Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment

Abstract

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin-induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL-60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL-60 cells which also were more sensitive and contained autophagic vesicles. Studies in Rho 0 cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti-proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance.

Noninvasive Real-Time Monitoring by AlamarBlue®DuringIn VitroCulture of Three-Dimensional Tissue-Engineered Bone Constructs

“…function of the cell type, and to make use of a reduced incubation time followed by replacing the AB solution by a fresh cell culture medium. 42,45 Our experiments suggest an incubation time of 1 h to be optimal for the tested bone TE constructs (Fig. 4).…”

“…However, this over-reduction would result in a signal decrease in function of time after the maximal intensity was reached rather than resulting in a plateau value 14 ; (3) Although the AB dye was often reported to be nontoxic for cells, even after longer incubation times, 19,43,44 several groups reported a negative effect of the AB assay on cell growth and cell viability. 28,42,45 However, the toxicity is dependent on the cell type, the AB concentration, and exposure time. function of the cell type, and to make use of a reduced incubation time followed by replacing the AB solution by a fresh cell culture medium.…”

“…Since toxicity is only reported after longer incubation times and the AB solution was replaced after each measurement, potential AB toxicity is not expected to have influenced our experiments. 45 To investigate the correlation between the metabolic activity and the cellularity upon cell seeding, the overall metabolic activity and the total DNA content of the seeded TE constructs were compared. Although the correlation of the metabolic activity expressed in function of the number of seeded cells (Fig.…”

Regulation and Quantification of Cellular Mitochondrial Morphology and Content

“…[38][39][40][181][182][183][184][185]). The strategy of isolating mitochondria from human and animal tissue/cell material has historically been very important, and the technique was pioneered by scientists such as Chance and Williams [186].…”

Monitoring cellular accumulation of 3′-deoxy-3′-fluorothymidine (FLT) and its monophosphate metabolite (FLT-MP) by LC–MS/MS as a measure of cell proliferation in vitro

“…Cell counting and direct measurements of tumor mass are the "gold-standard" measures of cell proliferation, but are not practical in most pharmaceutical settings. The most common alternative, indirect quantification of DNA synthesis via the incorporation of [ 3 H]-thymidine or bromodeoxyuridine (BrdU) into DNA, can offer a high degree of sensitivity, but available methods are time consuming and labor-intensive, and are susceptible to experimental artifacts induced by long labeling incubations [2][3][4][5][6][7]. Cellular processes that are associated with cell proliferation and tend to increase proportionally with the number of proliferating cells in a sample can be measured using higher throughput methods.…”