Isothiocyanates Potentiate Tazemetostat-Induced Apoptosis by Modulating the Expression of Apoptotic Genes, Members of Polycomb Repressive Complex 2, and Levels of Tri-Methylating Lysine 27 at Histone 3 in Human Malignant Melanoma Cells.

In this study, we utilized an in vitro model consisting of human malignant melanoma as well as non-tumorigenic immortalized keratinocyte cells with the aim of characterizing the therapeutic effectiveness of the clinical epigenetic drug Tazemetostat alone or in combination with various isothiocyanates. In doing so, we assessed markers of cell viability, apoptotic induction, and expression levels of key proteins capable of mediating the therapeutic response. Our data indicated, for the first time, that Tazemetostat caused a significant decrease in viability levels of malignant melanoma cells in a dose- and time-dependent manner via the induction of apoptosis, while non-malignant keratinocytes were more resistant. Moreover, combinatorial treatment protocols caused a further decrease in cell viability, together with higher apoptotic rates. In addition, a significant reduction in the Polycomb Repressive Complex 2 (PRC2) members [e.g., Enhancer of Zeste Homologue 2 (EZH2), Embryonic Ectoderm Development (EED), and suppressor of zeste 12 (SUZ12)] and tri-methylating lysine 27 at Histone 3 (H3K27me3) protein expression levels was observed, at least partially, under specific combinatorial exposure conditions. Reactivation of major apoptotic gene targets was determined at much higher levels in combinatorial treatment protocols than Tazemetostat alone, known to be involved in the induction of intrinsic and extrinsic apoptosis. Overall, we developed an optimized experimental therapeutic platform aiming to ensure the therapeutic effectiveness of Tazemetostat in malignant melanoma while at the same time minimizing toxicity against neighboring non-tumorigenic keratinocyte cells.

Naturally Derived Phenethyl Isothiocyanate Modulates Induction of Oxidative Stress via Its N-Acetylated Cysteine Conjugated form in Malignant Melanoma.

Phenethyl isothiocyanate (PEITC) is a secondary metabolic product yielded upon the hydrolysis of gluconasturtiin and it is highly accumulated in the flowers of watercress. The aim of the current study was to assess the role of a naturally derived PEITC-enriched extract in the induction of oxidative stress and to evaluate its anti-melanoma potency through the regulation of its metabolism with the concurrent production of the N-acetyl cysteine conjugated by-product. For this purpose, an in vitro melanoma model was utilized consisting of human primary (A375) cells as well as metastatic (COLO-679) malignant melanoma cells together with non-tumorigenic immortalized keratinocytes (HaCaT). Cytotoxicity was assessed via the Alamar Blue assay whereas the antioxidant/prooxidant activity of PEITC was determined via spectrophotometric assays. Finally, kinetic characterization of the end-product of PEITC metabolism was monitored via UPLC coupled to a tandem mass spectrometry (MS/MS). Our results indicate that although PhEF showed very minor antioxidant activity in a cell-free system, in a cell-based system, it can modulate the activity of key enzyme(s) involved in cellular antioxidant defense mechanism(s). In addition, we have shown that PhEF induces lipid and protein oxidation in a concentration-dependent manner, while its cytotoxicity is not only dependent on PEITC itself but also on its N-acetylated cysteine conjugated form.

A Naturally Derived Watercress Flower-Based Phenethyl Isothiocyanate-Enriched Extract Induces the Activation of Intrinsic Apoptosis via Subcellular Ultrastructural and Ca2+ Efflux Alterations in an In Vitro Model of Human Malignant Melanoma.

