ABSTRACT
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.
Subject(s)
Apoptosis , Cannabis , Leukemia, Myeloid, Acute , Mice, Inbred BALB C , Plant Oils , Animals , Leukemia, Myeloid, Acute/drug therapy , Cell Line, Tumor , Apoptosis/drug effects , Lebanon , Cannabis/chemistry , Plant Oils/pharmacology , Plant Oils/therapeutic use , Mice , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Male , Humans , Cell Survival/drug effectsABSTRACT
Combined immunodeficiency diseases (CID) represent the most severe forms of inborn errors of immunity. Defective T cell development and/or function, leading to an impairment in adaptive immunity are responsible for these diseases. The DNA polymerase δ complex is important for genome duplication and maintenance and consists of the catalytic subunit POLD1, and the accessory subunits POLD2 and POLD3 which stabilizes the complex. Mutations in POLD1 and POLD2 have been recently shown to be associated with a syndromic CID characterized by T cell lymphopenia with or without intellectual deficiency and sensorineural hearing loss. Here we report a homozygous POLD3 variant (NM_006591.3; p.Ile10Thr) in a Lebanese patient, the product of a consanguineous family, presenting with a syndromic severe combined immunodeficiency (SCID) with neurodevelopmental delay and hearing loss. The homozygous POLD3Ile10Thr variant abolishes POLD3 as well as POLD1 and POLD2 expression. Our findings implicate POLD3 deficiency as a novel cause of syndromic SCID.
Subject(s)
Hearing Loss , Severe Combined Immunodeficiency , Humans , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , Severe Combined Immunodeficiency/complications , Severe Combined Immunodeficiency/genetics , Mutation , Homozygote , PedigreeABSTRACT
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.
ABSTRACT
A novel platinum(II) complex 47OMESS(II) and its platinum(IV) derivative 47OMESS(IV) were synthesized and characterized. Cytotoxicity studies against mesenchymal cells (MCs) and lung (A549), breast (MDA-MB-231), and melanoma (A375) cancer cells demonstrated 7-20-fold superior activity for both complexes relative to cisplatin. Remarkably, 47OMESS(IV) demonstrated 17-22-fold greater selectivity toward the cancerous cells compared to the non-cancerous MCs. Western blot analysis on A549 cells showed the involvement of the intrinsic apoptotic pathway. Cellular fractionation and uptake experiments in A549 cells using ICP-mass spectrometry (MS) indicated that 47OMESS(II) and 47OMESS(IV) cross the cellular membrane predominantly via active transport mechanisms. The significant improvement in selectivity that is exhibited by 47OMESS(IV) is reported for the first time for this class of complexes.
Subject(s)
Antineoplastic Agents , Platinum , Humans , Platinum/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Cisplatin/pharmacology , Apoptosis , A549 Cells , Cell Line, TumorABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Cannabis/chemistry , Edema/drug therapy , Inflammation/drug therapy , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Animals , Anti-Inflammatory Agents/chemistry , Carrageenan/toxicity , Disease Models, Animal , Edema/blood , Edema/chemically induced , Edema/pathology , Flowers/chemistry , Formaldehyde/toxicity , Inflammation/blood , Inflammation/chemically induced , Inflammation/pathology , Lebanon , Lipopolysaccharides/toxicity , MAP Kinase Signaling System/drug effects , Male , Monocytes/drug effects , Monocytes/metabolism , Phytochemicals/chemistry , Phytochemicals/pharmacology , Phytochemicals/therapeutic use , Plant Extracts/chemistry , Primary Cell Culture , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVE AND DESIGN: Ghrelin has a key role in modulating energy metabolism and weight gain. The present study aimed at studying the potential role of ghrelin in the development and/or exacerbation of organ damage in a mouse model of diet-induced obesity. OBJECTIVE AND DESIGN: Adult mice were fed one of two diets for 20 weeks: standard high carbohydrate (HC) or high-fat high-sugar (HFHS). Starting week 17, the animals were given regular intraperitoneal ghrelin (160 µg/kg) or saline injections Abdominal fat, serum creatinine, and glucose levels, as well as kidney, liver and heart weight and pathology were assessed. RESULTS: Ghrelin-injected mice showed significant organ damage, which was more exacerbated in HFHS-fed animals. While the HFHS diet was associated with significant liver damage, ghrelin administration did not reverse it. Interestingly, ghrelin administration induced moderate kidney damage and significantly affected the heart by increasing perivascular and myocardium fibrosis, steatosis as well as inflammation. Moreover, serum creatinine levels were higher in the animal group injected with ghrelin. CONCLUSION: Ghrelin administration was associated with increased functional and structural organ damage, regardless of diet. The present study provides novel evidence of multi-organ physiologic alterations secondary to ghrelin administration.
