RESUMO
The side effects of cancer therapy continue to cause significant health and cost burden to the patient, their friends and family, and governments. A major barrier in the way in which these side effects are managed is the highly siloed mentality that results in a fragmented approach to symptom control. Increasingly, it is appreciated that many symptoms are manifestations of common underlying pathobiology, with changes in the gastrointestinal environment a key driver for many symptom sequelae. Breakdown of the mucosal barrier (mucositis) is a common and early side effect of many anti-cancer agents, known to contribute (in part) to a range of highly burdensome symptoms such as diarrhoea, nausea, vomiting, infection, malnutrition, fatigue, depression, and insomnia. Here, we outline a rationale for how, based on its already documented effects on the gastrointestinal microenvironment, medicinal cannabis could be used to control mucositis and prevent the constellation of symptoms with which it is associated. We will provide a brief update on the current state of evidence on medicinal cannabis in cancer care and outline the potential benefits (and challenges) of using medicinal cannabis during active cancer therapy.
Assuntos
Maconha Medicinal , Mucosite , Neoplasias , Humanos , Maconha Medicinal/efeitos adversos , Mucosite/tratamento farmacológico , Neoplasias/tratamento farmacológico , Náusea/induzido quimicamente , Náusea/tratamento farmacológico , Vômito , Microambiente TumoralRESUMO
White tea (WT) is one of six tea types originally derived from Fujian Province, China. White tea is known for its health-promoting properties. However, the neuroprotective and anti-aggregatory properties of WT against the hallmark toxic Alzheimer's protein, Aß have not been investigated. In this study, WT, green tea (GT), oolong tea (OT) and black tea (BT) were manufactured using tea leaves from the cultivar Camellia sinensis (Jin Guanyin). The protective effects of these tea extracts were then studied under oxidative stress conditions via t-bhp and H2O2 exposure, in addition to Aß treatment using a PC-12 cell model. Each tea type failed to rescue PC-12 cells from either t-bhp or H2O2-mediated toxicity, however each extract exerted significant protection against Aß-evoked neurotoxicity. Results of the Thioflavin T Kinetic (ThT) and TEM assay showed that Aß aggregate formation was inhibited by each tea type. Additionally, TEM also supported the different anti-aggregatory effect of WT by modifying Aß into an amorphous and punctate aggregate morphology. Higher accumulated precedent or potential neuroprotective compounds in WT, including ECG''3Me, 8-C-ascorbyl-EGCG, GABA and Gln, in addition to flavonol or flavone glycosides detected by using UPLC-QTOF-MS and UPLC-QqQ-MS, may contribute to a favourable anti-aggregative and neuroprotective effect of WT against Aß.
Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/química , Extratos Vegetais/química , Chá/química , Camellia sinensis/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Peróxido de Hidrogênio/química , Cinética , Neurônios/citologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Folhas de Planta/química , Análise de Componente Principal/métodos , Espectrometria de Massas em Tandem/métodos , Chá/parasitologiaRESUMO
OBJECTIVE: Little is understood regarding how disease progression alters immune and sensory nerve function in colitis. We investigated how acute colitis chronically alters immune recruitment and the impact this has on re-activated colitis. To understand the impact of disease progress on sensory systems we investigated the mechanisms underlying altered colonic neuro-immune interactions after acute colitis. DESIGN: Inflammation was compared in mouse models of health, acute tri-nitrobenzene sulphonic acid (TNBS) colitis, Remission and Reactivated colitis. Cytokine concentrations were compared by ELISA in-situ and in explanted colon tissue. Colonic infiltration by CD11b/F4-80 macrophage, CD4 THELPER (TH) and CD8 TCYTOTOXIC (TC) and α4ß7 expression on mesenteric lymph node (MLN) TH and TC was determined by flow cytometry. Cytokine and effector receptor mRNA expression was determined on colo-rectal afferent neurons and the mechanisms underlying cytokinergic effects on high-threshold colo-rectal afferent function were investigated using electrophysiology. RESULTS: Colonic damage, MPO activity, macrophage infiltration, IL-1ß and IL-6 concentrations were lower in Reactivated compared to Acute colitis. TH infiltration and α4ß7 expression on TH MLN was increased in Remission but not Acute colitis. IFN-γ concentrations, TH infiltration and α4ß7 expression on TH and TC MLN increased in Reactivated compared to Acute colitis. Reactivated explants secreted more IL-1ß and IL-6 than Acute explants. IL-6 and TNF-α inhibited colo-rectal afferent mechanosensitivity in Remission mice via a BKCa dependent mechanism. CONCLUSIONS: Acute colitis persistently alters immune responses and afferent nerve signalling pathways to successive episodes of colitis. These findings highlight the complexity of viscero-sensory neuro-immune interactions in painful remitting and relapsing diseases.
