The role of isothiocyanates as epigenetic modulators in malignant melanoma

Mitsiogianni, Melina (2020) The role of isothiocyanates as epigenetic modulators in malignant melanoma. Doctoral thesis, Northumbria University.

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Abstract

Malignant melanoma is one of the most aggressive types of human skin cancer, characterized by high mortality due to its resistance to current therapies. Thus, there is an imperative need to develop more effective therapeutic approaches. To this end, isothiocyanates (ITCs), the biologically active plant secondary metabolites of glucosinolates, have been shown to act as promising anti melanoma agents, mediating various biological effects, including modulation of detoxifying enzymes, induction of oxidative stress, apoptosis as well as modulation of the epigenome among others. This study aimed to characterize the effect of 5 ITCs [Sulforaphane (SFN); Iberin (IBN); Allyl- (AITC); Benzyl- (BITC) and Phenethyl- Isothiocyanate (PEITC)] on lysine acetylation and methylation marks as a potential epigenetic-induced anti melanoma modality. An in vitro model of malignant melanoma was utilized, consisting of (i) human (A375) and (ii) murine (B16-F10) malignant melanoma cells; human metastatic melanoma cells derived from (iii) brain (VMM1) and (iv) lymph node (Hs 294T); (v) non-melanoma epidermoid carcinoma (A431) cells and (vi) immortalized keratinocyte (HaCaT) cells. All cell lines were subjected to treatments with varying doses and time frames for each of the 5 ITCs and various cytotoxicity end points were determined [cell viability (by Resazurin assay), levels of reactive oxygen species (ROS), total glutathione (GSH), cell cycle distribution and apoptosis (by flow cytometry)]. Furthermore, western immunoblotting was used to determine the expression of specific histone acetyltransferases (HAT), deacetylases (HDAC) and methyltransferases (HMT) together with acetylation and methylation marks on histones. Overall, we demonstrate that all five ITCs were able to reduce cell viability, decrease GSH levels, and promote G2/M cell cycle arrest and apoptosis. Moreover, utilizing a real-time PCR microarray based gene expression profiling platfrom, we found several genes implicated in apoptosis-related processes. Finally, all 5 ITCs showed to influence histone acetylation and methylation patterns by modulating several key enzymes (HDACs, HATs and HMTs) that mediate these epigenetic processes and consequently the acetylation and methylation status of specific lysine residues on histones 3 and 4, in human malignant melanoma cells.

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