Modifications of Cytochrome <i>c</i> by Retinoic Acid Play a Crucial Role in Mitochondrial Dysfunction of Triple-Positive Human Breast Cancer Cells: Raman Spectroscopy and Imaging Study

Conventional assays for the assessment of the receptors on the surface and inside specific organelles in human breast cancer cells include immunohistochemistry (IHC) and in situ hybridization (ISH), both of which have limitations. We propose a novel Raman method to monitor modifications such as the redox status of cytochrome <i>c</i> and phosphorylation triggered in cells by retinoic acid. We showed that Raman imaging provides an effective assay for detecting the redox status of cytochrome <i>c</i> and tyrosine kinase activity in specific cell organelles of human triple-positive breast cancer cells MCF-7 upon incubation with retinoic acid. Therefore, in contrast to existing analytical technologies, Raman imaging can detect the full extent of cytochrome <i>c</i> localization and tyrosine activity inside and outside specific organelles. We found that retinoic acid has a spectacular impact on mitochondrial functional activity in cancer cells. Abnormal retinoic acid signaling in the mitochondria, cytoplasm, lipid droplets, endoplasmic reticulum, and nucleus was monitored by Raman signals at 1582 cm^-1, 1616 cm^-1, 3058 cm^-1, and 3072 cm^-1 in mitochondria, cytoplasm, lipid droplets, endoplasmic reticulum, and nucleus. In human breast cancer cells, it was found that the balance between Fe³⁺/Fe²⁺ heme forms of cytochrome <i>c</i> is spectacularly shifted toward the reduced form Fe²⁺ upon retinoic acid and tyrosine activity in mitochondria decreases with increasing concentration of retinoic acid. These results may have far-reaching implications for cancer therapy. A quantitative approach to the measurement of receptor protein expression of tyrosine activity may improve specificity in selecting patients for triple-positive breast cancer targeted therapy. In the current study, we have used retinol binding protein (RBP) and STRA6 protein expression and cytochrome <i>c</i> in breast cancer cells as a model to explore the potential utility of a novel immunodetection technique, using Raman spectroscopy and Raman imaging, which can be quantitatively constructed by using cluster analysis. The paper provides experimental support for the theoretical hypothesis of how retinoic acid catalyzes resonance energy transfer reactions and controls the activation/inactivation cycle of protein kinase PKCδ. It has been proposed that reversible phosphorylation of cytochrome <i>c</i> mediated by cell signaling pathways is a primary regulatory mechanism that determines mitochondrial respiration, electron transport chain (ETC) flux, proton gradient ΔΨ_m, ATP production, and ROS generation, linking oxidative phosphorylation to human cancer through a lack of energy, ROS production, cytochrome <i>c</i> release, and activation of apoptosis.