RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: The Cannabis sativa L. ssp. indica (Lam.) plant has been historically utilized as a natural herbal remedy for the treatment of several ailments. In Lebanon, cannabis extracts have long been traditionally used to treat arthritis, diabetes, and cancer. AIM OF THE STUDY: The current study aims to investigate the anti-cancer properties of Lebanese cannabis oil extract (COE) on acute myeloid leukemia using WEHI-3 cells, and a WEHI-3-induced leukemia mouse model. MATERIALS AND METHODS: WEHI-3 cells were treated with increasing concentrations of COE to determine the IC50 after 24, 48 and 72-h post treatment. Flow cytometry was utilized to identify the mode of cell death. Western blot assay was performed to assess apoptotic marker proteins. In vivo model was established by inoculating WEHI-3 cells in BALB/c mice, and treatment commencing 10 days post-inoculation and continued for a duration of 3 weeks. RESULTS: COE exhibited significant cytotoxicity with IC50 of 7.76, 3.82, and 3.34 µg/mL at 24, 48, and 72 h respectively post-treatment. COE treatment caused an induction of apoptosis through an inhibition of the MAPK/ERK pathway and triggering a caspase-dependent apoptosis via the extrinsic and intrinsic modes independent of ROS production. Animals treated with COE exhibited a significantly higher survival rate, reduction in spleen weight as well as white blood cells count. CONCLUSION: COE exhibited a potent anti-cancer activity against AML cells, both in vitro and in vivo. These findings emphasize the potential application of COE as a chemotherapeutic adjuvant in treatment of acute myeloid leukemia.

Asunto(s)

Apoptosis , Cannabis , Leucemia Mieloide Aguda , Ratones Endogámicos BALB C , Aceites de Plantas , Animales , Leucemia Mieloide Aguda/tratamiento farmacológico , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Líbano , Cannabis/química , Aceites de Plantas/farmacología , Aceites de Plantas/uso terapéutico , Ratones , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Masculino , Humanos , Supervivencia Celular/efectos de los fármacos

RESUMEN

The majority of drugs are metabolized by cytochrome P450 (CYP) enzymes, primarily belonging to the CYP1, CYP2 and CYP3 families. Genetic variations are the main cause of inter-individual differences in drug response, which constitutes a major concern in pharmacotherapy. G-quadruplexes (G4s), are non-canonical DNA and RNA secondary structures formed by guanine-rich sequences. G4s have been implicated in cancer and gene regulation. In this study, we investigated putative G4-forming sequences (PQSs) in the CYP genes. Our findings reveal a high density of PQSs in the full genes of CYP family 2. Moreover, we observe an increased density of PQSs in the promoters of CYP family 1 genes compared to non-CYP450 genes. Importantly, stable PQSs were also identified in all studied CYP genes. Subsequently, we assessed the impact of the most frequently reported genetic mutations in the selected genes and the possible effect of these mutations on G4 formation as well as on the thermodynamic stability of predicted G4s. We found that 4 SNPs overlap G4 sequences and lead to mutated DNA and RNA G4 forming sequences in their context. Notably, the mutation in the CYP2C9 gene, which is associated with impaired (S)-warfarin metabolism in patients, alters a G4 sequence. We then demonstrated that at least 10 of the 13 chosen cytochrome P450 G4 candidates form G-quadruplex structures in vitro, using a combination of spectroscopic methods. In conclusion, our findings indicate the potential role of G-quadruplexes in the regulation of cytochrome genes, and emphasize the importance of G-quadruplexes in drug metabolism.

Asunto(s)

G-Cuádruplex , Humanos , Regiones Promotoras Genéticas , ADN , ARN , Sistema Enzimático del Citocromo P-450/genética

RESUMEN

Daucus carota L., a member of the Apiaceae family, comprises 13 subspecies, with one being cultivated (D. carota L. ssp. sativus (Hoffm.) Arcang.) and the remaining being wild. Traditionally, the wild carrot has been recognized for its antilithic, diuretic, carminative, antiseptic, and anti-inflammatory properties and has been employed in the treatment of urinary calculus, cystitis, gout, prostatitis, and cancer. While extensive literature is available on the phytochemical, pharmacological, and therapeutic evaluations of the cultivated carrot, limited information has been published on the wild carrot. A thorough search was conducted on the phytochemical composition, folk-medicine uses, and pharmacological properties of wild carrot subspecies (Daucus carota L. ssp. carota). Various electronic databases were consulted, and the literature spanning from 1927 to early 2023 was reviewed. Thirteen wild Daucus carota subspecies were analyzed, revealing over 310 compounds, including terpenoids, phenylpropenoids, flavonoids, and phenolic acids, with 40 constituting more than 3% of the composition. This review also highlights the antioxidant, anticancer, antipyretic, analgesic, antibacterial, antifungal, hypolipidemic, and hepato- and gastroprotective properties of wild carrot subspecies. Existing in vitro and in vivo studies support their traditional uses in treating infections, inflammation, and cancer. However, further research on other subspecies is required to confirm additional applications. Well-designed preclinical and clinical trials are still necessary to establish the safety and efficacy of wild Daucus carota for human use.

