Cisplatin induced ATP depletion via mitochondrial damage and inhibition of glycolysis.

<p>(A) Intracellular localization of reactive oxygen species (ROS) by confocal microscopy. Visualization of mitochondria by Mito-tracker Red CMX (red, middle) demonstrated co-localization with mitochondrial ROS (mROS) as detected by the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (green, left) fluorescence dye. Magnification of the images was ×100 and scale bars represent 30 μm. (B) Quantification of mROS by the two-photon (TP) probe for the ratiometric detection of mitochondrial H₂O₂. The reaction between the SHP-Mito probe and H₂O₂ resulted in a change in the fluorescence from blue to yellow. Pseudo-colored green areas indicated mitochondria (blue fluorescence, left), and pseudo-colored red areas indicate mitochondrial H₂O₂ (yellow fluorescence, middle). Magnification of the images was ×63 and scale bars represent 26 μm. (C) mRNA expression levels of mitochondria-related genes (<i>OPA1</i>, <i>PDK1</i>, <i>BIK</i>, <i>RRM2B</i>, and <i>ENDOG</i>) determined after cisplatin treatment by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). (D) Expression levels of glycolysis-related enzymes of cisplatin-treated cells using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. (E) mRNA expression levels of glycolysis-related genes (<i>HIF1a</i>, <i>HK2</i>, <i>ACMSD</i>, and <i>PRKAG2</i>) determined after cisplatin treatment using qRT-PCR. (F) Quantification of intracellular glucose levels after cisplatin treatment. (G) Intracellular ATP levels were determined using LAS 1000 and a microplate reader. The mean ± S.D. of three independent experiments are presented; *<i>p</i> < 0.05, **<i>p</i> < 0.01, by paired <i>t</i> test.