Lebanese cannabis oil as a potential treatment for acute myeloid leukemia: In vitro and in vivo evaluations.

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.

Enhanced potency of a chloro-substituted polyaromatic platinum(II) complex and its platinum(IV) prodrug against lung cancer.

The present study investigates the anti-neoplastic activity of a platinum (II) complex, Pt(II)5ClSS, and its platinum (IV) di-hydroxido analogue, Pt(IV)5ClSS, against mesenchymal cells (MCs), lung (A549), melanoma (A375) and breast (MDA-MB-231) cancer cells. Both complexes exhibited up to 14-fold improved cytotoxicity compared to cisplatin. NMR was used to determine that ∼25 % of Pt(IV)5ClSS was reduced to Pt(II)5ClSS in the presence of GSH (Glutathione) after 72 h. The complex 1H NMR spectra acquired for Pt(II)5ClSS with GSH shows evidence of degradation and environmental effects (∼30 %). The prominence of the 195Pt peak at âˆ¼ -2800 ppm suggests that a significant amount of Pt(II)5ClSS remained in the mixture. Pt(II)5ClSS and Pt(IV)5ClSS have shown exceptional selectivity to cancer cells in comparison to MCs (IC50 > 150 µM). Western blot analysis of Pt(II)5ClSS and Pt(IV)5ClSS on A549 cells revealed significant upregulation of cleaved PARP-1, BAX/Bcl2 ratio, cleaved caspase 3 and cytochrome thus suggesting apoptosis was induced through the intrinsic pathway. Flow cytometry also revealed significant cell death by apoptosis. Treatment with Pt(II)5ClSS and Pt(IV)5ClSS also showed significant amounts of free radical production while the COMET assay showed that both complexes cause minimal DNA damage. Cellular uptake results via ICP-MS suggest a time-dependent active mode of transport for both complexes with Pt(II)5ClSS being transported at a higher rate compared to Pt(IV)5ClSS. A Dose Escalation Study carried out on BALB/c mice showed that Pt(II)5ClSS and Pt(IV)5ClSS were approximately 8- folds and 12.5-folds, respectively, more tolerated than cisplatin. The present study provides evidence that both complexes may have the characteristics of an efficient and potentially safe anti-tumor drug that could support NSCLC treatment.

The Wild Carrot (Daucus carota): A Phytochemical and Pharmacological Review.

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.

A Ru(II)-Strained Complex with 2,9-Diphenyl-1,10-phenanthroline Ligand Induces Selective Photoactivatable Chemotherapeutic Activity on Human Alveolar Carcinoma Cells via Apoptosis.

[Ru(bipy)2(dpphen)]Cl2 (where bipy = 2,2'-bipyridine and dpphen = 2,9-diphenyl-1,10-phenanthroline) (complex 1) is a sterically strained compound that exhibits promising in vitro photocytotoxicity on an array of cell lines. Since lung adenocarcinoma cancer remains the most common lung cancer and the leading cause of cancer deaths, the current study aims to evaluate the plausible effect and uptake of complex 1 on human alveolar carcinoma cells (A549) and mesenchymal stem cells (MSC), and assess its cytotoxicity in vitro while considering its effect on cell morphology, membrane integrity and DNA damage. MSC and A549 cells showed similar rates of complex 1 uptake with a plateau at 12 h. Upon photoactivation, complex 1 exhibited selective, potent anticancer activity against A549 cells with phototoxicity index (PI) values of 16, 25 and 39 at 24, 48 and 72 h, respectively. This effect was accompanied by a significant increase in A549-cell rounding and detachment, loss of membrane integrity and DNA damage. Flow cytometry experiments confirmed that A549 cells undergo apoptosis when treated with complex 1 followed by photoactivation. In conclusion, this present study suggests that complex 1 might be a promising candidate for photochemotherapy with photoproducts that possess selective anticancer effects in vitro. These results are encouraging to probe the potential activity of this complex in vivo.

