Application of a homogenous membrane potential assay to assess mitochondrial function - PubMed (original) (raw)

Comparative Study

Application of a homogenous membrane potential assay to assess mitochondrial function

Srilatha Sakamuru et al. Physiol Genomics. 2012.

Abstract

Decreases in mitochondrial membrane potential (MMP) have been associated with mitochondrial dysfunction that could lead to cell death. The MMP is generated by an electrochemical gradient via the mitochondrial electron transport chain coupled to a series of redox reactions. Measuring the MMP in living cells is commonly used to assess the effect of chemicals on mitochondrial function; decreases in MMP can be detected using lipophilic cationic fluorescent dyes. To identify an optimal dye for use in a high-throughput screening (HTS) format, we compared the ability of mitochondrial membrane potential sensor (Mito-MPS), 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide, rhodamine 123, and tetramethylrhodamine to quantify a decrease in MMP in chemically exposed HepG2 cells cultured in 1,536-well plates. Under the conditions used, the optimal dye for this purpose is Mito-MPS. Next, we developed and optimized a homogenous cell-based Mito-MPS assay for use in 1,536-well plate format and demonstrated the utility of this assay by screening 1,280 compounds in the library of pharmacologically active compounds in HepG2 cells using a quantitative high-throughput screening platform. From the screening, we identified 14 compounds that disrupted the MMP, with half-maximal potencies ranging from 0.15 to 18 μM; among these, compound clusters that contained tyrphostin and 3'-substituted indolone analogs exhibited a structure-activity relationship. Our results demonstrate that this homogenous cell-based Mito-MPS assay can be used to evaluate the ability of large numbers of chemicals to decrease mitochondrial function.

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Figures

Fig. 1.

Fig. 1.

Principle of the mitochondrial membrane potential sensor (Mito-MPS) assay. Based on the molecule structure of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo carbocyanine iodide (JC-1), Mito-MPS was modified to be more water soluble. In healthy cells (left, DMSO control), the mitochondria are polarized and Mito-MPS accumulates in the mitochondria as aggregates with red fluorescence (emission at 590 nm). In cells with lower mitochondrial membrane potential [MMP, right, 3.4 μM mesoxalonitrile 4-trifluoromethoxyphenylhydrazone (FCCP) treated], Mito-MPS remains in cytoplasm as the monomeric form that shows green fluorescence (emission at 535 nm). The dye undergoes a change in fluorescence emission from green to red when the MMP increases or vice versa. This process is reversible. The red/green ratio can be used to determine the mitochondrial function of the cells (25).

Fig. 2.

Fig. 2.

Assay performance using MMP dyes, Mito-MPS, JC-1, rhodamine 123, or tetramethylrhodamine ethyl ester (TMRE). MMP was measured after the cells were treated with various concentrations of FCCP (A), rotenone (B), or antimycin (C) for 1 h in 384-well plate format. The fold change was defined as the value of DMSO control divided by the value of compound treatment. Values are means ± SD from 3 experiments performed in quadruplicate.

Fig. 3.

Fig. 3.

Mito-MPS assay validation with DMSO plate. Column 1 was a concentration response curve of FCCP in duplicate. Column 2 was 9.2 μM FCCP. Column 3 was 3.5 μM (top half) and 6.9 μM (bottom half) FCCP, respectively. Columns 4–48 were treated with DMSO at a final concentration of 0.46%. Cells were treated with DMSO or FCCP for 1 h (A) or 5 h (B). Each value was expressed as the ratio of 590/535 nm emissions.

Fig. 4.

Fig. 4.

Tyrphostin compounds. A: concentration response curves of tyrphostin A9, tyrphostin AG-879, and tyrphostin 47 in Mito-MPS assay at 1 h (left) or 5 h (right) of treatment. B: structures of these compounds are shown with compound names, half-maximal inhibition (IC50) values in the Mito-MPS assay at 1 or 5 h of treatment. C: representative imagine of subcellular staining with Mito-MPS dye in the absence or in the presence of tyrphostin A9. Images acquired in ImageXpress microsystem using a ×20 objective. While red fluorescent aggregates are localized in the mitochondria, green fluorescent monomers are mainly in cytosol. The composed images were the merger of red and green fluorescence.

Fig. 5.

Fig. 5.

3′-Substituted indolone-related compounds. A: concentration response curves of (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO), indirubin-3′-oxime, and GW-5074 in Mito-MPS assay at 1 h (left) or 5 h (right) of treatment. B: structures of these compounds are shown with compound names, IC50 values in the Mito-MPS assay at 1 h or 5 h of treatment.

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