Assuntos
Colite/imunologia , Colo/metabolismo , Inflamação/imunologia , Neurônios Aferentes/imunologia , Doença Aguda , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Naturally-occurring flavonoids have well documented anti-aggregatory and neuroprotective properties against the hallmark toxic protein in Alzheimer's disease, amyloid ß (Aß). However the extensive diversity of flavonoids has limited the insight into the precise structure-activity relationships that confer such bioactive properties against the Aß protein. In the present study we have characterised the Aß binding properties, anti-aggregatory and neuroprotective effects of a discreet set of flavones, including the recently described novel protein sumoylation inhibitor 2',3',4'-trihydroxyflavone (2-D08). Quercetin, transilitin, jaceosidin, nobiletin and 2-D08 were incubated with human Aß1-42 for 48h in vitro and effects on Aß fibrillisation kinetics and morphology measured using Thioflavin T (ThT) and electron microscopy respectively, in addition to effects on neuronal PC12 cell viability. Of the flavones studied, only quercetin, transilitin and 2-D08 significantly inhibited Aß1-42 aggregation and toxicity in PC12 cells. Of those, 2-D08 was the most effective inhibitor. The strong anti-amyloid activity of 2-D08 indicates that extensive hydroxylation in the B ring is the most important determinant of activity against ß amyloid within the flavone scaffold. The lack of efficacy of jaceosidin and nobiletin indicate that extension of B ring hydroxylation with methoxyl groups result in an incremental loss of anti-fibrillar and neuroprotective activity, highlighting the constraint to vicinal hydroxyl groups in the B ring for effective inhibition of aggregation. These findings reveal further structural insights into anti-amyloid bioactivity of flavonoids in addition to a novel and efficacious anti-aggregatory and neuroprotective effect of the semi-synthetic flavone and sumoylation inhibitor 2',3',4'-trihydroxyflavone (2-D08). Such modified flavones may facilitate drug development targeting multiple pathways in neurodegenerative disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Flavonas/química , Fármacos Neuroprotetores/química , Fragmentos de Peptídeos/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Benzotiazóis , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Flavonas/metabolismo , Flavonas/farmacologia , Flavonoides/química , Flavonoides/metabolismo , Flavonoides/farmacologia , Humanos , Ligação de Hidrogênio , Microscopia Eletrônica de Transmissão , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Células PC12 , Fragmentos de Peptídeos/antagonistas & inibidores , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/metabolismoRESUMO
Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against ß amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter ß amyloid (Aß) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aß1-42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ(9)-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aß1-42. Aß1-42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aß-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aß fibrils and aggregates, there was no clear correlation between effects on Aß morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Canabinoides/farmacologia , Microglia/metabolismo , Neurônios/metabolismo , Estrutura Quaternária de Proteína/efeitos dos fármacos , Albuminas/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologiaRESUMO
BACKGROUND: Cannabis sativa L. (C. sativa) can efficiently synthesize of over 200 terpenes, including monoterpenes, sesquiterpenes and triterpenes that may contribute to the known biological activities of phytocannabinoids of relevance for the burgeoning access to medicinal cannabis formulations globally; however, to date have been uncharacterized. We assessed twelve predominant terpenes in C. sativa for neuroprotective and anti-aggregative properties in semi-differentiated PC12 neuronal cell line that is robust and validated as a cell model responsive to amyloid ß (Aß1-42) protein exposure and oxidative stress. METHODS: Cell viability was assessed biochemically using the MTT assay in the presence of myrcene, ß-caryophyllene, terpinolene, limonene, linalool, humulene, α-pinene, nerolidol, ß-pinene, terpineol, citronellol and friedelin (1-200 µM) for 24 hr. Sub-toxic threshold test concentrations of each terpene were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-BHP; 0-250 µM) or amyloid ß (Aß1-42; 0-1 µM) to assess neuroprotective effects. Direct effects of each terpene on Aß fibril formation and aggregation were also evaluated using the Thioflavin T (ThT) fluorometric kinetic assay and transmission electron microscopy (TEM) to visualize fibril and aggregate morphology. RESULTS: Terpenes were intrinsically benign to PC12 cells up to 50 µM, with higher concentrations of ß-caryophyllene, humulene and nerolidol inducing some loss of PC12 cell viability. No significant protective effects of terpenes were observed following t-BHP (0-200 µM) administration, with some enhanced toxicity instead demonstrated from both ß-caryophyllene and humulene treatment (each at 50 µM). α-pinene and ß-pinene demonstrated a significant neuroprotective effect against amyloid ß exposure. α-pinene, ß-pinene, terpineol, terpinolene and friedelin were associated with a variable inhibition of Aß1-42 fibril and aggregate density. CONCLUSIONS: The outcomes of this study underline a neuroprotective role of α-pinene and ß-pinene against Aß-mediated neurotoxicity associated with an inhibition of Aß1-42 fibrilization and density. This demonstrates the bioactive potential of selected terpenes for consideration in the development of medicinal cannabis formulations targeting neurodegenerative diseases.