The aim of the current study was to (i) extract isolated fractions of watercress flowers enriched in polyphenols, phenethyl isothiocyanate and glucosinolates and (ii) characterize the anticancer mode of action of non-lethal, sub-lethal and lethal concentrations of the most potent extract fraction in primary (A375) and metastatic (COLO-679) melanoma cells as well as non-tumorigenic immortalized keratinocyte (HaCaT) cells. Cytotoxicity was assessed via the Alamar Blue assay, whereas ultrastructural alterations in mitochondria and the endoplasmic reticulum were determined via transmission electron microscopy. Mitochondrial membrane depolarization was determined using Mito-MP dye, whereas apoptosis was evaluated through the activation of caspases-3, -8 and -9. Among all extract fractions, the phenethyl isothiocyanate-enriched one (PhEF) possessed significant cytotoxicity against A375 and COLO-679 cells, while HaCaT cells remained relatively resistant at sub-lethal and lethal concentrations. Additionally, ultrastructural subcellular alterations associated with apoptosis were observed by means of increased mitochondrial area and perimeter, decreased cristae density and a shorter distance of the endoplasmic reticulum to the mitochondria, all taking place during "early" time points (2-4 h) of exposure. Moreover, PhEF induced mitochondrial membrane depolarization associated with "late" time points (24 h) of exposure, thereby leading to the activation of intrinsic apoptosis. Finally, the inhibition of cytosolic Ca2+ efflux reduced levels of caspases-9 and -3 activity, suggesting the involvement of Ca2+ efflux in modulating the activation of intrinsic apoptosis. To conclude, our data demonstrate an association of "early" ultrastructural alterations in mitochondria and the endoplasmic reticulum with the "late" induction of intrinsic apoptosis via the modulation of Ca2+ efflux.

Chemical Profiling and Antioxidant and Anti-Amyloid Capacities of Salvia fruticosa Extracts from Greece.

An increasingly common ailment in elderly persons is Alzheimer's disease (AD), a neurodegenerative illness. Present treatment is restricted to alleviating symptoms; hence, there is a requirement to develop an effective approach to AD treatment. Salvia fruticosa (SF) is a medicinal plant with a documented neuroprotective potential. To identify extracts of increased neuroprotectivity, we partitioned the methanolic extract of SF aerial parts from Greece into several fractions, by employing solvents of different polarities. The fractions were chemically identified and evaluated for their antioxidancy and anti-neurotoxic potential against amyloid beta peptides 25-35 (Aß25-35). Carnosol and carnosic acid were among the prominent compounds, while all partitions showed significant antioxidant capacity, with the diethyl ether and ethyl acetate partitions being the most potent. These, along with the aqueous and the butanolic fractions, demonstrated statistically significant anti-neurotoxic potential. Thus, our findings further validate the neuroprotective potential of SF and support its ethnopharmacological usage as an antioxidant. The particular properties found define SF as a promising source for obtaining extracts or bioactive compounds, possibly beneficial for generating AD-related functional foods or medications. Finally, our results encourage plant extract partitioning for acquiring fractions of enhanced biological properties.

Catching up with tetrazines: coordination of Re(I) to 1,2,4-triazine facilitates an inverse electron demand Diels-Alder reaction with strained alkynes to a greater extent than in corresponding 1,2,4,5-tetrazines.

Inverse electron demand Diels Alder (IEDDA) reactions of 1,2,4-triazines are of interest to biorthogonal chemistry but suffer from slow kinetics. It is shown here that coordination of Re(I) to a 1,2,4-triazine ring speeds up the IEDDA reaction with bicyclooctyne (BCN) by a factor of 55. Comparative analysis with corresponding 1,2,4,5-tetrazine analogues reveals that the origin of the increased reactivity is markedly different and more profound than in tetrazine analogues. DFT calculations and subsequent analysis indicated the greater increase for the triazine than the tetrazines on coordination could be attributed to the triazine's lower distortion energy and more favourable interaction energy for the triazine, the latter attributable to lower Pauli repulsion than the tetrazines rather than to favourable frontier orbital energies.

Chemical Characterization and Biological Evaluation of Epilobium parviflorum Extracts in an In Vitro Model of Human Malignant Melanoma.

