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

Authors

Sotiris Kyriakou, The Cyprus Institute of Neurology & Genetics
Louiza Potamiti, The Cyprus Institute of Neurology & Genetics
Nikoletta Demosthenous, The Cyprus Institute of Neurology & Genetics
Tom Amery, The Watercress Company
Kyle Stewart, Watercress Research Limited
Paul G. Winyard, Watercress Research Limited
Rodrigo Franco, University of Nebraska-LincolnFollow
Aglaia Pappa, Democritus University of Thrace
Mihalis I. Panayiotidis, The Cyprus Institute of Neurology & Genetics

Document Type

Article

Date of this Version

9-18-2023

Citation

Kyriakou, S.; Potamiti, L.; Demosthenous, N.; Amery, T.; Stewart, K.; Winyard, P.G.; Franco, R.; Pappa, A.; Panayiotidis, M.I. A Naturally DerivedWatercress 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. Nutrients 2023, 15, 4044. https://doi.org/10.3390/ nu15184044

Comments

Open access.

Abstract

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 sublethal 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.