Assuntos
Monoterpenos Bicíclicos , Cannabis , Monoterpenos Cicloexânicos , Alucinógenos , Lupanos , Maconha Medicinal , Sesquiterpenos Monocíclicos , Fármacos Neuroprotetores , Sesquiterpenos Policíclicos , Sesquiterpenos , Animais , Ratos , Terpenos/farmacologia , Terpenos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Neuroproteção , Maconha Medicinal/farmacologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/químicaRESUMO
Irinotecan use is linked to the development of gastrointestinal toxicity and inflammation, or gastrointestinal mucositis. Selected phytocannabinoids have been ascribed anti-inflammatory effects in models of gastrointestinal inflammation, associated with maintaining epithelial barrier function. We characterised the mucoprotective capacity of the phytocannabinoids: cannabidiol, cannabigerol, cannabichromene and cannabidivarin in a cell-based model of intestinal epithelial stress occurring in mucositis. Transepithelial electrical resistance (TEER) was measured to determine changes in epithelial permeability in the presence of SN-38 (5 µM) or the pro-inflammatory cytokines TNFα and IL-1ß (each at 100 ng/mL), alone or with concomitant treatment with each of the phytocannabinoids (1 µM). The DCFDA assay was used to determine the ROS-scavenging ability of each phytocannabinoid following treatment with the lipid peroxidant tbhp (200 µM). Each phytocannabinoid provided significant protection against cytokine-evoked increases in epithelial permeability. Cannabidiol, cannabidivarin and cannabigerol were also able to significantly inhibit SN-38-evoked increases in permeability. None of the tested phytocannabinoids inhibited tbhp-induced ROS generation. These results highlight a novel role for cannabidiol, cannabidivarin and cannabigerol as inhibitors of SN-38-evoked increases in epithelial permeability and support the rationale for the further development of novel phytocannabinoids as supportive therapeutics in the management of irinotecan-associated mucositis.
Assuntos
Canabidiol , Canabinoides , Mucosa Intestinal , Irinotecano , Espécies Reativas de Oxigênio , Humanos , Células CACO-2 , Canabidiol/farmacologia , Canabinoides/farmacologia , Citocinas/metabolismo , Interleucina-1beta/metabolismo , Função da Barreira Intestinal , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Irinotecano/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Phytochemicals are often promoted generally as antioxidants and demonstrate variable levels of reactive oxygen species (ROS) sequestration in vitro, which attributes to their neuroprotective bioactivity. Sesquiterpenes from cannabis and essential oils may demonstrate bifunctional properties towards cellular oxidative stress, possessing pro-oxidant activities by generating ROS or scavenging ROS directly. Sesquiterpenes can also oxidize forming sesquiterpene oxides, however the relative contribution they make to the bioactivity or cytotoxicity of complex botanical extracts more generally is unclear, while selected cannabis-prevalent terpenes such as ß-caryophyllene may also activate cannabinoid receptors as part of their biological activity. In the present study, we investigated selected sesquiterpenes ß-caryophyllene and humulene and their oxidized forms (ß-caryophyllene oxide and zerumbone, respectively) against established antioxidants (ascorbic acid, α-tocopherol, and glutathione) and in the presence of cannabinoid receptor 1 and cannabinoid receptor 2 antagonists, to gain a better understanding of the molecular and cellular mechanisms of neuroprotection versus neurotoxicity in semi-differentiated rat neuronal phaeochromocytoma (PC12) cells. Our results demonstrate that the sesquiterpenes ß-caryophyllene, humulene and zerumbone possess concentration-dependent neurotoxic effects in PC12 cells. Both ß-caryophyllene- and humulene-evoked toxicity was unaffected by CB1 or CB2 receptor antagonism, demonstrating this occurred independently of cannabinoid receptors. Both glutathione and α-tocopherol were variably able to alleviate the concentration-dependent loss of PC12 cell viability from exposure to ß-caryophyllene, humulene and zerumbone. During 4-hour exposure to sesquiterpenes only modest increases in ROS levels were noted in PC12 cells, with glutathione co-incubation significantly inhibiting intracellular ROS production. However, significant increases in ROS levels in PC12 cells were demonstrated during 24-hour incubation with either antioxidants or sesquiterpenes individually, and with additive toxicity exhibited in combination. Overall, the results highlight a concentration-dependent profile of sesquiterpene neurotoxicity independent of cannabinoid receptors and dissociated from the formation of reactive oxygen species as a marker or correlate to the loss of cell viability.
