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Monitoring the dynamics of hemeoxygenase-1 activation in head and neck cancer cells in real-time using plasmonically enhanced Raman spectroscopy.

ABSTRACT: We report for the first time the usage of plasmonically enhanced Raman spectroscopy (PERS) to directly monitor the dynamics of pharmacologically generated hemeoxygenase-1 (HO-1) by evaluating the kinetics of formation of carbon monoxide (CO), one of the metabolites of HO-1 activation, in live cells during cisplatin treatment. Being an endogenous signaling molecule, CO plays an important role in cancer regression. Many aspects of HO-1's and CO's functions in biology are still unclear largely due to the lack of technological tools for the real-time monitoring of their dynamics in live cells and tissues. In this study, we found that, together with nuclear region-targeted gold nanocubes (AuNCs), cisplatin treatment can dramatically trigger the activation of HO-1 and thereby the rate and production of CO in mammalian cells in a dose-dependent manner. Though quantitative molecular data revealed that a lower concentration of cisplatin up-regulates HO-1 expression in cancer cells, PERS data suggest that it poorly facilitates the activation of HO-1 and thereby the production of CO. However, at a higher dose, cisplatin along with AuNCs could significantly enhance the activation of HO-1 in cancer cells, which could be probed in real-time by monitoring the CO generation by using PERS. Under the same conditions, the rate of formation of CO in healthy cells was relatively higher in comparison to the cancer cells. Additionally, molecular data revealed that AuNCs have the potential to suppress the up-regulation of HO-1 in cancer cells during cisplatin treatment at a lower concentration. As up-regulation of HO-1 has a significant role in cell adaptation to oxidative stress in cancer cells, the ability of AuNCs in suppressing the HO-1 overexpression will have a remarkable impact in the development of nanoformulations for combination cancer therapy. This exploratory study demonstrates the unique possibilities of PERS in the real-time monitoring of endogenously generated CO and thereby the dynamics of HO-1 in live cells, which could expedite our understanding of the signaling action of CO and HO-1 in cancer progression.

SUBMITTER: Panikkanvalappil SR

PROVIDER: S-EPMC6520930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Metabolomics is an emerging field of cell biology that aims at the comprehensive identification of metabolite levels in biological fluids or cells in a specific functional state. Currently, the major tools for determining metabolite concentrations are mass spectrometry coupled with chromatographic techniques and nuclear magnetic resonance, which are expensive, time consuming and destructive for the samples. Here, we report a time resolved approach to monitor metabolite dynamics in cell cultures, based on Surface Enhanced Raman Scattering (SERS). This method is label-free, easy to use and provides the opportunity to simultaneously study a broad range of molecules, without the need to process the biological samples. As proof of concept, NIH/3T3 cells were cultured in vitro, and the extracellular medium was collected at different time points to be analyzed with our engineered SERS substrates. By identifying individual peaks of the Raman spectra, we showed the simultaneous detection of several components of the conditioned medium, such as L-tyrosine, L-tryptophan, glycine, L-phenylalanine, L-histidine and fetal bovine serum proteins, as well as their intensity changes during time. Furthermore, analyzing the whole Raman data set with the Principal Component Analysis (PCA), we demonstrated that the Raman spectra collected at different days of culture and clustered by similarity, described a well-defined trajectory in the principal component plot. This approach was then utilized to determine indirectly the functional state of the macrophage cell line Raw 264.7, stimulated with the lipopolysaccharide (LPS) for 24 hours. The collected spectra at different time points, clustered by the PCA analysis, followed a well-defined trajectory, corresponding to the functional change of cells toward the activated pro-inflammatory state induced by the LPS. This study suggests that our engineered SERS surfaces can be used as a versatile tool both for the characterization of cell culture conditions and the functional state of cells over time.

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Monitoring the dynamics of hemeoxygenase-1 activation in head and neck cancer cells in real-time using plasmonically enhanced Raman spectroscopy.

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