RESUMEN

The present study deals with the assessment of acrylamide levels, dietary intake and toxicity associated with food products which constitute the main components of a Lebanese breakfast including bread, crackers, toast and kaak. Quantification of acrylamide levels was performed on a UPLC-MS/MS spectrometer and upon correlation with the results of a community survey, the carcinogenic and neurotoxic risks associated with the dietary intake of acrylamide were calculated. The average exposure to acrylamide from the investigated dietary products was found to be 5 times higher than the intake of 0.08 µg/kg-bw/day, as estimated by the NFCA (Norwegian Food Control Authority) and 3 times higher than the intake of 0.14 µg/kg-bw/day as set by the WHO (World Health Organization). MOEN and MOEC (Margin of Exposure for neurotoxic and carcinogenic risks) values ranged between 290 and 556, and between 449 and 861 respectively. Kaak, Crackers, and Toast appear to pose no neurotoxic or carcinogenic risk of concern among the entire population as well as the individual age groups. French bread and Lebanese bread pose different levels of carcinogenic risk among the entire population as well as various age groups. The results also indicate that 24% of children, 4% of young adults and 8% of adults are at both neurotoxic and carcinogenic risks.

Asunto(s)

Acrilamida , Síndromes de Neurotoxicidad , Acrilamida/toxicidad , Pan/análisis , Carcinógenos/toxicidad , Niño , Cromatografía Liquida , Contaminación de Alimentos/análisis , Humanos , Espectrometría de Masas en Tándem , Adulto Joven

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Cannabis sativa L. is an aromatic annual herb belonging to the family Cannabaceae and it is widely distributed worldwide. Cultivation, selling, and consumption of cannabis and cannabis related products, regardless of its use, was prohibited in Lebanon until April 22, 2020. Nevertheless, cannabis oil has been traditionally used unlawfully for many years in Lebanon to treat diseases such as arthritis, diabetes, cancer and few neurological disorders. AIM OF THE STUDY: The present study aims to evaluate the phytochemical and anti-inflammatory properties of a cannabis oil preparation that is analogous to the illegally used cannabis oil in Lebanon. MATERIALS AND METHODS: Dried Cannabis flowers were extracted with ethanol without any purification procedures to simulate the extracts sold by underground dealers in Lebanon. GC/MS was performed to identify chemical components of the cannabis oil extract (COE). In vivo anti-inflammatory effect of COE was evaluated by using carageenan- and formalin-induced paw edema rat models. TNF-α production were determined by using LPS-activated rat monocytes. Anti-inflammatory markers were quantified using Western blot. RESULTS: Chemical analysis of COE revealed that cannabidiol (CBD; 59.1%) and tetrahydrocannabinol (THC; 20.2%) were found to be the most abundant cannabinoids.Various monoterpenes (α-Pinene, Camphene, ß-Myrecene and D-Limonene) and sesquiterpenes (ß-Caryophyllene, α-Bergamotene, α-Humelene, Humulene epoxide II, and Caryophyllene oxide) were identified in the extract. Results showed that COE markedly suppressed the release of TNF-α in LPS-stimulated rat monocytes. Western blot analysis revealed that COE significantly inhibited LPS-induced COX-2 and i-NOS protein expressions and blocked the phosphorylation of MAPKs, specifically that of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 MAPK. COE displayed a significant inhibition of paw edema in both rat models. Histopathological examination revealed that COE reduced inflammation and edema in chronic paw edema model. CONCLUSION: The current findings demonstrate that COE possesses remarkable in vivo and in vitro anti-inflammatory activities which support the traditional use of the Lebanese cannabis oil extract in the treatment of various inflammatory diseases including arthritis.