RESUMO
BACKGROUND: Phytocannabinoids (pCBs) have been shown to inhibit the aggregation and neurotoxicity of the neurotoxic Alzheimer's disease protein beta amyloid (Aß). We characterized the capacity of six pCBs-cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidiol (CBD) and Δ9 -tetrahydrocannabinol (Δ9 -THC)-to disrupt Aß aggregation and protect against Aß-evoked neurotoxicity in PC12 cells. METHODS: Neuroprotection against lipid peroxidation and Aß-induced cytotoxicity was assessed using the MTT assay. Transmission electron microscopy was used to visualize pCB effects on Aß aggregation and fluorescence microscopy, with morphometrics and principal component analysis to assess PC12 cell morphology. RESULTS: CBD inhibited lipid peroxidation with no significant effect on Aß toxicity, whilst CBN, CBDV and CBG provided neuroprotection. CBC, CBG and CBN inhibited Aß1-42 -induced neurotoxicity in PC12 cells, as did Δ9 -THC, CBD and CBDV. CBC, CBN and CBDV inhibited Aß aggregation, whilst Δ9 -THC reduced aggregate density. Aß1-42 induced morphological changes in PC12 cells, including a reduction in neuritic projections and rounded cell morphology. CBC and CBG inhibited this effect, whilst Δ9 -THC, CBD and CBDV did not alter Aß1-42 effects on cell morphology. CONCLUSIONS: These findings highlight the neuroprotective activity of CBC, CBG and CBN as novel pCBs associated with variable effects on Aß-evoked neurite damage and inhibition of amyloid ß aggregation.
Assuntos
Canabidiol , Canabinoides , Síndromes Neurotóxicas , Ratos , Animais , Canabinol , Peptídeos beta-Amiloides/toxicidade , Células PC12 , Canabidiol/farmacologia , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/prevenção & controle , Dronabinol/farmacologiaRESUMO
Endocannabinoids are protective in animal colitis models. As endocannabinoids also form novel prostaglandin ethanolamides (prostamides) via COX-2, we investigated the effects of prostamides and other COX-2 mediators on tissue damage in an ex vivo human mucosal explant colitis model. Healthy human colonic mucosae were incubated with pro-inflammatory cytokines TNF-α and IL-1ß to elicit colitis-like tissue damage. The PGF-ethanolamide analogue, bimatoprost decreased colitis scores which were reversed by a prostamide-specific antagonist AGN 211334, but not the FP receptor antagonist AL-8810. PGF-ethanolamide and PGE-ethanolamide also reduced cytokine-evoked epithelial damage. Anandamide was protective in the explant colitis model; however COX-2 inhibition did not alter its effects, associated with a lack of COX-2 induction in explant mucosal tissue. These findings support an anti-inflammatory role for prostamides and endocannabinoids in the human colon.
Assuntos
Colite/prevenção & controle , Colo Sigmoide/efeitos dos fármacos , Dinoprostona/análogos & derivados , Adulto , Amidas/farmacologia , Ácidos Araquidônicos/farmacologia , Bimatoprost , Cloprostenol/análogos & derivados , Cloprostenol/farmacologia , Colite/metabolismo , Colo Sigmoide/metabolismo , Colo Sigmoide/patologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprosta/análogos & derivados , Dinoprosta/farmacologia , Dinoprostona/farmacologia , Endocanabinoides/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Interleucina-1beta/farmacologia , Masculino , Pessoa de Meia-Idade , Oxazóis/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/metabolismo , Sulfonamidas/farmacologia , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
BACKGROUND: Neurodegenerative diseases and dementia pose a global health challenge in an aging population, exemplified by the increasing incidence and prevalence of its most common form, Alzheimer's disease. Although several approved treatments exist for Alzheimer's disease, they only afford transient symptomatic improvements and are not considered disease-modifying. The psychoactive properties of Cannabis sativa L. have been recognized for thousands of years and now with burgeoning access to medicinal formulations globally, research has turned to re-evaluate cannabis and its myriad phytochemicals as a potential treatment and adjunctive agent for neurodegenerative diseases. PURPOSE: This review evaluated the neuroprotective potential of C. sativa's active constituents for potential therapeutic use in dementia and Alzheimer's disease, based on published studies demonstrating efficacy in experimental preclinical settings associated with neurodegeneration. STUDY DESIGN: Relevant information on the neuroprotective potential of the C. sativa's phytoconstituents in preclinical studies (in vitro, in vivo) were included. The collated information on C. sativa's component bioactivity was organized for therapeutic applications against neurodegenerative diseases. METHODS: The therapeutic use of C. sativa related to Alzheimer's disease relative to known phytocannabinoids and other phytochemical constituents were derived from online databases, including PubMed, Elsevier, The Plant List (TPL, www.theplantlist.org), Science Direct, as well as relevant information on the known pharmacological actions of the listed phytochemicals. RESULTS: Numerous C. sativa -prevalent phytochemicals were evidenced in the body of literature as having efficacy in the treatment of neurodegenerative conditions exemplified by Alzheimer's disease. Several phytocannabinoids, terpenes and select flavonoids demonstrated neuroprotection through a myriad of cellular and molecular pathways, including cannabinoid receptor-mediated, antioxidant and direct anti-aggregatory actions against the pathological toxic hallmark protein in Alzheimer's disease, amyloid ß. CONCLUSIONS: These findings provide strong evidence for a role of cannabis constituents, individually or in combination, as potential neuroprotectants timely to the emergent use of medicinal cannabis as a novel treatment for neurodegenerative diseases. Future randomized and controlled clinical studies are required to substantiate the bioactivities of phytocannabinoids and terpenes and their likely synergies.