Malignant melanoma is an aggressive type of skin cancer characterised by high metastatic capacity and mortality rate. On the other hand, Epilobium parviflorum is known for its medicinal properties, including its anticancer potency. In this context, we aimed to (i) isolate various extracts of E. parviflorum, (ii) characterize their phytochemical content, and (iii) determine their cytotoxic potential in an in vitro model of human malignant melanoma. To these ends, we utilized various spectrophotometric and chromatographic (UPLC-MS/MS) approaches to document the higher content of the methanolic extract in polyphenols, soluble sugars, proteins, condensed tannins, and chlorophylls -a and -b as opposed to those of dichloromethane and petroleum. In addition, the cytotoxicity profiling of all extracts was assessed through a colorimetric-based Alamar Blue assay in human malignant melanoma (A375 and COLO-679) as well as non-tumorigenic immortalized keratinocyte (HaCaT) cells. Overall, the methanolic extract was shown to exert significant cytotoxicity, in a time- and concentration-dependent manner, as opposed to the other extracts. The observed cytotoxicity was confined only to human malignant melanoma cells, whereas non-tumorigenic keratinocyte cells remained relatively unaffected. Finally, the expression levels of various apoptotic genes were assessed by qRT-PCR, indicating the activation of both intrinsic and extrinsic apoptotic cascades.

Sideritis scardica Extracts Demonstrate Neuroprotective Activity against Aß25-35 Toxicity.

Alzheimer's disease (AD) is the most prevalent neurodegenerative condition, primarily affecting seniors. Despite the significant time and money spent over the past few decades, no therapy has been developed yet. In recent years, the research has focused on ameliorating the cytotoxic amyloid beta (Aß) peptide aggregates and the increased elevated oxidative stress, two interconnected main AD hallmarks. Medicinal plants constitute a large pool for identifying bioactive compounds or mixtures with a therapeutic effect. Sideritis scardica (SS) has been previously characterized as neuroprotective toward AD. We investigated this ability of SS by generating eight distinct solvent fractions, which were chemically characterized and assessed for their antioxidant and neuroprotective potential. The majority of the fractions were rich in phenolics and flavonoids, and all except one showed significant antioxidant activity. Additionally, four SS extracts partly rescued the viability in Aß25-35-treated SH-SY5Y human neuroblastoma cells, with the initial aqueous extract being the most potent and demonstrating similar activity in retinoic-acid-differentiated cells as well. These extracts were rich in neuroprotective substances, such as apigenin, myricetin-3-galactoside, and ellagic acid. Our findings indicate that specific SS mixtures can benefit the pharmaceutical industry to develop herbal drugs and functional food products that may alleviate AD.

Chemical Profiling, Antiproliferative and Antimigratory Capacity of Haberlea rhodopensis Extracts in an In Vitro Platform of Various Human Cancer Cell Lines.

Haberlea rhodopensis is a Balkan endemic plant that belongs to the Gesneriaceae family, and is believed to have medicinal use and health-promoting properties. This study aimed to (i) prepare aqueous (HAE) and ethanolic (HEE) extracts from the leaves of H. rhodopensis from in vitro propagated plants, (ii) screen for their potential antiproliferative and antimigratory activities, and (iii) chemically characterize both HAE and HEE by identifying compounds which may contribute to their observed bioactivity thereby further supporting their potential use in biomedical applications. The antiproliferative activity of both extracts was assessed against six human cancer cell lines by employing the sulforhodamine-B (SRB) assay. HEE was found to be more potent in inhibiting cancer cell growth as compared to HAE. Therefore, HEE's antimigratory effects were further studied in hepatocellular carcinoma (HepG2) and non-small cell lung adenocarcinoma (A459) cell lines as they were among the most sensitive ones to its antiproliferative activity. HEE was found to exert significant antimigratory concentration-dependent effects in both cell lines assessed with the wound healing assay. Chemical characterization by UPLC-MS/MS analysis identified that HEE contains higher levels of flavonoids, phenolic compounds, pigments (chlorophyll-/-b, lycopene, and ß-carotene), monoterpenoids, and condensed tannins compared to HAE, while HAE, contains higher levels of soluble protein and sugars. Furthermore, HEE demonstrated remarkable antioxidant activity evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH●), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS●+) and ferric reducing/antioxidant power (FRAP) assays. We have obtained comprehensive results highlighting the potential of HEE as a source of bioactive compounds with anticancer properties. Future studies should aim at identifying the chemical constituents responsible for the bioactivities observed, and focus on investigating HEE's effects, in in vivo preclinical cancer models.

