Apoptosis induction of essential oils from Artemisia arborescens L. in human prostate cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer originates from cells inside a gland, which begin to grow out of control. In the world, prostate cancer is the most common cancer in the male population. New therapeutic strategies are needed for this tumor which still has a high mortality. A. arborescens leaves and aerial parts have various ethnopharmacological uses such as anti-spasmodic, and their decoctions were used to resolve urticaria, neuralgia and several lung diseases. Often this species has been also used to treat different inflammatory-related diseases such as cancer. AIM OF THE STUDY: In a continuation of our research on essential oils from medicinal plants, we have selected, two essential oils from Artemisia arborescens L. (Compositae), an aromatic shrub widely used in traditional medicine. We evaluated their pro-apototic effect on androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. In this study, we also evaluated the anti-Signal transducer and transcription factor 3 (STAT-3) activity of both essential oils in the human prostate cancer cell lines, and the treatment with Tumor necrosis factor (TNF)-Related Apoptosis (TRAIL). MATERIALS AND METHODS: The cells were exposed to essential oils for 72 h and cell viability and cell membrane integrity were evaluated. Genomic DNA and the activity of caspase-3 was tested to confirm the cell death for apoptosis. Western blot analysis was employed to evaluate the expression of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, Hsp70, STAT-3 and SOD proteins. Assays to evaluate reactive oxygen species (ROS) and GSH levels were also performed. RESULTS: The results showed the capacity of two essential oils to activate an apoptotic process increasing the inhibition of Hsp70 and STAT-3 protein expression. In addition, our natural products sensitize LNCaP cells to Tumor necrosis factor (TNF)-Related Apoptosis (TRAIL)-induced apoptosis. CONCLUSIONS: In summary, our study provides a further contribution to the hypothesis of the use of essential oils, from traditional medicinal plants, for the treatment of tumors, and suggests that the combination of our samples with other anti-prostate cancer therapies could be used to affect prostate cancer.

Green Synthesis of Near-Infrared Plasmonic Gold Nanostructures by Pomegranate Extract and Their Supramolecular Assembling with Chemo- and Photo-Therapeutics.

Au nanostructures exhibiting a localized surface plasmon resonance in the near-infrared spectral window are obtained in a single, green step at room temperature by pomegranate extract in the presence of a highly biocompatible ß-cyclodextrin branched polymer, without the need of preformed seeds, external reducing and sacrificial agents, and conventional surfactants. The polymeric component makes the Au nanostructures dispersible in water, stable for weeks and permits their supramolecular assembling with the chemotherapeutic sorafenib and a nitric oxide (NO) photodonor (NOPD), chosen as representative for chemo- and photo-therapeutics. Irradiation of the plasmonic Au nanostructures in the therapeutic window with 808 nm laser light results in a good photothermal response, which (i) is not affected by the presence of either the chemo- or the phototherapeutic guests and (ii) does not lead to their photoinduced decomposition. Besides, irradiation of the hybrid Au nanoassembly with the highly biocompatible green light results in the NO release from the NOPD with efficiency similar to that observed for the free guest. Preliminary biological experiments against Hep-G2 hepatocarcinoma cell lines are also reported.

A Supramolecular Nanoassembly of Lenvatinib and a Green Light-Activatable NO Releaser for Combined Chemo-Phototherapy.

The chemotherapeutic Lenvatinib (LVB) and a nitric oxide (NO) photodonor based on a rhodamine antenna (RD-NO) activatable by the highly compatible green light are supramolecularly assembled by a ß-cyclodextrin branched polymer (PolyCD). The poorly water-soluble LVB and RD-NO solubilize very well within the polymeric host leading to a ternary supramolecular nanoassembly with a diameter of ~55 nm. The efficiency of the NO photorelease and the typical red fluorescence of RD-NO significantly enhance within the polymer due to its active role in the photochemical and photophysical deactivation pathways. The co-presence of LVB within the same host does not affect either the nature or the efficiency of the photoinduced processes of RD-NO. Besides, irradiation of RD-NO does not lead to the decomposition of LVB, ruling out any intermolecular photoinduced process between the two guests despite sharing the same host. Ad-hoc devised Förster Resonance Energy Transfer experiments demonstrate this to be the result of the not close proximity of the two guests, which are confined in different compartments of the same polymeric host. The supramolecular complex is stable in a culture medium, and its biological activity has been evaluated against HEP-G2 hepatocarcinoma cell lines in the dark and under irradiation with visible green light, using LVB at a concentration well below the IC50. Comparative experiments performed using the polymeric host encapsulating the individual LVB and RD-NO components under the same experimental conditions show that the moderate cell mortality induced by the ternary complex in the dark increases significantly upon irradiation with visible green light, more likely as the result of synergism between the NO photogenerated and the chemotherapeutic.