Assuntos
Doença de Alzheimer , Cannabis , Maconha Medicinal , Fármacos Neuroprotetores , Idoso , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Antioxidantes , Cannabis/química , Flavonoides/química , Humanos , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Receptores de Canabinoides , Terpenos/farmacologiaRESUMO
The chemotherapeutic drug irinotecan and its active metabolite SN-38 have been linked to the development of off-target gastrointestinal toxicity and inflammation, termed gastrointestinal mucositis (GIM). Flavonoids possess antioxidant and anti-inflammatory effects in models of gastrointestinal inflammation; however, few studies have investigated their potential in ameliorating chemotherapy-induced GIM. Here, we characterised the intestinal epithelial barrier-protective and antioxidant capacity of the novel flavonoids 2',3',4'-trihydroxyflavone (2-D08) and transilitin in comparison with flavones myricetin and quercetin in vitro via viability and permeability assessments in Caco-2 epithelial monolayers exposed to 7-ethyl-10-hydroxycamptothecin (SN-38). Transilitin, 2-D08 and myricetin maintained barrier function in the presence of SN-38, with 2-D08 proving most effective. 2-D08 was the most effective inhibitor of cytokine-evoked increases in epithelial permeability, with myricetin providing modest protection; quercetin afforded no significant protection against either SN-38 or cytokine-evoked reductions in barrier integrity. Each flavonoid significantly reduced tert-butyl hydroperoxide (tbhp)-induced reactive oxygen species (ROS) generation, although 2-D08 was comparatively less effective. These results highlight a novel role for 2-D08 as an inhibitor of both SN-38 and cytokine-evoked increases in epithelial permeability, with lesser barrier protective roles ascribed to transilitin and myricetin and not correlated with antioxidant capacity. Such novel flavonoids as 2-D08 may have active or adjunctive roles in ameliorating chemotherapy and inflammation-evoked changes in intestinal barrier function.
Assuntos
Mucosite , Humanos , Irinotecano , Mucosite/induzido quimicamente , Mucosite/tratamento farmacológico , Citocinas , Células CACO-2 , Quercetina/farmacologia , Antioxidantes/farmacologia , Flavonoides/farmacologia , Permeabilidade , Inflamação , Mucosa IntestinalRESUMO
BACKGROUND: Terpenes form a diverse class of naturally occurring chemicals ascribed various biological activities. Cannabis contains over 400 different terpenes of varying chemical complexity which may add to the known biological activities of phytocannabinoids of relevance to the increasing use of medical cannabis; however, to date have been incompletely characterized. We assessed three terpenes predominant in cannabis: α-bisabolol, myrcene and ß-caryophyllene for neuroprotective and anti-aggregative properties in both undifferentiated and differentiated NSC-34 motorneuronal-like cells as a sensitive model for neurotoxicity to oxidative stress and amyloid ß (Aß1-42) protein exposure. METHODS: Cell viability was assessed biochemically using the MTT assay in the presence of either α-bisabolol, myrcene and ß-caryophyllene (1-1000 µM) for 48 hr. Sub-toxic threshold test concentrations of each terpene were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-BHP) or amyloid ß (Aß1-42; 0-1 µM) to assess neuroprotective effects. Direct effects of each terpene on Aß fibril formation and aggregation were also evaluated using the Thioflavin T (ThT) fluorometric kinetic assay, circular dichroism and transmission electron microscopy (TEM) to visualise fibril and aggregate morphology. RESULTS: Terpenes were intrinsically benign to NSC-34 cells up to 100 µM. No significant antioxidant effects were observed following t-BHP administration with myrcene and ß-caryophyllene, however α-bisabolol provided a modest but significant increase in cell viability in undifferentiated cells. α-bisabolol also demonstrated a significant neuroprotective effect against amyloid ß exposure, with ß-caryophyllene also providing a lesser, but significant increase in cell viability. Protective effects of terpenes were more pronounced in undifferentiated versus differentiated cells, attributable more so to an attenuated loss of cell viability in response to Aß1-42 following NSC-34 cell differentiation. Neuroprotection was associated with a direct inhibition of Aß1-42 fibril and aggregate density, evidenced by both attenuated ThT fluorescence kinetics and both spectral and microscopic evidence of altered and diminished density of Aß aggregates. While myrcene and ß-caryophyllene also elicited reductions in ThT fluorescence and alterations in Aß aggregation, these were less well associated with neuroprotective capacity. CONCLUSIONS: These findings highlight a neuroprotective role of α-bisabolol against Aß-mediated neurotoxicity associated with an inhibition of Aß fibrillization and modest antioxidant effect against lipid peroxidation, while ß-caryophyllene also provided a small but significant measure of protection to Aß-mediated neurotoxicity. Anti-aggregatory effects were not directly correlated with neuroprotective efficacy. This demonstrates that bioactivity of selected terpenes should be a consideration in the emergent use of medicinal cannabis formulations for the treatment of neurodegenerative diseases.
