Phenotypic Changes in Mitochondrial Membrane Potential (Δψ_m) during Valinomycin‐Induced Depolarisation and Apoptosis

Abstract

A large body of evidence has implicated mitochondria in control of cell death, where key apoptotic mechanisms involve change in mitochondrial membrane permeability and depolarisation of mitochondrial membrane potential (Δψm). Assessment of Δψm is traditionally conducted using the lipophilic cation JC-1 on the flow cytometer or by fluorescent microscopy. Here we assess JC-1 aggregation using the novel tool of digital texture analysis to establish mitochondrial phenotypic changes induced by the K+ ionophore, valinomycin in a unique model comprising SW480 and SW620 cell lines. This provides an opportunity to study these phenomena in the context of colorectal cancer. Valinomycin-induced apoptosis was detected using morphology and analysis of DNA content. Cells were treated with valinomycin, images digitally recorded on a calibrated video photometer and subjected to high resolution digital texture analysis. This demonstrated that the HARAM texture features (Mean of the Haralick texture features) were highly valuable in describing the transition of Δψm as the cell undergoes apoptosis. In Conclusion this study illustrates the potential of texture analysis as a novel and additional technique for quantifying JC-1 aggregation and revealing the spectrum of collapse of Δψm during apoptosis.

Acute Nitrite Exposure Induces Dysfunction and Oxidative Damage in Grass Carp Isolated Hemocytes

“…As the exposure time was extended, ▵Ψm showed a trend of rising initially and then falling. Previous research considered that external chemicals would decrease ▵Ψm and induce apoptosis of cells (Morrison et al 2016;Wu et al 2020). Similarly, a reduced ▵Ψm appeared in the 100-mg/L nitrite exposure group at 24 h in the present study; however, this reduction was not observed in the other nitrite exposure groups.…”

“…Previous research considered that external chemicals would decrease ▵Ψm and induce apoptosis of cells (Morrison et al. 2016; Wu et al. 2020).…”

Antitumor Potential of Conjugable Valinomycins Bearing Hydroxyl Sites: In Vitro Studies

“…Although these mechanistic aspects are under investigation, flow cytometry experiments run on MCF-7 cells stained with the mitochondria-specific fluorescent probe JC-1 indicate that HyVLMs, similarly to VLM, induce ΔΨ m dissipation, an event that is known to precede the VLM-triggered apoptosis. ,, In particular, we found that the percentage of JC-1-loaded MCF-7 cells with depolarized mitochondria, upon exposure to compounds 1 – 4 for 72 h, were 16, 8.9, 12.1, and 15.6%, respectively, as opposed to a 1% of MCF-7 control cells. It is noteworthy that these percent values strictly parallel the order of cytotoxicity of compounds 1 – 4 (IC 50 values of Table 1), as well as their relative order of efficiency ( 1 > 3 ≈ 4 > 2 ) in depolarizing isolated rat-liver mitochondria …”

“…To highlight the antiproliferative activity of compounds 1 – 4 , we evaluate their ability to interfere with the cells cycle progression of MCF-7 cells by flow cytometry. It is well-known that VLM is a potent apoptotic inducer. ,, However, in our preliminary experiments, cells treated with compounds 1 – 4 showed only a marked accumulation in S phase after 24 h of incubation, irrespective to the utilized concentrations (Figure ; Table S1, Supporting Information). As already suggested by Iguchi et al, an accumulation in S phase could be correlated to apoptosis through the modulation of MAPK and mevalonate pathways. , After 72 h of incubation, only VLM showed a marked accumulation of cells in the G2/M phase, which might be compatible with an induction of apoptosis.…”

“…Chemically, it consists of a three-repeating sequence of the tetramer d -α-hydroxyisovaleryl- d -valyl- l -lactyl- l -valyl ( d -Hyi- d -Val- l -Lac- l -Val) cyclically arranged to form a 36-membered macro-ring with an internal cavity designed to specifically accommodate K + , which can hence be safely shuttled across membranes . This causes dissipation of mitochondrial transmembrane potential (ΔΨ m ) and induction of apoptosis, which has been shown in several mammalian cell types, , including a number of tumor cell lines. ,, Because of its unique properties, VLM has captured the attention of many searchers devoted to various fields of science, so that studies detailing its mechanism of action, − as well as concerning the synthesis of analogues have continued unabated over the past decades …”