Oregano (Origanum vulgare L.) essential oil provides anti-inflammatory activity and facilitates wound healing in a human keratinocytes cell model.

Skin acts as a protective barrier between the body and the external environment. Skin wounds are a common inflammatory disorder for the solution of which plants and essential oils have been applied as a medical option for centuries. Origanum vulgare essential oil (OEO) is largely used in folk medicine, but its molecular mechanisms of action are not fully known. In this study, we evaluated the anti-inflammatory/antioxidant activity as well as wound healing capacity of a well-characterized OEO on human keratinocytes NCTC 2544 treated with interferon-gamma (IFN-γ) and histamine (H) or subjected to a scratch test. The expression of pro-inflammatory mediators such as reactive oxygen species (ROS), inter-cellular adhesion molecule (ICAM)-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 were verified. The DNA damage was shown by the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) and activation of proliferating cell nuclear antigen (PCNA). Moreover, the abnormal modification of extracellular matrix components (ECM) was examined by determining matrix metalloproteinase (MMP)-1, and -12. Compared to untreated control, OEO showed efficacy in supporting and enhancing the cell motility. In IFN-γ and H treated cells, OEO displayed a significant reduction of ROS, ICAM-1, iNOS, COX-2, 8-OHdG, MMP-1, and MMP-12. OEO proved useful to treat inflammation and support cell motility during wound healing.

Moscatilin, a bibenzyl derivative from the orchid Dendrobium loddigesii, induces apoptosis in melanoma cells.

The use of orchids in herbal medicine has a very long history. Dendrobium species are known to produce a variety of secondary metabolites such as phenanthrens, bibenzyls, fluorenones and sesquiterpenes, and alkaloids and are responsible for their wide variety of medicinal properties. For decades, bibenzyls, which are the main bioactive components derived from Dendrobium species, have been subjected to extensive investigation as likely candidates for cancer treatment. The present study was undertaken to investigate the effect of moscatilin, a bibenzyl derivative from the orchid Dendrobium loddigesii on human melanoma cells. In A375 cells compound moscatilin showed a clear dose-response relationship in the range of 6.25-50 µM concentrations. In addition, we demonstrated an apoptotic response after treatment of cancer cells with this bibenzyl compound at 6.25 and 12.5 µM concentrations that probably involves PTEN activity, inhibition of Hsp70 expression and reactive oxygen species production. Alternatively, the inhibition of the caspase cascade at higher concentrations, 25 and 50 µM, correlated with additional reactive oxygen species increase, probably switched the mode of moscatilin-induced cell death from apoptosis to necrosis.

Isocordoin analogues promote apoptosis in human melanoma cells via Hsp70.

Isocordin 1 and a series of 4-oxyalkyl-isocordoin analogues 2-8 were evaluated for their cytotoxicity effect against human melanoma cells (A2058). Analogues 4, 5, and 6 showed a higher inhibitory activity with IC50 values of 12.91 ± 0.031, 24.88 ± 0.013, and 11.62 ± 0.017, respectively. These analogues, 4, 5, and 6, also induced an apoptotic response at 12.5- and 25-µM concentrations. They inhibited the expression of antiapoptotic proteins Bcl-2 and Hsp70, a critical factor that promotes tumour cell survival. In contrast, Bax and caspase-9 expression, and caspase-3 enzyme resulted activated. These results were correlated to a DNA fragmentation typical of apoptosis and an increase of intracellular reactive oxygen species (ROS) levels. Alternatively, at higher concentration (50 µM), when the capacity of the cells to sustain Hsp70 synthesis is reduced, our results seem to indicate that necrosis was induced by a further increase in ROS production. Therefore, the central finding in the present study is that these molecules downregulates Hsp70 expression. Altogether, these results suggest that 4-oxyalkyl-isocordoin analogues 4, 5, and 6 deserve to be deeply investigated for a possible application as Hsp70 inhibitor in the management of melanoma.

Antigrowth activity and induction of apoptosis in human melanoma cells by Drymis winteri forst extract and its active components.