Assuntos
Cannabis , Alucinógenos , Fármacos Neuroprotetores , Síndromes Neurotóxicas , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Sesquiterpenos Monocíclicos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/prevenção & controle , Células PC12 , Fragmentos de Peptídeos/toxicidade , Ratos , Terpenos/toxicidadeRESUMO
Nutlin-3a is a p53 activator and potential cyclotherapy approach that may also mitigate side effects of chemotherapeutic drugs in the treatment of colorectal cancer. We investigated cell proliferation in a panel of colorectal cancer (CRC) cell lines with wild-type or mutant p53, as well as a non-tumorigenic fetal intestinal cell line following Nutlin-3a treatment (10 µM). We then assessed apoptosis at 24 and 48 h following administration of the active irinotecan metabolite, SN-38 (0.001 µM - 1 µM), alone or following pre-treatment with Nutlin-3a (10 µM). Nutlin-3a treatment (10 µM) significantly reduced proliferation in wild-type p53 expressing cell lines (FHS 74 and HCT116+/+) at 72 and 96 h, but was without effect in cell lines with mutated or deleted p53 (Caco-2, SW480, and HCT 116-/-). SN-38 treatment induced significant apoptosis in all cell lines after 48 h. Nutlin-3a unexpectedly increased cell death in the p53 wild-type CRC cell line, HCT116+/+, while Nutlin-3a pre-treatment provided protection from SN-38 in the p53 wild-type normal cell line, FHs 74. These results demonstrate Nutlin-3a's selective growth-arresting efficacy in p53 wild-type non-malignant intestinal cell lines, enabling the selective targeting of malignant cells with chemotherapy drugs. These studies highlight the potential of Nutlin-3a to minimise intestinal mucosal damage following chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Imidazóis/farmacologia , Irinotecano/farmacologia , Piperazinas/farmacologia , Proteína Supressora de Tumor p53 , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colo/citologia , Neoplasias Colorretais/metabolismo , Células Epiteliais/metabolismo , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Brown seaweed (Phaeophyceae) polyphenolics such as phlorotannins are ascribed various biological activities, including neuroprotection. Of these seaweeds, Ecklonia radiata (E. radiata) is found abundantly along South Australian coastal regions; however it has not been explored for various biological activities relative to any component phlorotannins previously ascribed neuroprotective capacity. In the present study, we evaluated neuroprotective activity against the neurotoxic amyloid ß protein (Aß1-42) of an ethanol extract of E. radiata compared with various additional solvent-solubilised fractions in a neuronal PC-12 cell line. The ethyl acetate fraction comprising 62% phlorotannins demonstrated the most efficacious neuroprotective activity, inhibiting neurotoxicity at all Aß1-42 concentrations. In addition, this fraction demonstrated a significant reduction in Aß aggregate density, but did not alter overall aggregate morphology. Centrifugal partitioning chromatography was used to isolate the major component, eckol, in high yield and liquid chromatography-mass spectrometry was used to characterize the major components of the ethyl acetate fraction. Our results demonstrate that the prevalence of eckol-type phlorotannins are associated with neuroprotective bioactivity of E. radiata, suggestive of potential nutraceutical and biopharmaceutical uses of this brown seaweed phlorotannin in dementia.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Dioxinas/farmacologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Phaeophyceae/química , Extratos Vegetais/farmacologia , Peptídeos beta-Amiloides/toxicidade , Animais , Austrália , Produtos Biológicos/química , Sobrevivência Celular/efeitos dos fármacos , Humanos , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/tratamento farmacológico , Células PC12 , Fragmentos de Peptídeos/toxicidade , Extratos Vegetais/química , Ratos , Alga Marinha/química , Taninos/farmacologiaRESUMO