Asunto(s)

Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Cannabis/química , Edema/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Animales , Antiinflamatorios/química , Carragenina/toxicidad , Modelos Animales de Enfermedad , Edema/sangre , Edema/inducido químicamente , Edema/patología , Flores/química , Formaldehído/toxicidad , Inflamación/sangre , Inflamación/inducido químicamente , Inflamación/patología , Líbano , Lipopolisacáridos/toxicidad , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fitoquímicos/química , Fitoquímicos/farmacología , Fitoquímicos/uso terapéutico , Extractos Vegetales/química , Cultivo Primario de Células , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

The present study aims to determine the carcinogenic and neurotoxic risks associated with acrylamide intake from cereal products. Analysis on a UPLC-MS/MS spectrometer revealed that oat-based and mixed cereals contain the highest amount of acrylamide among cereal products with levels as high as 271 and 348 µg/kg, respectively. Children were shown to exhibit both carcinogenic and neurotoxic risks regardless of the type of cereal product consumed. For adults above 50 years of age, only consumers of oat-based cereal products seem to exhibit carcinogenic and neurotoxic risks. To avoid a carcinogenic and neurotoxic risk among the Lebanese population, we propose that food processors set the maximum tolerable concentration for acrylamide in cereal products at 94.8 µg/kg product, a value which is threefolds lower than the average acrylamide levels found in this study. Alternatively, and unreasonably, the average Lebanese population and children among the Lebanese population may choose to cut down on cereal consumption by 1.7- and 7.2-folds respectively, should they want to avoid a health hazard as a result of acrylamide intake. The industry should also respond by optimizing the production process in a way to reduce acrylamide levels in cereals.

RESUMEN

Current treatment of patients with coronavirus 2019 (COVID-19) involves repurposed drugs that inhibit viral infection by either binding to their respective targets or via modulating cellular signal transduction. However, there is still a great deal of efficacy enhancement through combination therapy and derivatization. Combination therapy should involve agents with significant activity and different mechanisms of action. The structural map of the interaction between a drug and its target protein will help guide drug discovery for devising safe and effective ways to treat COVID-19. Herein, we report numerous synthetic designs based on enhanced affinity to the viral carbohydrate-rich protein spikes and protein-binding sites of COVID-19.

Asunto(s)

Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Reposicionamiento de Medicamentos , SARS-CoV-2/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Antivirales/efectos adversos , Sitios de Unión , COVID-19/metabolismo , COVID-19/mortalidad , COVID-19/virología , Ensayos Clínicos como Asunto , Quimioterapia Combinada , Medicina Basada en la Evidencia , Interacciones Huésped-Patógeno , Humanos , Ligandos , Terapia Molecular Dirigida , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Resultado del Tratamiento

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Cedrus libani A. Rich (C. libani) is majestic evergreen Mediterranean conifer growing in the mountains of Lebanon. The ethnobotanical and traditional uses of cedar wood oil traces back to ancient times for the treatment of various ailments including cancer. Previous work in our laboratories revealed that himachalol (7-HC), a major sesquiterpene isolated from C. libani, possesses potent cytotoxic activity against various human cancer cell lines as well as promising anti-inflammatory effect in isolated rat monocytes. AIM OF THE STUDY: The present study aims to elucidate the mechanism of action behind the cytotoxic activity of 7-HC against murine melanoma cells (B16F-10) and evaluates its chemopreventive effect against chemically-induced skin carcinogenesis in mice. MATERIALS AND METHODS: 7-HC was extracted and purified from Cedrus libani wood. Cell viability was evaluated using WST-1 kit. Cell cycle analysis and apoptosis were assessed by Flow cytometry using propidium iodide (PI) and fluorescein Isothiocyanate (FITC)-conjugated Annexin V/PI staining respectively. Apoptosis related protein were quantified using western blot. The chemopreventive activity of 7-HC was evaluated for 20 weeks using a DMBA/TPA induced skin carcinogenesis model in Balb/c mice. RESULTS: 7-HC displayed a potent anti-proliferative activity against the melanoma cells with an IC50 of 8.8 µg/ml and 7.3 µg/ml at 24 and 48 h, respectively. Co-treatment with Cisplatin did not show any synergistic or additive effect on cell viability. Flow cytometry analysis using PI revealed that 7-HC treatment (5 and 10 µg/ml) induces the accumulation of cells in the sub-G1 phase and causes a decline in cell populations in the S and G2/M phases. Annexin/PI staining also reveals that 7-HC treatment significantly increases the percentage of cells undergoing early and late apoptosis. Western blot analysis shows that 7-HC treatment decreases the level of the anti-apoptotic protein Bcl-2 and increases the level of the pro-apoptotic protein Bax. A reduction in the level of phosphorylated Erk and Akt was also observed. 7-HC via topical (2.5%), intraperitoneal (10, 25 and 50 mg/kg) or gavage (50 mg/kg) treatment revealed a significant decrease in papilloma volume with no adverse effect on liver and kidney function. CONCLUSIONS: The present study demonstrates that 7-HC treatment protects against chemically-induced skin carcinogenesis, promotes cell cycle arrest and induces apoptosis partially through an inhibition of both the MAPK/Erk and PI3K/Akt pathways.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Benzocicloheptenos/farmacología , Melanoma Experimental/tratamiento farmacológico , Sesquiterpenos Policíclicos/farmacología , Neoplasias Cutáneas/prevención & control , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Benzocicloheptenos/administración & dosificación , Benzocicloheptenos/aislamiento & purificación , Cedrus/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasa/metabolismo , Sesquiterpenos Policíclicos/administración & dosificación , Sesquiterpenos Policíclicos/aislamiento & purificación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Tiempo