Melanoma is a highly invasive cancer that resists most conventional treatments. Therefore, there is an urgent need to identify alternative anticancer agents able to affect new molecular targets. Drimys winteri (Winteraceae) is a medicinal plant, employed in Brazil and many countries, in folk medicine against a variety of ailments, especially for the treatment of fevers, ulcers, pains, affections of respiratory tract and cancers. Previous phytochemical studies have isolated and identified the presence of diverse classes of secondary metabolites in this plant such as sesquiterpenes. In an ongoing to identify new natural anticancer compounds for the treatment and/or prevention of melanoma, we study the effects of Drimys winteri bark ethyl acetate extract and its sesquiterpenes drimenol, nordrimenone, isonordrimenone and polygodial on human melanoma cells. The treatment of melanoma cells with extract, drimenol, isordrimenone and polygodial resulted in a significant reduction in cell viability. But, polygodial showed the highest inhibitory growth activity. In addition, we reported an apoptotic response after treatment with drimenol, isordrimenone and polygodial that probably involves the reduction of Hsp70 expression and reactive oxygen species production. Alternatively, the inhibition of caspase cascade at higher concentrations, correlated with additional reactive oxygen species increase, probably switches natural product-induced cell death from apoptosis to necrosis. Therefore, this evidence provides a scientific support for the anticancer employ of Drimys winteri in traditional medicinal and suggests that active molecules can be considered potential candidates to be tested also in in vivo models, alone or in combination with chemotherapy agents, for the management of melanoma.

Nanotechnological Approach to Increase the Antioxidant and Cytotoxic Efficacy of Crocin and Crocetin.

Crocin and crocetin are two interesting constituents of saffron (Crocus sativus) that possess important biological activities. Their use as therapeutic agents is strongly compromised by a scarce stability, poor absorption, and low bioavailability. Therefore, to improve these unfavorable features, the aim of the present work has been to apply a nanotechnological approach based on the formulation of solid lipid nanoparticles containing crocin and crocetin. Solid lipid nanoparticles were formulated according to crocin and crocetin chemical properties, using a variation of the quasi-emulsion solvent diffusion method to formulate crocin-solid lipid nanoparticles, while crocetin-solid lipid nanoparticles were prepared following the solvent diffusion method. Morphology and dimensional distribution of solid lipid nanoparticles have been characterized by differential scanning calorimetry and photon correlation spectroscopy, respectively, while the effect of drug incorporation versus time has been studied by Turbiscan technology. In order to verify the role of the nanotechnological approach on the biological activities of crocin and crocetin, the antioxidant and antiproliferative effects of these carotenoids once incorporated in lipid nanoparticles have been evaluated. For this aim, the oxygen radical absorbance capacity assay and the MTT test were used, respectively.The results pointed out the formulation of nanometric dispersions endowed with high homogeneity and stability, with an encapsulation efficiency ranging from 80 (crocetin-solid lipid nanoparticles) to 94% (crocin-crocetin). The oxygen radical absorbance capacity assay evidenced an interesting and prolonged antioxidant activity of crocin and crocetin once encapsulated in solid lipid nanoparticles, while the nanoencapsulation strategy showed a different mechanism in ameliorating the cytotoxic effect of these two substances.

Melaleuca styphelioides Sm. Polyphenols Modulate Interferon Gamma/Histamine-Induced Inflammation in Human NCTC 2544 Keratinocytes.

Melaleuca styphelioides, known as the prickly-leaf tea tree, contains a variety of bioactive compounds. The purposes of this study were to characterize the polyphenols extracted from Melaleuca styphelioides leaves and assess their potential antioxidant and anti-inflammatory effects. The polyphenol extracts were prepared by maceration with solvents of increasing polarity. The LC/MS-MS technique was used to identify and quantify the phenolic compounds. An assessment of the radical scavenging activity of all extracts was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS⁺), and ferric reducing antioxidant power (FRAP) assays. The anti-inflammatory activity was determined on interferon gamma (IFN-γ)/histamine (H)-stimulated human NCTC 2544 keratinocytes by Western blot and RT-PCR. Compared to other solvents, methanolic extract presented the highest level of phenolic contents. The most frequent phenolic compounds were quercetin, followed by gallic acid and ellagic acid. DPPH, ABTS⁺, and FRAP assays showed that methanolic extract exhibits strong concentration-dependent antioxidant activity. IFN-γ/H treatment of human NCTC 2544 keratinocytes induced the secretion of high levels of the pro-inflammatory mediator inter-cellular adhesion molecule-1 (ICAM-1), nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB), which were inhibited by extract. In conclusion, the extract of Melaleuca styphelioides leaves is rich in flavonoids, and presents antioxidant and anti-inflammatory proprieties. It can be proposed as a useful compound to treat inflammatory skin diseases.