Novel Platinum(II) and Platinum(IV) Antitumor Agents that Exhibit Potent Cytotoxicity and Selectivity.

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.

Drug-free phototherapy of superficial tumors: White light at the end of the tunnel.

Visible light has long been recognized as a treatment for many diseases and an essential component of photo-induced chemotherapy. While previous data proved its inherent cytotoxicity, this study is the first to explore the use of a commercially available, high-intensity white LED light (24.5 mW.cm-2) as a treatment for skin tumors. After a 9-h exposure in vitro, the viability of Human Malignant Melanoma cells (A375) decreased by around 70%. Western blot analysis suggested an apoptotic cell death confirmed by the upregulation of Bax, cleaved PARP/caspase-3/8, cytochrome c, and t-bid. Additionally, cellular ROS accumulation and DNA damage were induced upon irradiation with blue light. When tested on a DMBA/TPA skin carcinogenesis model, a 90-min exposure to white light thrice weekly resulted in a significant decrease in tumor volumes/incidence compared to control and cisplatin groups, and restored normal morphological features, as confirmed by histopathology. Toxicological evaluation of ight-treated animals indicated a 100% survival rate, no skin irritation, no signs of discomfort or changes in body weight/behavior, and no toxicities to vital organs. Although these results must be confirmed by further studies, this research showed that short-exposure by commercially available high-intensity white LED light irradiation may be a promising approach for the treatment of superficial malignancies.

A photoactivatable chemotherapeutic Ru(II) complex bearing bathocuproine ligand efficiently induces cell death in human malignant melanoma cells through a multi-mechanistic pathway.

Photoactivated chemotherapy (PACT) is an emerging strategy for targeted cancer therapy. Strained Ru complexes with pseudo-octahedral geometry may undergo photo-induced ligand dissociation, forming aquated photoproducts that are significantly more cytotoxic compared to the precursor complex. The complexes investigated were the strained complex [Ru(bpy)2BC]Cl2 (where bpy = 2,2'-bipyridine and BC = bathocuproine) and its unstrained control [Ru(bpy)2phen]Cl2 (where phen = 1,10-phenanthroline). The uptake of [Ru(bpy)2BC]Cl2, assessed by ICP/MS, started immediately post-incubation and plateaued after 24 h. Active transport was found as the main mode of intracellular transport. Cell viability assays on A375 cells indicated a mean phototoxicity index of 340-fold, and the effect was shown to be primarily mediated by the aquated photoproducts rather than the dissociating ligands. A significant increase in ROS production and DNA damage was also observed. Flow cytometry confirmed the induction of early apoptosis at 48 h that proceeds to late apoptosis/necrosis by 72 h post-treatment. Western blot analysis of pro- and anti-apoptotic proteins revealed that apoptosis was mediated through an interplay between the intrinsic and extrinsic pathways, as well as autophagy and via inhibition of the MAPK and PI3K pathways. In conclusion, this study demonstrates that [Ru(bpy)2BC]Cl2 is a multi-mechanistic PACT drug which exhibits promising anticancer potential.

A photoactivatable Ru (II) complex bearing 2,9-diphenyl-1,10-phenanthroline: A potent chemotherapeutic drug inducing apoptosis in triple negative human breast adenocarcinoma cells.

The photoactivatable Ru (II) complex 1 [Ru(bipy)2(dpphen)]Cl2 (where bipy = 2,2'-bipyridine and dpphen = 2,9-diphenyl-1,10-phenanthroline) has been shown to possess promising anticancer activity against triple negative adenocarcinoma MDA-MB-231 cells. The present study aims to elucidate the plausible mechanism of action of the photoactivatable complex 1 against MDA-MB-231 cells. Upon photoactivation, complex 1 exhibited time-dependent cytotoxic activity with a phototoxicity index (P Index) of >100 after 72 h. A significant increase in cell rounding and detachment, loss of membrane integrity, ROS accumulation and DNA damage was observed. Flow cytometry and a fluorescent apoptosis/necrosis assay showed an induction of cell apoptosis. Western blot analysis revealed the induction of intrinsic and extrinsic pathways and inhibition of the MAPK and PI3K pathways. The photoproduct of complex 1 showed similar effects on key apoptotic protein expression confirming that it is behind the observed cell death. In conclusion, the present study revealed that complex 1 is a potent multi-mechanistic photoactivatable chemotherapeutic drug that may serve as a potential lead molecule for targeted cancer chemotherapy.