RESUMEN

Cedrus libani is a majestic evergreen tree native to the Mediterranean mountains of Lebanon, Syria and Turkey. In this study, the tree heart wood was extracted using hexane to produce C. libani oil extract (CLOE) as a dark oil. GCMS analysis of CLOE identified up to 30 compounds whereby 2-himachalen-7-ol (7-HC) was the most abundant (40%). 7-HC was isolated using column chromatography and the identity of the white crystalline solid was confirmed via NMR spectroscopy and X-Ray Crystallography. 7-HC demonstrated potent cytotoxic activity against several human cancer cell lines including brain (SF-268, IC50 8.1 µg/mL) and colon (HT-29, IC50 10.1 µg/mL; Caco-2, IC50 9.9 µg/mL) with ovarian (Sk-OV-3, IC50 > 50 µg/mL) cells being the most resistant. However, while HT-29 displayed resistance to Cisplatin, 7-HC was 8-10 folds more potent. Co-treatment with 7-HC and Cisplatin showed a significant synergistic anti-proliferative effect against SF-268, HT-29 and Caco-2 cells. 7-HC also exhibited significant anti-inflammatory effect in formalin-induced paw edema in rats. Western blot analysis revealed that 7-HC displayed dose dependent inhibition of LPS-induced COX-2 protein expression in isolated rat monocytes. The present study demonstrates that 7-HC possesses promising anticancer and anti-inflammatory activities, and may serve as a lead molecule in cancer therapy.

Asunto(s)

Antiinflamatorios/farmacología , Antineoplásicos Fitogénicos/farmacología , Cedrus/química , Extractos Vegetales/farmacología , Animales , Antiinflamatorios/química , Antineoplásicos Fitogénicos/química , Células CACO-2 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Edema/prevención & control , Células HT29 , Miembro Posterior/efectos de los fármacos , Miembro Posterior/patología , Humanos , Masculino , Estructura Molecular , Extractos Vegetales/química , Ratas Sprague-Dawley

RESUMEN

ß-2-himachalen-6-ol (HC), a major sesquiterpene isolated from the Lebanese wild carrot umbels, was shown to possess remarkable in vitro and in vivo anticancer activities. The present study investigates the anti-metastatic activity of HC post 4T1 breast cancer cells inoculation in a murine model. The effect of HC on 4T1 cell viability was assessed using WST-1 kit, while cell cycle analysis was performed using flow cytometry. Tumor development and metastasis were evaluated by injecting 4T1 cells in the mice mammary gland region followed by either HC or cisplatin treatment. The 6-thioguanine assay was used for the quantification of metastatic cells in the blood. HC treatment caused a dose-dependent decrease in cell viability with IC50 and IC90 values of 7 and 28 µg/mL respectively. Concomitant treatment with cisplatin significantly reduced cell viability when compared to cells treated with cisplatin or HC alone. Flow cytometry revealed a significant increase (p˂0.05) in cell count in the Sub-G1 phase at HC 10 µg/mL, and total DNA fragmentation (p˂0.001) at HC 25 µg/mL. Annexin/PI staining showed early and late apoptotic mode of cell death upon treatment with HC. Histopathological evaluation revealed less incidence of primary and metastatic tumor/inflammation in the HC and cisplatin treated groups. Tumor size and colony-forming units were significantly decreased in the HC treated group. HC treatment induced cell cycle arrest, promoted apoptosis and reduced the incidence of primary and metastatic lesions caused by 4T1 cells. The present findings suggest that HC has an anti-metastatic potential against aggressive types of cancer.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Sesquiterpenos/farmacología , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cisplatino/farmacología , Cisplatino/uso terapéutico , Fragmentación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Sesquiterpenos/uso terapéutico , Piel/patología , Trasplante Homólogo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología

