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

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 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.

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

“…For this reason, the activity of SOD, CAT, GPx, GR and GST to metabolize ROS was studied. To this end, A375 cells were subjected to PhEF at non-toxic (below EC 50 ; 0.75% v/v), sub-toxic (at EC 50 ; 1.75% v/v) and toxic (above EC 50 ; 2.5% v/v) concentrations as previously characterized by our group [15]. Overall, it was shown that SOD activity is proportional to the concentration of both PhEF and PoEF since the highest activity was noticed at their highest concentrations (Figure 2B).…”

“…In addition, PEITC was shown to induce anticancer potency in various cancer types including cervical, breast, lung, prostate, melanoma and leukemia [10][11][12][13][14]. Recently, a study demonstrated that a naturally derived watercress flowers-based PEITC-enriched extract (PhEF) was capable of inducing the activation of intrinsic apoptosis via subcellular ultrastructural alterations and perturbations in Ca 2+ efflux in malignant melanoma [15]. Furthermore, it appears that the PEITC-induced cytotoxicity was associated with its electrophilic carbon center which can react with cellular nucleophiles (e.g., thiols and amines) to form thiocarbamates/dithiocarbamates and thioureas [16,17].…”

“…The aim of the current study was to investigate the capacity of PhEF, previously characterized by our group [3,15], to cause perturbations in oxidative stress status in malignant melanoma. To this end, we have aimed to investigate the potential role of PEITC-NAC in inducing cytotoxicity to human malignant melanoma (A375, COLO-679) as opposed to neighboring, non-tumorigenic immortalized keratinocyte (HaCaT) cells.…”