We investigated the contribution of cyclo-oxygenase enzyme inhibition and prostamide agonism on human colonic contractility in vitro. The effects of the non-specific COX inhibitor diclofenac were compared against selective COX-2 inhibition via nimesulide, the prostanoid EP(1) receptor antagonist SC19220 or the prostaglandin prodrug/prostamide receptor agonist bimatoprost, on potency of contraction to acetylcholine in human colonic circular and longitudinal muscle strips. Pre-treatment with either nimesulide (10(-5)M) or diclofenac (10(-6)M) caused a significant decrease in the potency of acetylcholine-evoked longitudinal muscle contraction, but did not inhibit acetylcholine-evoked circular muscle contraction. Pre-treatment with the EP(1) receptor antagonist SC19220 (10(-5)M) similarly decreased cholinergic potency in longitudinal muscle, without influence on circular muscle contraction. The prostamide agonist bimatoprost (10(-6)M) increased basal circular and longitudinal muscle tone, but did not alter cholinergic potency in either muscle layer. In conclusion, colonic longitudinal muscle contraction is augmented by COX-2 activity, most likely via PGE(2) acting at EP(1) receptors. While colonic contraction is tonically modulated by bimatoprost, it does not share the same functional properties attributed to other endogenous COX-2 metabolites on colonic contractile function.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Receptores de Prostaglandina E/antagonistas & inibidores , Acetilcolina/farmacologia , Amidas/farmacologia , Bimatoprost , Cloprostenol/análogos & derivados , Cloprostenol/farmacologia , Diclofenaco/farmacologia , Humanos , Técnicas In Vitro , Receptores de Prostaglandina E Subtipo EP1 , Sulfonamidas/farmacologiaRESUMO
We investigated the effects of the small molecule flavorants diacetyl, 2,3-pentanedione and acetoin on neuronal cell viability and ß amyloid aggregation and morphology. Two neuroblastoma cell lines, SH-SY5Y and Neuro 2a (N2a) were exposed to diacetyl, 2,3-pentanedione and acetoin, while Thioflavin T fluorescence kinetics and transmission electron microscopy were used to assess effects on Aß1-42 fibril and aggregate formation and morphology respectively. Diacetyl was intrinsically toxic to both SH-SY5Y and N2a cells, with time and concentration-dependent reductions in cell viability occurring over 24 h and 48 h incubation periods. 2.3-Pentanedione evoked a similar concentration-dependent loss of cell viability in N2a cells at 48 h, but exhibited lessened toxicity in SH-SY5Y cells over 24 h, and minimal loss of cell viability by 48 h. Diacetyl inhibited Aß1-42 aggregation kinetics, reduced aggregate and fibril density and rendered Aß1-42 into amorphous small aggregates. 2,3-Pentanedione also reduced overall aggregate formation, but to a lesser extent than diacetyl and retaining the presence of a meshwork of Aß1-42 aggregates and fibrils. Acetoin was innocuous to neuronal cells and did not alter Aß1-42 fibril density or morphology. These findings highlight the intrinsic neurotoxicity of small molecule diketone flavorants. While providing further insight into their molecular interactions with amyloidogenic proteins, the neurotoxicity of such flavorants is a significant finding and warrants further investigation.
Assuntos
Acetoína/toxicidade , Peptídeos beta-Amiloides/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Aromatizantes/toxicidade , Neuroblastoma/induzido quimicamente , Síndromes Neurotóxicas/etiologia , Pentanonas/toxicidade , Células Cultivadas , HumanosRESUMO
1. The endogenous cannabinoid (endocannabinoid) system is emerging as a key modulator of intestinal physiology, influencing motility, secretion, epithelial integrity and immune function in the gut, in addition to influencing satiety and emesis. 2. Accumulating evidence suggests that the endocannabinoid system may play a pivotal role in the pathophysiology of gastrointestinal disease, particularly in the light of recent studies demonstrating an effect of endocannabinoids on the development of experimental inflammation and linkages with functional clinical disorders characterized by altered motility. 3. The predominant endocannabinoids, anandamide and 2-arachidonoylglycerol, not only mediate their effects via two recognized cannabinoid receptor subtypes, namely CB(1) and CB(2), but emerging evidence now shows they are also substrates for cyclo-oxygenase (COX)-2, generating a distinct and novel class of prostaglandin ethanolamides (prostamides) and prostaglandin glycerol esters. These compounds are bioactive and may mediate an array of biological effects distinct to those of conventional prostanoids. 4. The effects of prostamides on gastrointestinal motility, secretion, sensation and immune function have not been characterized extensively. Prostamides may play an important role in gastrointestinal inflammation, particularly given the enhanced expression of both COX-2 and endocannabinoids that occurs in the inflamed gut. 5. Further preclinical studies are needed to determine the therapeutic potential of drugs targeting the endocannabinoid system in functional and inflammatory gut disorders, to assist with the determination of feasibility for clinical translation.