Involvement of Bax and Bcl-2 in Induction of Apoptosis by Essential Oils of Three Lebanese Salvia Species in Human Prostate Cancer Cells.

Prostate cancer is one of the most common forms of cancer in men, and research to find more effective and less toxic drugs has become necessary. In the frame of our ongoing program on traditionally used Salvia species from the Mediterranean Area, here we report the biological activities of Salvia aurea, S. judaica and S. viscosa essential oils against human prostate cancer cells (DU-145). The cell viability was measured by 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) test and lactate dehydrogenase (LDH) release was used to quantify necrosis cell death. Genomic DNA, caspase-3 activity, expression of cleaved caspase-9, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X (Bax) proteins were analyzed in order to study the apoptotic process. The role of reactive oxygen species in cell death was also investigated. We found that the three essential oils, containing caryophyllene oxide as a main constituent, are capable of reducing the growth of human prostate cancer cells, activating an apoptotic process and increasing reactive oxygen species generation. These results suggest it could be profitable to further investigate the effects of these essential oils for their possible use as anticancer agents in prostate cancer, alone or in combination with chemotherapy agents.

Effect of Three Centaurea Species Collected from Central Anatolia Region of Turkey on Human Melanoma Cells.

Centaurea is the largest genus within the Asteraceae family. Many members of this genus are used in traditional folk medicine, such as Centaurea pulchella used to treat skin problems such as to resolve the abscess. Although biological activities of many Centaurea species have been investigated in different countries and Turkey, cytotoxic effect of C. patula, C. pulchella and C. tchihatcheffii has not been studied yet. Melanoma is one of the most invasive and deadly forms of skin cancer. Therefore, in an ongoing effort to identify new natural anticancer products for the treatment and/or prevention of melanoma cancer, the present study was undertaken to investigate the effect of these Centaurea species, collected from Central Anatolia region of Turkey on cell growth and death in human melanoma cell line, A375.The results revealed that all extracts were able to inhibit, after 48 h of treatment, the growth of cancer cells, that could be related to an overall action of the phenolic compounds present. In fact, C. pulchella, with the highest level of phenolics, showed a major activity followed by C. patula and C. tchihatcheffii. Our data also demonstrate that these natural products induce apoptotic cell death. In conclusion, the study of plant extracts for their cytotoxic and apoptotic properties has shown that medicinal herbs from Centaurea species might have also importance in the prevention and treatment of melanoma.

Psoralea glandulosa as a potential source of anticancer agents for melanoma treatment.

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on melanoma cancer, the present study was undertaken to investigate the biological activity of the resinous exudate of aerial parts from Psoralea glandulosa, and its active components (bakuchiol (1), 3-hydroxy-bakuchiol (2) and 12-hydroxy-iso-bakuchiol (3)) against melanoma cells (A2058). In addition, the effect in cancer cells of bakuchiol acetate (4), a semi-synthetic derivative of bakuchiol, was examined. The results obtained show that the resinous exudate inhibited the growth of cancer cells with IC50 value of 10.5 µg/mL after 48 h of treatment, while, for pure compounds, the most active was the semi-synthetic compound 4. Our data also demonstrate that resin is able to induce apoptotic cell death, which could be related to an overall action of the meroterpenes present. In addition, our data seem to indicate that the apoptosis correlated to the tested products appears, at least in part, to be associated with an increase of reactive oxygen species (ROS) production. In summary, our study provides the first evidence that P. glandulosa may be considered a source of useful molecules in the development of analogues with more potent efficacy against melanoma cells.

Clinical evaluation of Moro (Citrus sinensis (L.) Osbeck) orange juice supplementation for the weight management.

In the last years, several studies have recently evaluated the beneficial effects of red orange juice (Citrus sinensis (L.) Osbeck) and its active components in weight management and obesity. Moro orange is a cultivar of red orange, particularly rich in active compounds such as anthocyanins, hydroxycinnamic acids, flavone glycosides and ascorbic acid, which displays anti-obesity effects in in vitro and in vivo studies. In this clinical study, the effect of a Moro juice extract (Morosil(®), 400 mg/die) supplementation was evaluated in overweight healthy human volunteers for 12 weeks. Results showed that Moro juice extract intake was able to induce a significant reduction in body mass index (BMI) after 4 weeks of treatment (p < 0.05). Moreover, in subjects treated with Moro extract, body weight, BMI, waist and hip circumference were significantly different from the placebo group (p < 0.05). In conclusion, it could be suggested that the active compounds contained in Moro juice have a synergistic effect on fat accumulation in humans and Moro juice extract can be used in weight management and in the prevention of human obesity.

