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.

Sulforaphane and iberin are potent epigenetic modulators of histone acetylation and methylation in malignant melanoma.

OBJECTIVE(S): Growing evidence supports that isothiocyanates exert a wide range of bioactivities amongst of which is their capacity to interact with the epigenetic machinery in various cancers including melanoma. Our aim was to characterise the effect of sulforaphane and iberin on histone acetylation and methylation as a potential anti-melanoma strategy. METHODS: We have utilised an in vitro model of malignant melanoma [consisting of human (A375, Hs294T, VMM1) and murine (B16F-10) melanoma cell lines as well as a non-melanoma (A431) and a non-tumorigenic immortalised keratinocyte (HaCaT) cell line] exposed to sulforaphane or iberin. Cell viability was evaluated by the Alamar blue assay whilst total histone deacetylases and acetyltransferases activities were determined by the Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively. The expression levels of specific histone deacetylases and acetyltransferases together with those of lysine acetylation and methylation marks were obtained by western immunoblotting. RESULTS: Overall, both sulforaphane and iberin were able to (1) reduce cell viability, (2) decrease total histone deacetylase activity and (3) modulate the expression levels of various histone deacetylases as well as acetyl and methyl transferases thus modulating the acetylation and methylation status of specific lysine residues on histones 3 and 4 in malignant melanoma cells. CONCLUSIONS: Our findings highlight novel insights as to how sulforaphane and iberin differentially regulate the epigenetic response in ways compatible with their anticancer action in malignant melanoma.

Allyl isothiocyanate regulates lysine acetylation and methylation marks in an experimental model of malignant melanoma.

OBJECTIVE(S): Isothiocyanates (ITCs) are biologically active plant secondary metabolites capable of mediating various biological effects including modulation of the epigenome. Our aim was to characterize the effect of allyl isothiocyanate (AITC) on lysine acetylation and methylation marks as a potential epigenetic-induced anti-melanoma strategy. METHODS: Our malignant melanoma model consisted of (1) human (A375) and murine (B16-F10) malignant melanoma as well as of human; (2) brain (VMM1) and lymph node (Hs 294T) metastatic melanoma; (3) non-melanoma epidermoid carcinoma (A431) and (4) immortalized keratinocyte (HaCaT) cells subjected to AITC. Cell viability, histone deacetylases (HDACs) and acetyltransferases (HATs) activities were evaluated by the Alamar blue, Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively, while their expression levels together with those of lysine acetylation and methylation marks by western immunoblotting. Finally, apoptotic gene expression was assessed by an RT-PCR-based gene expression profiling methodology. RESULTS: AITC reduces cell viability, decreases HDACs and HATs activities and causes changes in protein expression levels of various HDACs, HATs, and histone methyl transferases (HMTs) all of which have a profound effect on specific lysine acetylation and methylation marks. Moreover, AITC regulates the expression of a number of genes participating in various apoptotic cascades thus indicating its involvement in apoptotic induction. CONCLUSIONS: AITC exerts a potent epigenetic effect suggesting its potential involvement as a promising epigenetic-induced bioactive for the treatment of malignant melanoma.

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.

DNA-dependent protein kinase: Epigenetic alterations and the role in genomic stability of cancer.

DNA-dependent protein kinase (DNA-PK), a member of phosphatidylinositol-kinase family, is a key protein in mammalian DNA double-strand break (DSB) repair that helps to maintain genomic integrity. DNA-PK also plays a central role in immune cell development and protects telomerase during cellular aging. Epigenetic deregulation due to endogenous and exogenous factors may affect the normal function of DNA-PK, which in turn could impair DNA repair and contribute to genomic instability. Recent studies implicate a role for epigenetics in the regulation of DNA-PK expression in normal and cancer cells, which may impact cancer progression and metastasis as well as provide opportunities for treatment and use of DNA-PK as a novel cancer biomarker. In addition, several small molecules and biological agents have been recently identified that can inhibit DNA-PK function or expression, and thus hold promise for cancer treatments. This review discusses the impact of epigenetic alterations and the expression of DNA-PK in relation to the DNA repair mechanisms with a focus on its differential levels in normal and cancer cells.

The Role of Isothiocyanates as Cancer Chemo-Preventive, Chemo-Therapeutic and Anti-Melanoma Agents.