RESUMEN

The use of ruthenium complexes as chemotherapeutic agents has been recently explored as one of the alternatives to conventional treatments. In the present study, two Ru(ii) polypyridyl complexes were synthesized and characterized: a strained [Ru(bipy)2(BC)]Cl2 (complex 1) where [bipy = 2,2'-bipyridine and BC = bathocuproine] along with the unstrained control [Ru(bipy)2(phen)]Cl2 (complex 2) where [phen = 1,10-phenanthroline]. The photophysical and photochemical analyses proved that unlike the photostable complex 2, complex 1 ejected both bipy and BC ligands at a ratio of 3 : 1 respectively. Results showed that the activity of complex 1 was significantly enhanced upon photoactivation. The response was however particularly significant in B16-F10 melanoma cells where phototoxicity index (PI = IC50 dark/IC50 light) was >900. When compared to cisplatin, the photoproducts were more potent against all tested cell lines, implying that the complex acquired significant chemotherapeutic potential upon irradiation. Cellular uptake of complex 1 and the free BC ligand were found to be significantly facilitated as evidenced by 400-600 fold increase in concentration of the compounds inside the cells relative to the extracellular culture medium. Complex 2 exhibited 35 times lower cellular concentration relative to complex 1. Flow cytometry and plasmid DNA gel electrophoresis measurements showed that complex 1 interacts with DNA inducing apoptosis in the dark and either late-apoptosis or necrosis upon irradiation. These findings corroborate the importance of lipophilic ligands such as BC to enhance uptake and subsequently improve the photochemotherapy potential of Ru(ii) polypyridyl complexes.

RESUMEN

The present study aims to quantify acrylamide in caffeinated beverages including American coffee, Lebanese coffee, espresso, instant coffee and hot chocolate, and to determine their carcinogenic and neurotoxic risks. A survey was carried for this purpose whereby 78% of the Lebanese population was found to consume at least one type of caffeinated beverages. Gas Chromatography Mass Spectrometry analysis revealed that the average acrylamide level in caffeinated beverages is 29,176 µg/kg sample. The daily consumption of acrylamide from Lebanese coffee (10.9 µg/kg-bw/day), hot chocolate (1.2 µg/kg-bw/day) and Espresso (7.4 µg/kg-bw/day) was found to be higher than the risk intake for carcinogenicity and neurotoxicity as set by World Health Organization (WHO; 0.3-2 µg/kg-bw/day) at both the mean (average consumers) and high (high consumers) dietary exposures. On the other hand, American coffee (0.37 µg/kg-bw/day) was shown to pose no carcinogenic or neurotoxic risks among the Lebanese community for consumers with a mean dietary exposure. The study shows alarming results that call for regulating the caffeinated product industry by setting legislations and standard protocols for product preparation in order to limit the acrylamide content and protect consumers. In order to avoid carcinogenic and neurotoxic risks, we propose that WHO/FAO set acrylamide levels in caffeinated beverages to 7000 µg acrylamide/kg sample, a value which is 4-folds lower than the average acrylamide levels of 29,176 µg/kg sample found in caffeinated beverages sold in the Lebanese market. Alternatively, consumers of caffeinated products, especially Lebanese coffee and espresso, would have to lower their daily consumption to 0.3-0.4 cups/day.

Asunto(s)

Acrilamida/toxicidad , Bebidas/análisis , Cafeína/química , Carcinógenos/toxicidad , Exposición Dietética/efectos adversos , Contaminación de Alimentos/análisis , Síndromes de Neurotoxicidad/epidemiología , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Líbano/epidemiología , Masculino , Persona de Mediana Edad , Síndromes de Neurotoxicidad/etiología , Medición de Riesgo , Adulto Joven

RESUMEN

ß-2-himachalen-6-ol (HC), a novel sesquiterpene derived from Lebanese wild carrot, was shown to possess a remarkable anticancer activity. The present study investigates the in vitro anticancer activity of HC and its effect on papillomas induced using a DMBA/TPA skin carcinogenesis mouse model. HaCaT-ras II-4 epidermal squamous cell viability was assessed using WST-1 kit. Cell cycle was analyzed by flow cytometry, and pro/anti-apoptotic proteins were measured using western blot. Mice papillomas were induced by DMBA and promoted with TPA for 18 weeks. At week 12, animals were divided into four groups: HC topically treated (5%Top), HC intraperitoneally treated (25 mg/kg; HC25), Cisplatin treated (2.5 mg/kg), and control (DMSO treated). Papilloma yield, volume, histology, and mice weight and liver function were assessed. HC treatment decreased significantly cell survival (IC50 = 7 and IC90 = 40 µg/ml) and increased significantly cells undergoing late apoptosis and necrosis. It also significantly decreased the levels of pro-caspase-3, p53, Bcl-2, p-Erk/Erk and p-Akt/Akt and increased p21 and Bax proteins. Treatment with HC25, HC5%Top or Cisplatin showed a significant decrease in papilloma yield and volume. Only Cisplatin treatment caused a significant decrease in body weight and increase in serum ALT. In conclusion, ß-2-himachalen-6-ol induced significant tumor shrinkage, an effect partly mediated via promoting apoptosis through inhibition of the MAPK/ERK and PI3K/AKT pathways, with no significant toxicity to laboratory mice.