Polyphenolics, glucosinolates and isothiocyanates profiling of aerial parts of Nasturtium officinale (Watercress).

Watercress (Nasturtium officinale) is a rich source of secondary metabolites with disease-preventing and/or health-promoting properties. Herein, we have utilized extraction procedures to isolate fractions of polyphenols, glucosinolates and isothiocyanates to determine their identification, and quantification. In doing so, we have utilized reproducible analytical methodologies based on liquid chromatography with tandem mass spectrometry by either positive or negative ion mode. Due to the instability and volatility of isothiocyanates, we followed an ammonia derivatization protocol which converts them into respective ionizable thiourea derivatives. The analytes' content distribution map was created on watercress flowers, leaves and stems. We have demonstrated that watercress contains significantly higher levels of gluconasturtiin, phenethyl isothiocyanate, quercetin-3-O-rutinoside and isorhamnetin, among others, with their content decreasing from flowers (82.11 ± 0.63, 273.89 ± 0.88, 1459.30 ± 12.95 and 289.40 ± 1.37 ng/g of dry extract respectively) to leaves (32.25 ± 0.74, 125.02 ± 0.52, 1197.86 ± 4.24 and 196.47 ± 3.65 ng/g of det extract respectively) to stems (9.20 ± 0.11, 64.7 ± 0.9, 41.02 ± 0.18, 65.67 ± 0.84 ng/g of dry extract respectivbely). Pearson's correlation analysis has shown that the content of isothiocyanates doesn't depend only on the bioconversion of individual glucosinolates but also on other glucosinolates of the same group. Overall, we have provided comprehensive analytical data of the major watercress metabolites thereby providing an opportunity to exploit different parts of watercress for potential therapeutic applications.

Assessment of Methodological Pipelines for the Determination of Isothiocyanates Derived from Natural Sources.

Isothiocyanates are biologically active secondary metabolites liberated via enzymatic hydrolysis of their sulfur enriched precursors, glucosinolates, upon tissue plant disruption. The importance of this class of compounds lies in their capacity to induce anti-cancer, anti-microbial, anti-inflammatory, neuroprotective, and other bioactive properties. As such, their isolation from natural sources is of utmost importance. In this review article, an extensive examination of the various parameters (hydrolysis, extraction, and quantification) affecting the isolation of isothiocyanates from naturally-derived sources is presented. Overall, the effective isolation/extraction and quantification of isothiocyanate is strongly associated with their chemical and physicochemical properties, such as polarity-solubility as well as thermal and acidic stability. Furthermore, the successful activation of myrosinase appears to be a major factor affecting the conversion of glucosinolates into active isothiocyanates.

Evaluation of Bioactive Properties of Lipophilic Fractions of Edible and Non-Edible Parts of Nasturtium officinale (Watercress) in a Model of Human Malignant Melanoma Cells.

Watercress is an enriched source of phenethyl isothiocyanate (PEITC), among other phytochemicals, with an antioxidant capacity. The aim of this study was to (i) chemically characterize and (ii) biologically evaluate the profile of the main health-promoting compounds contained in edible (i.e., mixture of leaves and lateral buds) and non-edible (i.e., stems) parts of watercress in an in vitro model of malignant melanoma consisting of human malignant melanoma (A375), non-melanoma (A431) and keratinocyte (HaCaT) cells. The extraction of the main constituents of watercress was performed by subjecting the freeze-dried edible and non-edible samples through different extraction protocols, whereas their concentration was obtained utilizing analytical methodologies. In addition, cell viability was evaluated by the Alamar Blue assay, whereas levels of oxidative stress and apoptosis were determined by commercially available kits. The edible watercress sample contained a higher amount of various nutrients and phytochemicals in the hexane fraction compared to the non-edible one, as evidenced by the presence of PEITC, phenolics, flavonoids, pigments, ascorbic acid, etc. The cytotoxicity potential of the edible watercress sample in the hexane fraction was considerably higher than the non-edible one in A375 cells, whereas A431 and HaCaT cells appeared to be either more resistant or minimally affected, respectively. Finally, levels of oxidative stress and apoptotic induction were increased in both watercress samples, but the magnitude of the induction was much higher in the edible than the non-edible watercress samples. Herein, we provide further evidence documenting the potential development of watercress extracts (including watercress waste by-products) as promising anti-cancer agent(s) against malignant melanoma cells.