The anti-cancer effect of series of strained photoactivatable Ru(II) polypyridyl complexes on non-small-cell lung cancer and triple negative breast cancer cells.

Ruthenium complexes have been recently reported as potential chemotherapeutic agents that offer tumor selectivity and low tumor resistance. This study investigates the photochemistry and the effect of four strained photoactivatable polypyridyl ruthenium(II) complexes on non-small-cell lung cancer (A549) and triple negative breast cancer (MDA-MB-231) cells. All four ruthenium(II) complexes, [Ru(bpy)2dmbpy]Cl2 (C1) where (bpy = 2,2'-bipyridine and dmbpy = 6,6'-dimethyl-2,2'-bipyridine), [Ru(phen)2dmbpy]Cl2 (C2) where (phen = 1,10-phenanthroline), [Ru(dpphen)2dmbpy]Cl2 (C3) (where dpphen = 4,7-diphenyl-1,10-phenanthroline) and [Ru(BPS)2dmbpy]Na2 (C4) where (BPS = bathophenanthroline disulfonate) eject the dmbpy ligand upon activation by blue light. Determination of the octanol-water partition coefficient (log P) revealed that C3 was the only lipophilic complex (log P = 0.42). LC-MS/MS studies showed that C3 presented the highest cellular uptake. The cytotoxic effect of the complexes was evaluated with and without blue light activation using WST-1 kit. Data indicated that C3 exhibited the highest cytotoxicity after 72 h (MDA-MB-231, IC50 = 0.73 µM; A549, IC50 = 1.26 µM) of treatment. The phototoxicity indices of C3 were 6.56 and 4.64 for MDA-MB-230 and A549, respectively. Upon light activation, C3 caused significant ROS production and induced apoptosis in MDA-MB-231 cells as shown by flow cytometry. It also significantly increased Bax/Bcl2 ratio and PERK levels without affecting caspase-3 expression. C3 exhibited poor dark toxicity (IC50 = 74 µM) on rat mesenchymal stem cells (MSCs). In conclusion, the physical property of the complexes dictated by the variable ancillary ligands influenced cellular uptake and cytotoxicity. C3 may be considered a promising selective photoactivatable chemotherapeutic agent that induces ROS production and apoptosis.

In vivo and in vitro anti-inflammatory activity evaluation of Lebanese Cannabis sativa L. ssp. indica (Lam.).

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.

Rapid quantification of ruthenium(ii) polypyridyl anti-cancer drugs using a selective ligand dissociation LC-MS/MS method.

Research on Ru anti-cancer drugs is on the rise with many complexes in clinical trials. Inductively coupled plasma-mass spectrometry (ICP-MS) has been the standard technique for bioanalytical studies on Ru and Pt complexes in biological media. Tedious ICP-MS methods rely on detecting and quantifying the element while lacking important structural information of the original complexes. Despite being equally sensitive, more accessible, and highly selective to the target species, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has not been validated for the analysis of Ru drugs. Using USFDA guidelines, we report here the optimization and validation of a facile LC-MS/MS method for the detection and quantification of three Ru(ii) polypyridyl complexes in cells, plasma, and urine matrices. Importantly, a fast (10 min), single-step procedure was efficient for both extraction and sample purification, and analytes were rapidly eluted over a 3 min simple isocratic run. Specific parent ions were differentially fragmented by tandem MS, thus forming a unique and rational ligand dissociation chemistry that exhibits high selectivity to the target species with no measurable interferences or matrix effects. The developed LC-MS/MS method was advantageous vis-à-vis the prototypical ICP-MS based techniques both in vitro and in vivo, paving the way for its utilization in elaborate cellular uptake, pharmacokinetics, and pharmacodynamics studies.

