Characterization of the molecular mechanism(s) of cannabinoid-induced paraptosis in breast cancer cells

Natural products have been extensively studied for their anticancer potential, and several widely used anticancer drugs have natural origins. Many studies have demonstrated that natural compounds can induce paraptosis in various tumour cell lines. Paraptosis is a caspase-independent cell death mechanism characterised by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and the mitochondria. The molecular mechanism of paraptosis is unclear; however, the literature indicates that dysregulation of calcium signalling plays an important role in paraptosis induction, particularly calcium-mediated signalling between the ER and mitochondria. This study aimed to identify and characterise the mechanism of cell death induced by a phytocannabinoid ratio which induced significant cytoplasmic vacuolation in the MCF7 breast cancer cell line. Several techniques were employed to address the aim of the study. The crystal violet assay was used to detect changes in viability. Several pathway inhibitors, as well as fluorescent staining and analysis using high-content screening (HCS), were used to measure the induction of various cell death mechanisms. Morphological changes were investigated using light and transmission electron microscopy. The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, the same trend was not observed in the MCF10A non-tumourigenic breast cell line. No cell cycle arrest, apoptosis, necrosis, autophagy, or ferroptosis induction was detected in MCF7 cells, suggesting that an alternative mechanism of cell death was induced. Vacuolation and cell death induced by the phytocannabinoid ratio were inhibited by cycloheximide, suggesting a dependence on protein synthesis, which is characteristic of paraptosis induction. The mechanism of paraptosis induction by the phytocannabinoid ratio was investigated, and it was found that treatment 1) induced ER dilation and mitochondrial swelling; 2) induced significant ER stress, mitochondrial calcium ...