Assuntos
Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/patologia , Trato Gastrointestinal/patologia , Trato Gastrointestinal/fisiologia , Animais , Moduladores de Receptores de Canabinoides/farmacologia , Moduladores de Receptores de Canabinoides/uso terapêutico , Trato Gastrointestinal/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Receptores de Canabinoides/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Aggregation of α-Synuclein (αS) protein to amyloid fibrils is a neuropathological hallmark of Parkinson's disease (PD). Growing evidence suggests that extracellular αS aggregation plays a pivotal role in neurodegeneration found in PD in addition to the intracellular αS aggregates in Lewy bodies (LB). Here, we identified and compared a diverse set of molecules capable of mitigating protein aggregation and exogenous toxicity of αSA53T, a more aggregation-prone αS mutant found in familial PD. For the first time, we investigated the αS anti-amyloid activity of semi-synthetic flavonoid 2', 3', 4' trihydroxyflavone or 2-D08, which was compared with natural flavones myricetin and transilitin, as well as such structurally diverse polyphenols as honokiol and punicalagin. Additionally, two novel synthetic compounds with a dibenzyl imidazolidine scaffold, Compound 1 and Compound 2, were also investigated as they exhibited favorable binding with αSA53T. All seven compounds inhibited αSA53T aggregation as demonstrated by Thioflavin T fluorescence assays, with modified fibril morphology observed by transmission electron microscopy. Ion mobility-mass spectrometry (IM-MS) was used to monitor the structural conversion of native αSA53T into amyloidogenic conformations and all seven compounds preserved the native unfolded conformations of αSA53T following 48 h incubation. The presence of each test compound in a 1:2 molar ratio was also shown to inhibit the neurotoxicity of preincubated αSA53T using phaeochromocytoma (PC12) cell viability assays. Among the seven tested compounds 2-D08, honokiol, and the synthetic Compound 2 demonstrated the highest inhibition of aggregation, coupled with neuroprotection from preincubated αSA53T in vitro. Molecular docking predicted that all compounds bound near the lysine-rich region of the N-terminus of αSA53T, where the flavonoids and honokiol predominantly interacted with Lys 23. Overall, these findings highlight that (i) restricted vicinal trihydroxylation in the flavone B-ring is more effective in stabilizing the native αS conformations, thus blocking amyloidogenic aggregation, than dihydroxylation aggregation in both A and B-ring, and (ii) honokiol, punicalagin, and the synthetic imidazolidine Compound 2 also inhibit αS amyloidogenic aggregation by stabilizing its native conformations. This diverse set of molecules acting on a singular pathological target with predicted binding to αSA53T in the folding-prone N-terminal region may contribute toward novel drug-design for PD.
RESUMO
BACKGROUND: Inflammatory Bowel Disease (IBD) is characterized by overt inflammation of the intestine and is typically accompanied by symptoms of bloody diarrhea, abdominal pain and cramping. The Colonic Migrating Motor Complex (CMMC) directs the movement of colonic luminal contents over long distances. The tri-nitrobenzene sulphonic acid (TNBS) model of colitis causes inflammatory damage to enteric nerves, however it remains to be determined whether these changes translate to functional outcomes in CMMC activity. We aimed to visualize innate immune cell infiltration into the colon using two-photon laser scanning intra-vital microscopy, and to determine whether CMMC activity is altered in the tri-nitro benzene sulphonic (TNBS) model of colitis. METHODS: Epithelial barrier permeability was compared between TNBS treated and healthy control mice in-vitro and in-vivo. Innate immune activation was determined by ELISA, flow cytometry and by 2-photon intravital microscopy. The effects of TNBS treatment and IL-1ß on CMMC function were determined using a specialized organ bath. RESULTS: TNBS colitis increased epithelial barrier permeability in-vitro and in-vivo. Colonic IL-1ß concentrations, colonic and systemic CD11b+ cell infiltration, and the number of migrating CD11b+ cells on colonic blood vessels were all increased in TNBS treated mice relative to controls. CMMC frequency and amplitude were inhibited in the distal and mid colon of TNBS treated mice. CMMC activity was not altered by superfusion with IL-1ß. CONCLUSIONS: TNBS colitis damages the epithelial barrier and increases innate immune cell activation in the colon and systemically. Innate cell migration into the colon is readily identifiable by two-photon intra-vital microscopy. CMMC are inhibited by inflammation, but this is not due to direct effects of IL-1ß.