Synthesis, biological evaluation and modeling of hybrids from tetrahydro-1H-pyrazolo[3,4-b]quinolines as dual cholinestrase and COX-2 inhibitors.

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.

Himachalol induces apoptosis in B16-F10 murine melanoma cells and protects against skin carcinogenesis.

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.

In Vitro and In Vivo Evaluation of the Anticancer and Anti-inflammatory Activities of 2-Himachelen-7-ol isolated from Cedrus Libani.

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.

ß-2-Himachalen-6-ol inhibits 4T1 cells-induced metastatic triple negative breast carcinoma in murine model.

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.

Ghrelin modulates intracellular signalling pathways that are critical for podocyte survival.

Damage to podocytes is a key event in glomerulopathies. While energy dense food can contribute to kidney damage, the role of the orixegenic hormone "ghrelin" in podocyte biology is still unknown. In the present study, we investigated the effect of ghrelin on podocyte survival as well as the signalling pathways mediating ghrelin effect in immortalized cultured rat podocytes. RT-PCR analysis revealed that GHS-R1 is expressed in rat podocytes. Western blot analysis showed that ghrelin upregulated COX-2 protein expression in a time and dose-dependent manner. Additionally, ghrelin activated P38 MAPK, AKT, and ERK1/2 pathways and also induced P38 MAPK phosphorylation in high glucose conditions. Ghrelin induced ROS release and dose dependently reduced podocyte survival. Ghrelin mediated podocyte cell death was partially reversed by pharmacologically inhibiting P38 MAPK or phospholipase C (PLC). Furthermore, PLC inhibitor (U73122) inhibited ghrelin induced P38 MAPK activation. While PI3K inhibitor (LY294002) was without effect on cell survival or P38 MAPK activation, it inhibited ghrelin induced ERK1/2 phosphorylation. Finally, ghrelin induced TAU phosphorylation was reversed by pharmacologic inhibitors of either P38 MAPK or PKA. In conclusion, ghrelin activated harmful molecular pathways in podocytes that can be damaging to the glomerular filtration barrier SIGNIFICANCE OF THE STUDY: Endocrine derangements secondary to obesity are major players in the aetiology of renal injuries. Furthermore, energy dense diet is thought to be the major element in developing obesity. Appetite and increase in energy intake are regulated by complex hormonal pathways which mainly include the orexigenic hormone "ghrelin" in addition to leptin. To date no study have highlighted a significant role for ghrelin in kidney biology, and therefore, it is thought that its endocrine effect is mostly limited to adipose tissue metabolism and appetite regulation. In this study, we first showed that ghrelin receptor is expressed on glomerular podocytes. Also, ghrelin showed negative impact on podocyte survival through modulating signalling pathways such as P38 MAPK and AKT known to play a key role in podocyte health. Moreover, the negative effects of ghrelin on podocytes were further exacerbated in hyperglycemic conditions. Of note, podocytes contribute to the formation and the maintenance of the glomerular filtration barrier and thus are important for normal renal function. Therefore, ghrelin secretion in the context of obesity could be involved in the aetiology of kidney injury, a well-known hallmark found in obese patients.

Enhanced cellular uptake and photochemotherapeutic potential of a lipophilic strained Ru(ii) polypyridyl complex.

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.

The chemotherapeutic effect of ß-2-himachalen-6-ol in chemically induced skin tumorigenesis.

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.

ß-2-himachalen-6-ol protects against skin cancer development in vitro and in vivo.

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.

Antitumor activity of ß-2-himachalen-6-ol in colon cancer is mediated through its inhibition of the PI3K and MAPK pathways.

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.