Subject(s)
Abdominal Fat , Ghrelin/metabolism , Kidney/pathology , Liver/pathology , Myocardium/pathology , Animals , Diet, High-Fat , Glucose/metabolism , Male , Mice, Inbred C57BL , Obesity/metabolism , Obesity/pathology , Weight GainABSTRACT
New tetrahydro-1H-pyrazolo[3,4-b]quinoline derivatives were designed, synthesized and characterized as dual anticholinestrase and cyclooxygenase-2 inhibitors. The in vitro and in vivo anti-cholinesterase evaluation exhibited promising activities with lower hepatotoxicity for many candidates compared to tacrine as a reference. Furthermore, their anti-inflammatory activity using in vitro (COX-1/COX-2) inhibitory assay demonstrated superior activity to celecoxib with higher selectivity indices for some compounds. In addition, some candidates showed extended anti-inflammatory activity by inhibiting COX-2 protein induction. Besides, in silico docking experiments of the active compounds against hAChE rationalized the observed in vitro AChE inhibitory activity. In conclusion, this work provides an extension of the chemical space of tetrahydro-1H-pyrazolo[3,4-b]quinoline chemotype for the anticholinestrase and anti-inflammatory activity. This would aid to minimize the possible neuroinflammation linked to the pathogenesis of Alzheimer's disease.
Subject(s)
Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Cyclooxygenase 2 Inhibitors/chemistry , Cyclooxygenase 2 Inhibitors/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Acetylcholinesterase/metabolism , Alzheimer Disease/drug therapy , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Anura , Cholinesterase Inhibitors/chemical synthesis , Cyclooxygenase 2/metabolism , Cyclooxygenase 2 Inhibitors/chemical synthesis , Drug Design , Humans , Liver/drug effects , Molecular Docking Simulation , Pyrazoles/chemical synthesis , Quinolines/chemical synthesisABSTRACT
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⯵g/ml and 7.3⯵g/ml at 24 and 48â¯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⯵g/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 50â¯mg/kg) or gavage (50â¯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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Benzocycloheptenes/pharmacology , Melanoma, Experimental/drug therapy , Polycyclic Sesquiterpenes/pharmacology , Skin Neoplasms/prevention & control , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Benzocycloheptenes/administration & dosage , Benzocycloheptenes/isolation & purification , Cedrus/chemistry , Cell Cycle Checkpoints/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Inbred BALB C , Phosphatidylinositol 3-Kinase/metabolism , Polycyclic Sesquiterpenes/administration & dosage , Polycyclic Sesquiterpenes/isolation & purification , Proto-Oncogene Proteins c-akt/metabolism , Time FactorsABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cedrus/chemistry , Plant Extracts/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Caco-2 Cells , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Edema/prevention & control , HT29 Cells , Hindlimb/drug effects , Hindlimb/pathology , Humans , Male , Molecular Structure , Plant Extracts/chemistry , Rats, Sprague-DawleyABSTRACT
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⯵g/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 (pË0.05) in cell count in the Sub-G1 phase at HC 10⯵g/mL, and total DNA fragmentation (pË0.001) at HC 25⯵g/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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Sesquiterpenes/pharmacology , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Line, Tumor , Cell Movement/drug effects , Cisplatin/pharmacology , Cisplatin/therapeutic use , DNA Fragmentation/drug effects , Disease Models, Animal , Female , G1 Phase Cell Cycle Checkpoints/drug effects , Lung/pathology , Mice , Mice, Inbred BALB C , Sesquiterpenes/therapeutic use , Skin/pathology , Transplantation, Homologous , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathologyABSTRACT
