradation; and an expansive variety of cannabinoid receptors. Hypofunction or deregulation of the ECS is related to pathological conditions. Consequently, endogenous enhancement of endocannabinoid levels and/or regulation of their metabolism represent promising therapeutic approaches. Several major strategies have been suggested for the modulation of the ECS: (1) blocking endocannabinoids degradation, (2) inhibition of endocannabinoid cellular uptake, and (3) pharmacological modulation of cannabinoid receptors as potential therapeutic targets. Here, we focused in this review on degradation/reuptake inhibitors over cannabinoid receptor modulators in order to provide an updated synopsis of contemporary evidence advancing mechanisms of endocannabinoids as pharmacological tools with therapeutic properties for the treatment of several disorders. For this purpose, we revisited the available literature and reported the latest advances regarding the biomedical properties of fatty acid amide hydrolase and monoacylglycerol lipase inhibitors in pre-clinical and clinical studies. We also highlighted anandamide and 2-arachidonoylglycerol reuptake inhibitors with promising results in pre-clinical studies using in vitro and animal models as an outlook for future research in clinical trials.

URB597 Prevents the Short-Term Excitotoxic Cell Damage in Rat Cortical Slices: Role of Cannabinoid 1 Receptors.

Endocannabinoid-based therapies constitute an emerging tool for the potential treatment of neurodegenerative disorders, requiring characterization at the experimental level. The effects of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH), were tested against the quinolinic acid (QUIN)-induced early toxic effects in rat cortical slices, and compared with those effects exerted by the endocannabinoid anandamide (AEA). URB597 prevented the QUIN-induced loss of mitochondrial function/cell viability and lipid peroxidation, while reduced necrosis, and to a lesser extent, apoptosis. The protective effects of URB597 were mediated by activation of cannabinoid receptor 1 (CB1r), as evidenced by their inhibition by the selective CB1r antagonist AM281. Similar effects were observed when testing AEA against QUIN toxicity. Our findings demonstrate the neuroprotective properties of URB597 during the early stages of excitotoxic damage to cortical tissue, suggesting that these properties are mediated by FAAH inhibition, and might be linked to the protective effects of AEA, or the combination of endocannabinoids.

Oleamide Induces Cell Death in Glioblastoma RG2 Cells by a Cannabinoid Receptor-Independent Mechanism.

The endocannabinoid system has been associated with antiproliferative effects in several types of tumors through cannabinoid receptor-mediated cell death mechanisms. Oleamide (ODA) is a CB1/CB2 agonist associated with cell growth and migration by adhesion and/or ionic signals associated with Gap junctions. Antiproliferative mechanisms related to ODA remain unknown. In this work, we evaluated the effects of ODA on cell viability and morphological changes in a rat RG2 glioblastoma cell line and compared these effects with primary astrocyte cultures from 8-day postnatal rats. RG2 and primary astrocyte cultures were treated with ODA at increasing concentrations (25, 50, 100, and 200 µM) for different periods of time (12, 24, and 48 h). Changes in RG2 cell viability and morphology induced by ODA were assessed by viability/mitochondrial activity test and phase contrast microscopy, respectively. The ratios of necrotic and apoptotic cell death, and cell cycle alterations, were evaluated by flow cytometry. The roles of CB1 and CB2 receptors on ODA-induced changes were explored with specific receptor antagonists. ODA (100 µM) induced somatic damage, detachment of somatic bodies, cytoplasmic polarization, and somatic shrinkage in RG2 cells at 24 and 48 h. In contrast, primary astrocytes treated at the same ODA concentrations exhibited cell aggregation but not cell damage. ODA (100 µM) increased apoptotic cell death and cell arrest in the G1 phase at 24 h in the RG2 line. The effects induced by ODA on cell viability of RG2 cells were independent of CB1 and CB2 receptors or changes in intracellular calcium transient. Results of this novel study suggest that ODA exerts specific antiproliferative effects on RG2 glioblastoma cells through unconventional apoptotic mechanisms not involving canonical signals.