Asunto(s)

Antineoplásicos Fitogénicos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Sesquiterpenos/uso terapéutico , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/tratamiento farmacológico , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Antineoplásicos Fitogénicos/farmacología , Carcinogénesis/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos BALB C , Sesquiterpenos/farmacología , Neoplasias Cutáneas/metabolismo , Resultado del Tratamiento

RESUMEN

BACKGROUND: Previous studies in our laboratory showed that Daucus carota oil extract (DCOE) possesses remarkable in-vitro anticancer activity and antitumour promoting effect against DMBA/TPA skin carcinogenesis in mice. Chemical analysis of DCOE led to the isolation of the ß-2-himachalen-6-ol (HC), major sesquiterpene with a potent anticancer activity against various colon, breast, brain and skin cancer cells. This study investigated the anticancer activity of HC against invasive epidermal squamous cell carcinoma cells and evaluated its effect in a DMBA/TPA skin carcinogenesis Balb/c murine model. METHODS: HaCaT-ras II-4 epidermal squamous cells were treated with HC (1, 5, 10, 25 and 50 µg/ml), and cell viability was evaluated with WST 1 assay kit. Cell cycle analysis was carried out by flow cytometry, and pro/anti-apoptotic proteins were measured using Western blot. The effect of topical and intraperitoneal (IP) treatment with HC in mice was assessed using the DMBA/TPA skin carcinogenesis model. Cisplatin (2.5 mg/kg; IP) was used as a positive control. Papilloma incidence, yield and volume were monitored, and isolated papillomas were assessed for their pro/anti-apoptotic proteins and morphology. RESULTS: ß-2-himachalen-6-ol showed a dose-dependent decrease in cell survival with an IC50 and IC90 of 8 and 30 µg/ml, respectively. Flow cytometry analysis revealed that treatment with 10 µg/ml HC significantly increased the number of cells undergoing late apoptosis (28%), while 25 µg/ml caused a larger cell shift towards late apoptosis (46.6%) and necrosis (39%). A significant decrease in protein levels of p53 and Bcl-2 and a significant increase in p21 and Bax were observed. Also, there was a significant decrease in p-Erk and p-Akt protein levels. The treatment of mice (IP and topical) with HC caused a significant decrease in papilloma yield, incidence and volume. Similar effects were observed with cisplatin treatment, but HC-treated groups exhibited twofold to threefold increase in survival rates. Similar patterns in the pro- and anti-apoptotic proteins were observed in mice treated with HC, except for a significant increase in p53 protein. CONCLUSIONS: In conclusion, HC treatment induced cell cycle arrest (low dose) and promoted apoptosis partly via inhibition of the MAPK/ERK and PI3K/AKT pathways with no significant toxicity to laboratory mice.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Sesquiterpenos/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daucus carota/química , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Sesquiterpenos/administración & dosificación , Sesquiterpenos/aislamiento & purificación , Neoplasias Cutáneas/patología

RESUMEN

Previous studies in our laboratory showed that Daucus carota oil extract (DCOE) possesses in vitro and in vivo anticancer activities. Chemical analysis of DCOE led to the isolation of ß-2-himachalen-6-ol (HC) which exhibited potent anticancer activity against colon, breast, brain and skin cancer cells. The present study investigates the anticancer activity of HC against SW1116 colon cancer cell lines, and evaluates its effect in a 1,2-dimethylhydrazine (DMH) colon carcinogenesis black6 mice model. The SW1116 colon cancer cell line was treated with HC (1, 5, 10 and 25 µg/ml) and cell viability was evaluated with WST 1 assay kit. Cell cycle analysis was carried out by flow cytometry, and pro/anti-apoptotic proteins were measured using western blot. The effect of intraperitoneal (IP) treatment with HC (10, 25 and 50 µg/ml) in mice was assessed using the DMH colon carcinogenesis model with Cisplatin (2.5 µg/kg; IP) as a positive control. Blood samples were collected for assessment of liver toxicity and colon tumor incidence and size were studied histologically. HC showed a dose-dependent decrease in cell survival with an IC50 of 18 and 14.5 µg/ml after 24 and 48 h respectively. Flow cytometry analysis revealed that 10 µg/ml HC increased the number of cells undergoing necrosis (18.05%) and late apoptosis (15.66%). At HC 25 µg/ml more cells shifted toward necrosis (58.01%) and late apoptosis (30.47%). Western blot analysis revealed a significant decrease in p-Erk, p-Akt, pro-caspase-3 and Bcl-2 and an increase in p53, p21, Bax and PARP proteins. Mice treatment (IP) with HC caused a significant decrease in tumor incidence and size. Similar effects were observed with cisplatin treatment. In conclusion, HC treatment (low dose) induced cell cycle arrest and promoted apoptosis via inhibition of the MAPK/ERK and PI3K/AKT pathways. HC treatment also had antitumor effect in vivo with no significant toxicity to laboratory mice.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Sesquiterpenos/toxicidad , Transducción de Señal/efectos de los fármacos , 1,2-Dimetilhidrazina , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Daucus carota/química , Daucus carota/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sesquiterpenos/química , Sesquiterpenos/uso terapéutico

