Background

Natural products are small molecules that are isolated from natural sources. Over the last century, these molecules have been instrumental in treating human diseases, especially inspired chemotherapeutics. Metabolites like taxol, vincristine, and doxorubicin have revolutionized how we treat malign cancers and other natural products, for example rapamycin, oligomycin, and bafilomycin, are used as molecular probes and enable molecular studies of biochemical and cellular processes in the laboratory. While studying the mechanism of action of the cytotoxic natural product mensacarcin, we found that a fluorescently labeled mensacarcin probe localizes to a great extent in mitochondria (Plitzko et al., 2017). To investigate if mensacarcin’s cytotoxic properties might be derived from interference with mitochondrial function, we sought to examine mensacarcin’s effects on cellular bioenergetics. Using a Seahorse Extracellular Flux Analyzer, we monitored cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) in real time as measures of mitochondrial respiration and glycolysis, respectively (Wu et al., 2007; Serill et al., 2015). The Seahorse XF24 Extracellular Flux Analyzer allows continuous direct quantification of mitochondrial respiration and glycolysis of living cells. The instrument uses a sensor cartridge in a 24-well plate format with each sensor being equipped with two embedded fluorophores: one which is quenched by oxygen (O₂) and the other that is sensitive to change in pH. During measurements, the sensor cartridge is lowered 200 µm above the cell monolayer, forming a micro-chamber of about 2 µl. The Seahorse instrument contains fiber optic bundles that emit light, excite the fluorophores, and then measures the change in the fluorophore’s emission. The very small test volume formed by the transient micro chamber allows for sensitive, precise, and nondestructive measurements of parameters in real time. Changes in oxygen concentration and pH are automatically calculated and reported as Oxygen Consumption Rate (OCR) and Extra Cellular Acidification Rate (ECAR). Once a measurement is completed, the sensors lift which allows the larger medium volume above to mix with the medium in the transient micro chamber, restoring values to baseline. The sensor cartridge contains ports that allow sequential addition of up to four compounds per well during the assay measurements.

With the described protocol we assessed the energy metabolism of three cell lines (HCT-116, SK-Mel-28, and SK-Mel-5) (Figure 6). Addition of mensacarcin was found to have pronounced effect on the basal OCR of melanoma cells and no increasing effect on ECAR. An increase in glycolysis is often observed as a compensatory response. Mitochondria are essential for the energy metabolism of cells and have a key role in apoptotic cell death. Alteration of the mitochondrial respiration or the equilibrium between the pro-apoptotic and anti-apoptotic proteins can induce mitochondrial failure. To gain insights into the induced mitochondrial impairment in melanoma cells, we assessed key parameters of mitochondrial respiration by consecutively exposing cells to well described mitochondria perturbing reagents. Following addition of our test compound mensacarcin, we sequentially added oligomycin, FCCP, and lastly rotenone and antimycin A (Figure 5). Oligomycin inhibits ATP synthase and reduces OCR, FCCP uncouples oxygen consumption from ATP production and raises OCR to a maximal value, and antimycin A and rotenone target the electron transport chain and reduce OCR to a minimal value. The mitochondria stress test protocol provides information on basal respiration, ATP-linked respiration, proton leak, maximal respiration capacity, and non-mitochondrial respiration of cells. Therefore, this assay can be used to provide insight on the mechanism of action of compounds that directly target mitochondrial respiration.

Traditional measurements of mitochondrial function or glycolysis require an oxygen electrode, or kits and dyes that utilize colorimetric or fluorimetric detection (Li and Graham, 2012; TeSlaa and Teitell, 2014). Most of these methods are invasive and cumbersome methods that only allow low sample throughput. In contrast, the Seahorse analyzer assay with its sensor cartridge system enables measurement of mitochondrial respiration and glycolysis in real time and in a non-invasive manner that does not require any dyes or labels. Cellular energy metabolism research is highly topical in all fields of mammalian cell biology. The following protocol was developed for researchers in cancer biology but with approaches that suit studies of energy metabolism in all mammalian cell systems.