Chemical and Biological Characterization of the Anticancer Potency of Salvia fruticosa in a Model of Human Malignant Melanoma.

Malignant melanoma is one of the most aggressive types of skin cancer with an increasing incidence worldwide. Thus, the development of innovative therapeutic approaches is of great importance. Salvia fruticosa (SF) is known for its anticancer properties and in this context, we aimed to investigate its potential anti-melanoma activity in an in vitro model of human malignant melanoma. Cytotoxicity was assessed through a colorimetric-based sulforhodamine-B (SRB) assay in primary malignant melanoma (A375), non-malignant melanoma epidermoid carcinoma (A431) and non-tumorigenic melanocyte neighbouring keratinocyte (HaCaT) cells. Among eight (8) different fractions of S. fruticosa extracts (SF1-SF8) tested, SF3 was found to possess significant cytotoxic activity against A375 cells, while A431 and HaCaT cells remained relatively resistant or exerted no cytotoxicity, respectively. In addition, the total phenolic (Folin-Ciocalteu assay) and total flavonoid content of SF extracts was estimated, whereas the antioxidant capacity was measured via the inhibition of tert-butyl hydroperoxide-induced lipid peroxidation and protein oxidation levels. Finally, apoptotic cell death was assessed by utilizing a commercially available kit for the activation of caspases - 3, - 8 and - 9. In conclusion, the anti-melanoma properties of SF3 involve the induction of both extrinsic and intrinsic apoptotic pathway(s), as evidenced by the increased activity levels of caspases - 8, and - 9, respectively.

Benzyl and phenethyl isothiocyanates as promising epigenetic drug compounds by modulating histone acetylation and methylation marks in malignant melanoma.

Melanoma is an aggressive skin cancer with increasing incidence rates globally. On the other hand, isothiocyanates are derived from cruciferous vegetables and are known to exert a wide range of anti-cancer activities including, among others, their ability to interact with the epigenome in order to supress cancer progression. The aim of this study was to determine the role of phenethyl and benzyl isothiocyanates in modulating histone acetylation and methylation as a potential epigenetic therapeutic strategy in an in vitro model of malignant melanoma. We report that both isothiocyanates induced cytotoxicity and influenced acetylation and methylation status of specific lysine residues on histones H3 and H4 by modulating the expression of various histone acetyltransferases, deacetylases and methyltransferases in malignant melanoma cells. Our data highlight novel insights on the interaction of isothiocyanates with components of the histone regulatory machinery in order to exert their anti-cancer action in malignant melanoma.

An Evaluation of the Anti-Carcinogenic Response of Major Isothiocyanates in Non-Metastatic and Metastatic Melanoma Cells.

Malignant melanoma is one of the most deadly types of solid cancers, a property mainly attributed to its highly aggressive metastatic form. On the other hand, different classes of isothiocyanates, a class of phytochemicals, present in cruciferous vegetables have been characterized by considerable anti-cancer activity in both in vitro and in vivo experimental models. In the current study, we investigated the anti-cancer response of five isothiocyanates in an in vitro model of melanoma consisting of non-metastatic (A375, B16F-10) and metastatic (VMM1, Hs294T) malignant melanoma as well as non-melanoma epidermoid carcinoma (A431) and non-tumorigenic melanocyte-neighboring keratinocyte (HaCaT) cells. Our aim was to compare different endpoints of cytotoxicity (e.g., reactive oxygen species, intracellular glutathione content, cell cycle growth arrest, apoptosis and necrosis) descriptive of an anti-cancer response between non-metastatic and metastatic melanoma as well as non-melanoma epidermoid carcinoma and non-tumorigenic cells. Our results showed that exposure to isothiocyanates induced an increase in intracellular reactive oxygen species and glutathione contents between non-metastatic and metastatic melanoma cells. The distribution of cell cycle phases followed a similar pattern in a manner where non-metastatic and metastatic melanoma cells appeared to be growth arrested at the G2/M phase while elevated levels of metastatic melanoma cells were shown to be at sub G1 phase, an indicator of necrotic cell death. Finally, metastatic melanoma cells were more sensitive apoptosis and/or necrosis as higher levels were observed compared to non-melanoma epidermoid carcinoma and non-tumorigenic cells. In general, non-melanoma epidermoid carcinoma and non-tumorigenic cells were more resistant under any experimental exposure condition. Overall, our study provides further evidence for the potential development of isothiocyanates as promising anti-cancer agents against non-metastatic and metastatic melanoma cells, a property specific for these cells and not shared by non-melanoma epidermoid carcinoma or non-tumorigenic melanocyte cells.