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 (25â¯mg/kg; HC25), Cisplatin treated (2.5â¯mg/kg), and control (DMSO treated). Papilloma yield, volume, histology, and mice weight and liver function were assessed. HC treatment decreased significantly cell survival (IC50â¯=â¯7 and IC90â¯=â¯40⯵g/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.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Carcinogenesis/drug effects , Sesquiterpenes/therapeutic use , Skin Neoplasms/chemically induced , Skin Neoplasms/drug therapy , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Carcinogenesis/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Female , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mice , Mice, Inbred BALB C , Sesquiterpenes/pharmacology , Skin Neoplasms/metabolism , Treatment OutcomeABSTRACT
The focus of this research was on UVB radiation (280-320 nm) responsible for cellular changes in skin of acute and chronically exposed individuals. This study investigated the acute cellular damages triggered by UVB exposure of cultured human fibroblasts and keratinocyte cells immediately and 24 h post exposure in order to understand damage onset and progression. The study evaluated a number of cellular parameters including mitochondria, lysosomes, cell membrane, DNA damages as well as pro and anti-apoptotic protein expression levels. Cellular organelle damages were assessed by a battery of in vitro toxicological assays using MTS and Neutral red cytotoxicity assays. Cell membrane damages were also assessed by measuring lactate dehydrogenase (LDH) enzyme leakage from UVB exposed cells. Lastly DNA damages was assessed using the comet assay while protein expression was evaluated using Western Blot. In this study we reported in all our assay systems (MTS, NR and LDH) that cellular damages were UVB dose dependent with damages amplified 24 h post exposure. Our results also indicated that incubation of exposed cells for a period of 24 h increased the sensitivity of the assay systems used. The increased sensitivity in detecting early cytotoxic damages was manifested though organelle damage measurement at very low doses which were not manifested immediately post exposure. The data also indicated that HaCaT cells were most sensitive in detecting UVB triggered damages immediately and 24 h post exposure using the MTS assay. We also established upregulation and downregulation of various apoptotic proteins at various time points post exposure. The presented data clearly indicated the need for a comprehensive assessment of UVB damages 4 and 24 h post exposure due to the different assay sensitivities in addition to various signaling mechanisms activated at different time points post exposure.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Squamous Cell/drug therapy , Sesquiterpenes/pharmacology , Skin Neoplasms/drug therapy , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Carcinoma, Squamous Cell/pathology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Daucus carota/chemistry , Dose-Response Relationship, Drug , Female , Flow Cytometry , Humans , Inhibitory Concentration 50 , Mice , Mice, Inbred BALB C , Sesquiterpenes/administration & dosage , Sesquiterpenes/isolation & purification , Skin Neoplasms/pathologyABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Sesquiterpenes/toxicity , Signal Transduction/drug effects , 1,2-Dimethylhydrazine , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Apoptosis Regulatory Proteins/metabolism , Caspase 3/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Colonic Neoplasms/chemically induced , Colonic Neoplasms/drug therapy , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Daucus carota/chemistry , Daucus carota/metabolism , Enzyme Activation/drug effects , Humans , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Mice , Phosphatidylinositol 3-Kinases/metabolism , Plant Extracts/chemistry , Plant Extracts/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Sesquiterpenes/chemistry , Sesquiterpenes/therapeutic useABSTRACT
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.