Comparison of essential oil components and in vitro anticancer activity in wild and cultivated Salvia verbenaca.

The objectives of our research were to study the chemical composition and the in vitro anticancer effect of the essential oil of Salvia verbenaca growing in natural sites in comparison with those of cultivated (Sc) plants. The oil from wild (Sw) S. verbenaca presented hexadecanoic acid (23.1%) as the main constituent, while the oil from Sc plants contained high quantities of hexahydrofarnesyl acetone (9.7%), scarce in the natural oil (0.7%). The growth-inhibitory and proapoptotic effects of the essential oils from Sw and Sc S. verbenaca were evaluated in the human melanoma cell line M14, testing cell vitality, cell membrane integrity, genomic DNA fragmentation and caspase-3 activity. Both the essential oils were able to inhibit the growth of the cancer cells examined inducing also apoptotic cell death, but the essential oil from cultivated samples exhibited the major effects.

A multifunctional bichromophoric nanoaggregate for fluorescence imaging and simultaneous photogeneration of RNOS and ROS.

We report the design, preparation, and properties of a nanoaggregate formed in phosphate buffer solution by a porphyrin-ß-cyclodextrin (ß-CD) conjugate and a nitric oxide photodonor tailored to fit the ß-CD cavity. The small nanoassembly with a diameter of about 13 nm exhibits the typical red fluorescence of the porphyrin chromophore. The empty cavity of the ß-CD unit in the nanoaggregate is able to accommodate the NO photodonor, thereby forming a supramolecular bichromophoric aggregate with diameter of about 16 nm. This nanoconstruct preserves the fluorescence of the porphyrin core and is able to generate nitric oxide and singlet oxygen under illumination with visible light. The nanoassembly internalizes in melanoma cells, can be mapped therein by fluorescence microscopy, and induces a significant level of cell mortality, probably due to the combined action of reactive nitrogen oxide species (RNOS) and reactive oxygen species (ROS).

An engineered nanoplatform for bimodal anticancer phototherapy with dual-color fluorescence detection of sensitizers.

A multifunctional nanoplatform with four-in-one photoresponsive functionalities has been achieved through the co-encapsulation of two chromo-fluorogenic components within biocompatible polymeric nanoparticles. This engineered nanoconstruct efficiently delivers different photosensitizers in melanoma cells, which can be detected through their dual-color fluorescence, and induces amplified cell mortality due to the simultaneous photogeneration of singlet oxygen and nitric oxide.

Protective effects of an extract from Citrus bergamia against inflammatory injury in interferon-γ and histamine exposed human keratinocytes.

AIMS: The present work evaluated the anti-inflammatory/antioxidant activity of a well characterized extract from Citrus bergamia Risso and Poiteau (CBE), containing neoeriocitrin, naringin, neohesperidin and other flavonoids, on human NCTC 2544 keratinocytes treated with interferon-gamma (IFN-γ) and histamine (H). MAIN METHODS: High performance liquid chromatography (HPLC) coupled with diode array detectors was used to characterize and quantify phenolic compounds in CBE. Anti-inflammatory/antioxidant ability on keratinocytes was determined through evaluation of inter-cellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) expression by Western blot, production of nitric oxide (NO) with Griess reagent and concentration of reactive oxygen species (ROS) by fluorescent quantitative analysis with 2',7'-dichlorfluorescein-diacetate (DCFH-DA). Cell viability was assessed using 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Antioxidant activity was also measured by oxygen radical absorbance capacity (ORAC) assay. Glycosaminoglycans (GAGs) were quantified using 1.9-dimethyl methylene blue (DMB). KEY FINDINGS: CBE exhibited high antioxidant activity confirmed by elevated ORAC values related to high capacity in oxygen radical scavenging. The assays on keratinocytes demonstrated that CBE does not inhibit cell proliferation and is shown to significantly reduce dose-dependently ICAM-1, iNOS, NO, ROS and GAG production in cells exposed to IFN-γ and H. SIGNIFICANCE: Our study demonstrates that the pools of compounds of an extract from C. bergamia efficiently block the proinflammatory actions induced by IFN-γ and H on human keratinocytes. CBE may be used for topic employment in some inflammatory diseases of the skin and to represent an important opportunity for the essential oil processing industries.