A novel methylated analogue of L-Mimosine exerts its therapeutic potency through ROS production and ceramide-induced apoptosis in malignant melanoma.

Melanoma is an aggressive and highly metastatic type of skin cancer where the design of new therapies is of utmost importance for the clinical management of the disease. Thus, we have aimed to investigate the mode of action by which a novel methylated analogue of L-Mimosine (e.g., L-SK-4) exerts its therapeutic potency in an in vitro model of malignant melanoma. Cytotoxicity was assessed by the Alamar Blue assay, oxidative stress by commercially available kits, ROS generation, caspase 3/7 activation and mitochondrial membrane depolarisation by flow cytometry, expression of apoptosis-related proteins by western immunoblotting and profiling of lipid biosynthesis by a metabolomic approach. Overall, higher levels of ROS, sphingolipids and apoptosis were induced by L-SK-4 suggesting that the compound's therapeutic potency is mediated through elevated ROS levels which promote the upregulation of sphingolipid (ceramide) biosynthesis thus leading to the activation of both extrinsic and intrinsic apoptosis, in an experimental model of malignant melanoma.

Design and evaluation of bi-functional iron chelators for protection of dopaminergic neurons from toxicants.

While the etiology of non-familial Parkinson's disease (PD) remains unclear, there is evidence that increased levels of tissue iron may be a contributing factor. Moreover, exposure to some environmental toxicants is considered an additional risk factor. Therefore, brain-targeted iron chelators are of interest as antidotes for poisoning with dopaminergic toxicants, and as potential treatment of PD. We, therefore, designed a series of small molecules with high affinity for ferric iron and containing structural elements to allow their transport to the brain via the neutral amino acid transporter, LAT1 (SLC7A5). Five candidate molecules were synthesized and initially characterized for protection from ferroptosis in human neurons. The promising hydroxypyridinone SK4 was characterized further. Selective iron chelation within the physiological range of pH values and uptake by LAT1 were confirmed. Concentrations of 10-20 µM blocked neurite loss and cell demise triggered by the parkinsonian neurotoxicants, methyl-phenyl-pyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) in human dopaminergic neuronal cultures (LUHMES cells). Rescue was also observed when chelators were given after the toxicant. SK4 derivatives that either lacked LAT1 affinity or had reduced iron chelation potency showed altered activity in our assay panel, as expected. Thus, an iron chelator was developed that revealed neuroprotective properties, as assessed in several models. The data strongly support the role of iron in dopaminergic neurotoxicity and suggests further exploration of the proposed design strategy for improving brain iron chelation.

Anticancer activity of a novel methylated analogue of L-mimosine against an in vitro model of human malignant melanoma.

The anticancer activity of a series of novel synthesized, hydroxypyridone-based metal chelators (analogues of L-mimosine) was evaluated in an in vitro model of melanoma consisting of malignant melanoma (A375), non-melanoma epidermoid carcinoma (A431) and immortalized non-malignant keratinocyte (HaCaT) cells. More specifically, we have demonstrated that the L-enantiomer of a methylated analogue of L-mimosine (compound 22) can exert a potent anticancer effect in A375 cells when compared to either A431 or HaCaT cells. Moreover, we have demonstrated that this analogue has the ability to i) promote increased generation of reactive oxygen species (ROS), ii) activate both intrinsic and extrinsic apoptosis and iii) induce perturbations in cell cycle growth arrest. Our data highlights the potential of compound 22 to act as a promising therapeutic agent against an in vitro model of human malignant melanoma.