Subject(s)
Cell Proliferation/drug effects , Daucus carota/chemistry , Keratinocytes/cytology , Protective Agents/administration & dosage , Skin Neoplasms/prevention & control , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Apoptosis/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , G1 Phase/drug effects , Humans , Keratinocytes/drug effects , Male , Mice , Mice, Inbred BALB C , Skin Neoplasms/chemically induced , Skin Neoplasms/metabolism , Skin Neoplasms/physiopathology , Tetradecanoylphorbol Acetate/toxicity , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/chemical synthesis , Antineoplastic Agents/chemical synthesis , Cyclooxygenase Inhibitors/chemical synthesis , Pyrazoles/chemistry , Pyrazoles/chemical synthesis , Pyrimidines/chemistry , Pyrimidines/chemical synthesis , Animals , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclooxygenase 1/chemistry , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/chemistry , Cyclooxygenase 2/metabolism , Cyclooxygenase Inhibitors/metabolism , Cyclooxygenase Inhibitors/pharmacology , Diclofenac/pharmacology , Drug Design , Humans , Inhibitory Concentration 50 , Lipopolysaccharides/pharmacology , MCF-7 Cells , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Pyrazoles/metabolism , Pyrazoles/pharmacology , Pyrimidines/metabolism , Pyrimidines/pharmacology , RatsABSTRACT
Background and Aims: Hypertension and obesity are important risk factors of cardiovascular disease. They are both associated with high leptin levels and have been shown to promote vascular hypertrophy, through the RhoA/ROCK and ERK1/2 phosphorylation. Calcineurin/NFAT activation also induces vascular hypertrophy by upregulating various genes. This study aimed to decipher whether a crosstalk exists between the RhoA/ROCK pathway, Ca2+/calcineurin/NFAT pathway, and ERK1/2 phosphorylation in the process of mechanical stretch-induced vascular smooth muscle cell (VSMC) hypertrophy and leptin synthesis. Methods and Results: Rat portal vein (RPV) organ culture was used to investigate the effect of mechanical stretch and exogenous leptin (3.1 nM) on VSMC hypertrophy and leptin synthesis. Results showed that stretching the RPV significantly upregulated leptin secretion, mRNA, and protein expression, which were inhibited by the calcium channel blocker nifedipine (10 µM), the selective calcineurin inhibitor FK506 (1 nM), and the ERK1/2 inhibitor PD98059 (1 µM). The transcription inhibitor actinomycin D (0.1 µM) and the translation inhibitor cycloheximide (1 mM) significantly decreased stretch-induced leptin protein expression. Mechanical stretch or leptin caused an increase in wet weight changes and protein synthesis, considered as hypertrophic markers, while they were inhibited by FK506 (0.1 nM; 1 nM). In addition, stretch or exogenous leptin significantly increased calcineurin activity and MCIP1 expression whereas leptin induced NFAT nuclear translocation in VSMCs. Moreover, in response to stretch or exogenous leptin, the Rho inhibitor C3 exoenzyme (30 ng/mL), the ROCK inhibitor Y-27632 (10 µM), and the actin depolymerization agents Latrunculin B (50 nM) and cytochalasin D (1 µM) reduced calcineurin activation and NFAT nuclear translocation. ERK1/2 phosphorylation was inhibited by FK506 and C3. Conclusions: Mechanical stretch-induced VSMC hypertrophy and leptin synthesis and secretion are mediated by Ca2+/calcineurin/NFAT activation. RhoA/ROCK and ERK1/2 activation are critical for mechanical stretch-induced calcineurin activation.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Daucus carota/chemistry , Neoplasms/drug therapy , Plant Extracts/pharmacology , Sesquiterpenes/pharmacology , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/toxicity , Apoptosis/drug effects , Caco-2 Cells , Cell Adhesion/drug effects , Cell Movement/drug effects , Dose-Response Relationship, Drug , Extracellular Signal-Regulated MAP Kinases/metabolism , Gas Chromatography-Mass Spectrometry , Humans , Lethal Dose 50 , Magnetic Resonance Spectroscopy , Melanoma, Experimental/drug therapy , Melanoma, Experimental/pathology , Neoplasm Invasiveness , Neoplasms/pathology , Phosphorylation , Phytotherapy , Plant Extracts/isolation & purification , Plants, Medicinal , Proto-Oncogene Proteins c-akt/metabolism , Sesquiterpenes/isolation & purification , Sesquiterpenes/toxicity , Signal Transduction/drug effects , Time FactorsABSTRACT
The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels) and reduced into smaller fat pieces of around 1-3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in "Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent" [1].