RESUMEN

BACKGROUND: Previous studies in our laboratory showed that the Lebanese Daucus carota ssp. carota (wild carrot) oil extract possesses in vitro and in vivo anticancer activities. The present study aims to examine the cytotoxic effect of Daucus carota oil fractions on human epidermal keratinocytes and evaluate the chemopreventive activity of the pentane diethyl ether fraction on DMBA/TPA induced skin carcinogenesis in mice. METHODS: Wild carrot oil extract was chromatographed to yield four fractions (F1, 100% pentane; F2, 50:50 pentane:diethyl ether; F3, 100% diethyl ether; F4 93:7 chloroform:methanol). The cytotoxic effect of fractions (10, 25, 50 and 100 µg/mL) was tested on human epidermal keratinocytes (non-tumorigenic HaCaT cells and tumorigenic HaCaT-ras variants) using WST a ssay. Cell cycle phase distribution of tumorigenic HaCaT-ras variants was determined by flow cytometry post-treatment with F2 fraction. Apoptosis related proteins were also assessed using western blot. The antitumor activity of F2 fraction was also evaluated using a DMBA/TPA induced skin carcinoma in Balb/c mice. RESULTS: All fractions exhibited significant cytotoxicity, with HaCaT cells being 2.4-3 times less sensitive than HaCaT-ras A5 (benign tumorigenic), and HaCaT-ras II4 (malignant) cells. GC-MS analysis revealed the presence of a major compound (around 60%) in the pentane/diethylether fraction (F2), identified as 2-himachalen-6-ol. Treatment of HaCaT-ras A5 and HaCaT-ras II4 cells with F2 fraction resulted in the accumulation of cells in the sub-G1 apoptotic phase and decreased the population of cells in the S and G2/M phases. Additionally, F2 fraction treatment caused an up-regulation of the expression of pro-apoptotic (Bax) and down-regulation of the expression of anti-apoptotic (Bcl2) proteins. A decrease in the phosphorylation of AKT and ERK was also observed. Intraperitoneal treatment with F2 fraction (50 or 200 mg/kg) in the DMBA/TPA skin carcinogenesis mouse model showed a significant inhibition of papilloma incidence (mice with papilloma), yield (number of papilloma/mouse) and volume (tumor relative size) at weeks 15, 18 and 21. CONCLUSION: The present data reveal that F2 fraction has a remarkable antitumor activity against DMBA/TPA-induced skin carcinogenesis, an effect that may be mediated through inhibition of the MAPK/ERK and PI3K/AKT pathways.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Daucus carota/química , Queratinocitos/citología , Sustancias Protectoras/administración & dosificación , Neoplasias Cutáneas/prevención & control , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Apoptosis/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Fase G1/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/fisiopatología , Acetato de Tetradecanoilforbol/toxicidad , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

This study reports the synthesis of two series of new purine bioisosteres comprising a pyrazolo[3,4-d]pyrimidine scaffold linked to piperazine moiety through different amide linkages. The newly synthesized compounds were evaluated for anticancer activity against four cell lines (MDA-MB-231, MCF-7, SF-268, B16F-10) and cyclooxygenase (COX-2) protein expression inhibition in lipopolysaccharide (LPS)-activated rat monocytes. The results revealed that most of the synthesized compounds showed moderate-to-high cytotoxic activity against at least one cell line, with compound 10b being the most active against all used cell lines (IC50 values 5.5-11 µg/ml) comparable to cisplatin. In addition, six of these compounds (7b, 10a-d, and 12c) demonstrated inhibition of LPS-induced COX-2 protein expression at low concentration (25 µg/ml) as compared to the control non-stimulated cells and showed a COX-2 selectivity index range comparable to diclofenac sodium. The overall results indicate that many of these pyrazolopyrimidine derivatives possess in vitro anti-inflammatory and anticancer activities at varying doses, and the most active compounds will be subjected to in vivo pharmacological evaluation.