Many studies have shown evidence in support of the beneficial effects of phytochemicals in preventing chronic diseases, including cancer. Among such phytochemicals, sulphur-containing compounds (e.g., isothiocyanates (ITCs)) have raised scientific interest by exerting unique chemo-preventive properties against cancer pathogenesis. ITCs are the major biologically active compounds capable of mediating the anticancer effect of cruciferous vegetables. Recently, many studies have shown that a higher intake of cruciferous vegetables is associated with reduced risk of developing various forms of cancers primarily due to a plurality of effects, including (i) metabolic activation and detoxification, (ii) inflammation, (iii) angiogenesis, (iv) metastasis and (v) regulation of the epigenetic machinery. In the context of human malignant melanoma, a number of studies suggest that ITCs can cause cell cycle growth arrest and also induce apoptosis in human malignant melanoma cells. On such basis, ITCs could serve as promising chemo-therapeutic agents that could be used in the clinical setting to potentiate the efficacy of existing therapies.

From chemo-prevention to epigenetic regulation: The role of isothiocyanates in skin cancer prevention.

Skin cancer incidence is rapidly growing over the last decades and is generally divided into malignant melanoma and non-melanoma (NMSC) with the latter being subdivided into squamous (SCC) and basal cell carcinoma (BCC). Among them, melanoma is the most aggressive type with high mortality rates. On the other hand, aberrant gene expression is a critical step towards malignant transformation. To this end, epigenetic modifications like changes in DNA methylation patterns and miRNA expression profile as well as histone modifications are all capable of inducing an altered gene expression profile involved in various cellular cascades including cell cycle, proliferation and apoptosis. In general, there is an interest about the beneficiary effect of various phytochemicals in the prevention and treatment of skin malignancies. Among them, glucosinolates are an important type of compounds, abundant in cruciferous vegetables, which are hydrolysed by an endogenous enzyme called myrosinase to a range of bioactive compounds including isothiocyanates (ITCs). These are the major biologically active products capable of mediating the anti-cancer effect of cruciferous vegetables. Their chemo-preventive action is mainly attributed to a plurality of anti-cancer properties including regulation of the epigenetic machinery. Current evidence supports the view that ITCs are potent compounds in interacting with the epigenome in order to restore the normal epigenetic landscape in malignant cells. This review article summarizes the current state of knowledge on the epigenetic modifications that lead to malignant transformation and the role of ITCs with respect to their ability to restore the epigenetic landscape that contributes to skin carcinogenesis.

Gold nanoparticles, radiations and the immune system: Current insights into the physical mechanisms and the biological interactions of this new alliance towards cancer therapy.

Considering both cancer's serious impact on public health and the side effects of cancer treatments, strategies towards targeted cancer therapy have lately gained considerable interest. Employment of gold nanoparticles (GNPs), in combination with ionizing and non-ionizing radiations, has been shown to improve the effect of radiation treatment significantly. GNPs, as high-Z particles, possess the ability to absorb ionizing radiation and enhance the deposited dose within the targeted tumors. Furthermore, they can convert non-ionizing radiation into heat, due to plasmon resonance, leading to hyperthermic damage to cancer cells. These observations, also supported by experimental evidence both in vitro and in vivo systems, reveal the capacity of GNPs to act as radiosensitizers for different types of radiation. In addition, they can be chemically modified to selectively target tumors, which renders them suitable for future cancer treatment therapies. Herein, a current review of the latest data on the physical properties of GNPs and their effects on GNP circulation time, biodistribution and clearance, as well as their interactions with plasma proteins and the immune system, is presented. Emphasis is also given with an in depth discussion on the underlying physical and biological mechanisms of radiosensitization. Furthermore, simulation data are provided on the use of GNPs in photothermal therapy upon non-ionizing laser irradiation treatment. Finally, the results obtained from the application of GNPs at clinical trials and pre-clinical experiments in vivo are reported.

Development of a Novel Experimental In Vitro Model of Isothiocyanate-induced Apoptosis in Human Malignant Melanoma Cells.

BACKGROUND: Isothiocyanates are constituents of cruciferous vegetables which have been associated with reduced cancer risk partially through their ability to induce apoptosis in malignant cells including melanoma. MATERIALS AND METHODS: We have utilized human malignant melanoma (A375), epidermoid carcinoma (A431) and immortalized keratinocyte (HaCaT) cells exposed to various isothiocyanates, under different experimental conditions. RESULTS: An experimental in vitro model utilizing low isothiocyanate concentrations (0.1-5 µM for 48 h with all treatments being refreshed after 24h) was shown to be (i) most efficient in exerting an anti-cancer effect when compared to higher concentrations (5-100 µM for 24 or 48 h added as a single bolus) and (ii) specific to A375 cells while A431 and HaCaT cells remained unaffected. Such effect involved the activation of several caspases including (iii) initiator caspases 8, 9, 4 (indicating the involvement of intrinsic, extrinsic and endoplasmic reticulum-based pathways) and (iv) effector caspases 3, 7 and 6. CONCLUSION: Utilization of low isothiocyanate concentrations (under the conditions described herein) exerts an anti-cancer effect specific to human malignant melanoma cells thus providing a therapeutic basis for their utilization in management of the disease.