Fluorescence microscopy is a useful tool to characterize the metabolic properties of normal and cancerous cells and tissue (1
). The primary oxidation-reduction (redox) reactions in cells to generate energy in the form of ATP are the conversion of NAD+
to its reduced form NAD(P)H (henceforth referred to as NADH) and the oxidation of flavin adenine dinucleotide (FAD) to FADH2
, a process known as oxidative phosphorylation. Both NADH and FAD are autofluorescent and have distinct excitation and emission maxima. The optical redox ratio can be determined by calculating the ratio of the measured fluorescence intensities of NADH and FAD (NADH/FAD; ref. 3
Alterations in cellular metabolism are an important hallmark of carcinogenesis (4
). Cancer cell metabolism is often shifted from oxidative phosphorylation to aerobic glycolysis as the primary generator of cellular ATP. Although the exact mechanisms for the switch to aerobic glycolysis and altered cellular metabolism are variable and complex (4
), it presents clear advantages for tumor growth. These advantages include resistance to fluctuations in the local oxygen concentration (6
) and alterations in the tumor microenvironment that support tumor cell migration and invasion (7
). This shift, which gives rise to enhanced production of lactate in the presence of high oxygen, has long been known as the “Warburg effect” (9
). In aerobic glycolysis, glucose is metabolized into two pyruvate molecules, which are then converted into lactate. This results in the production of two molecules of ATP and two NADH. During oxidative phosphorylation, one molecule of glucose is converted to carbon dioxide and water, resulting in the production of 30 to 36 ATP molecules and the oxidation of 10 NADH molecules to NAD+
. Thus, the switch from oxidative phosphorylation to aerobic glycolysis results in a net increase in NADH.
Estrogens and estrogen receptors (ER) have been shown to play a role in numerous aspects of cellular metabolism in a number of organ systems, including the liver, pancreas, brain, muscle, and breast (10
). Estrogens/ER have been shown to increase glucose transport and glycolysis (12
). For example, estrogen exposure has been shown to increase the expression of a number of glucose transporter (GLUT) proteins (12
). Estrogen (but not the ER antagonist tamoxifen) increased glucose uptake and lactate production in MCF-7 xenografts as measured by C13
nuclear magnetic resonance imaging (13
). Additionally, estrogens/ER regulate gene expression of proteins involved in the citric acid cycle and oxidative phosphorylation (10
). Although not specifically studied in the breast, estrogen/ER have been shown to regulate citrate synthase, aconitase, and isocitrate dehydrogenase (17
). Notably, isocitrate dehydrogense activity results in the reduction of NAD+
to NADH, which is expected to cause an increase in the optical redox ratio. Furthermore, ER has been shown to localize to the mitochondria in a variety of cell types (10
), and it has been proposed that mitochondrial localization of ER is important for the transcriptional regulation of numerous mitochondrial DNA–encoded genes.
Previous studies have shown that the optical redox ratio is statistically different between cancer and normal epithelial cells, with cancer cells exhibiting higher redox ratios (2
). For example, in a study comparing normal keratinocytes to human papillomavirus (HPV)–transformed cells, the authors found that HPV-transformed cells had a higher overall intensity of NADH and a lower overall intensity of FAD, which resulted in a statistically significant difference in the optical redox ratio (22
). However, the optical redox ratio of NADH to FAD has not been quantified for different biological subtypes of breast cancer, nor has its relationship to breast cancer ER status been assessed.
Based on previously published reports in the literature, our primary hypotheses tested in this study were that the optical redox ratio can differentiate between malignant and nonmalignant breast cells and between ER(+) and ER(−) breast cancer cell lines. A secondary hypothesis is that the optical redox ratio can specifically monitor response to antiestrogen therapies. To test our hypotheses, we determined the optical redox ratio using a confocal microscopy approach. NADH and FAD intensities were acquired from a panel of normal mammary epithelial and breast cancer cell lines following excitation at 351 and 488 nm, respectively. The optical redox ratio differentiated normal mammary epithelial cells from breast cancer cells and also stratified breast cancer cell lines based on ER expression, which was associated with an increased optical redox ratio. Further, treatment of ER(+) breast cancer cell lines with antiestrogens resulted in a decrease in the optical redox ratio. This effect was not observed in ER(−) cell lines.
ER has proved to be a successful target of antitumor therapy in ER(+) breast tumors. However, resistance to antiestrogen therapies is a serious clinical problem for the treatment of breast cancer. Whereas ER expression is a good predictor of response to antiestrogen therapies, not all ER(+) tumors respond to therapy and some develop resistance after initially responding to therapy. Therefore, the optical redox ratio may serve as an important biomarker to differentially identify ER (+) breast cancers and monitor response to antiestrogen therapy, with applications in drug discovery and screening as well as clinical assessment of response to antiestrogen therapies.