Asunto(s)

Antiinflamatorios/síntesis química , Antineoplásicos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Pirazoles/química , Pirazoles/síntesis química , Pirimidinas/química , Pirimidinas/síntesis química , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 1/química , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/química , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Diclofenaco/farmacología , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Lipopolisacáridos/farmacología , Células MCF-7 , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Pirazoles/metabolismo , Pirazoles/farmacología , Pirimidinas/metabolismo , Pirimidinas/farmacología , Ratas

RESUMEN

Daucus carota ssp. carota, also known as wild carrot, is a commonly used herb in Lebanese folk medicine to treat several ailments including cancer. Previous studies in our laboratories showed that the Daucus carota oil extract (DCOE) and subsequent fractions exhibit antioxidant, anti-inflammatory and anti-cancer activities. In this study, we report the isolation and identification of the major compound responsible for the anti-cancer activity of DCOE along with the mechanism of action involved. GC-MS and NMR spectroscopy revealed the identity of the major compound as ß-2-himachalen-6-ol, a novel sesquiterpene unique to the Lebanese wild carrot. ß-2-Himachalen-6-ol demonstrated potent anti-cancer activity against B16F-10, Caco-2, MB-MDA-231, A549 and SF-268 cancer cells (IC50 13-4µg/ml; 58-18µM), with SF-268 cells being the most sensitive. The sesquiterpene was shown to induce cell death through apoptosis (flow cytometry), decrease 2D cell motility (wound healing assay) and 3D invasion, as well as increase cell adhesion in SF-268 cells. Additionally, ß-2-himachalen-6-ol showed very low toxicity in mice with an LD50>6000mg/kg body weight. In conclusion, the present data demonstrate that ß-2-himachalen-6-ol is a potential multi-mechanistic chemotherapeutic drug with high potency and safety.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Daucus carota/química , Neoplasias/tratamiento farmacológico , Extractos Vegetales/farmacología , Sesquiterpenos/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/toxicidad , Apoptosis/efectos de los fármacos , Células CACO-2 , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Dosificación Letal Mediana , Espectroscopía de Resonancia Magnética , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Invasividad Neoplásica , Neoplasias/patología , Fosforilación , Fitoterapia , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/toxicidad , Transducción de Señal/efectos de los fármacos , Factores de Tiempo

RESUMEN

CONTEXT: Wild carrot, Daucus carota L. ssp. carota (Apiacae), is widely distributed throughout the world and has various uses in traditional medicine in Lebanon. OBJECTIVE: The present study aimed to fractionate and analyze the chemical composition of the Daucus carota oil extract (DCOE) fractions and to evaluate their antioxidant and hepatoprotective properties in vitro and in vivo. MATERIALS AND METHODS: DCOE was chromatographed on silica gel column to produce four fractions: pentane (F1), 50:50 pentane:diethyl ether (F2), diethyl ether (F3), and 93:7 chloroform: methanol (F4). Qualitative and quantitative analyses of oil fractions were performed by GC-MS and HPLC techniques. The in vitro antioxidant properties were assessed using DPPH, FIC, and ferric-reducing antioxidant power (FRAP) assays. The hepatoprotective property was determined by examining the levels of serum markers (alanine transaminase (ALT) and aspartate transaminase (AST)) and hepatic antioxidant (superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST)) enzymes in CCl4-intoxicated mice pretreated with intraperitoenal 50, 100, or 200 mg/kg b.w. of the oil fractions for 5 d. RESULTS: GCMS analysis of F2 revealed the presence of 2-himachalen-6-ol (61.4%) which is reported for the first time in Daucus carota species. F3 and F4 were rich in phenolics and flavonoids and demonstrated significant DPPH activity (IC50 = 0.29 and 0.38 mg/ml, respectively) and high FRAP values (225.11 and 437.59 µmol FeSO4/g, respectively). The sesquiterpene-rich fraction F1 had the highest FIC ability (IC50 = 0.28 mg/ml). Pretreatment with F1 and F4 reversed the CCl4-induced decrease in SOD, CAT, and GST levels and reduced significantly hepatic damage. DISCUSSION AND CONCLUSION: The current results suggested that wild carrot oil fractions exhibited a unique chemical composition and possessed significant antioxidant activities as well as hepatoprotective effects against CCl4-induced hepatotoxicity.

Asunto(s)

Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Daucus carota , Hígado/efectos de los fármacos , Extractos Vegetales/farmacología , Aceites de Plantas/farmacología , Animales , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Biomarcadores/sangre , Compuestos de Bifenilo/química , Tetracloruro de Carbono , Fraccionamiento Químico , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cromatografía Líquida de Alta Presión , Citoprotección , Daucus carota/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Enzimas/sangre , Cromatografía de Gases y Espectrometría de Masas , Hígado/enzimología , Hígado/patología , Ratones Endogámicos BALB C , Oxidación-Reducción , Fitoterapia , Picratos/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Aceites de Plantas/química , Aceites de Plantas/aislamiento & purificación , Plantas Medicinales