Delayed Luminescence in cancer research

The evaluation of optical properties of biological samples is gaining increasing interest both in scientific and applicable field due to the ability of used optical techniques to provide information on the biological status of the system under analysis in a fast and non-destructive way. In this context the Delayed Luminescence (DL) appears to be an excellent candidate for the development of a reliable and economical optical biopsy techniques. The term Delayed Luminescence (DL) refers to the photo-induced ultra-weak luminescence emitted by systems, also biological, after the illumination source has been switched off. The DL has a spectral emission lying from the optical range to near infrared and its intensity is various orders of magnitude (103-105) lower than the usual Fluorescence or Phosphorescence. DL is by nature extremely polyphasic. Its lifetime spectrum extends from 10-7 to more several seconds after the stimulation end. Indeed it is well known that relaxation from non-equilibrium state towards equilibrium of complex systems can be approximated by a power law, being such an approximation consistent with the idea of a distribution for relaxation kinetics [1]. Due to the low level of DL, we used the dedicated equipment for fast ultraweak luminescence analysis ARETUSA developed at the National Southern Laboratories of the National Nuclear Physics Institute (LNS-INFN), in Catania, Italy. The samples were excited by a nitrogen laser source emitting pulses at 337 nm. The re-emitted photons, in the wavelength range 350–850, nm are collected by a photomultiplier tube working in single photon counting mode. Spectral measurements were carried out by using broadband bandpass filters centered at 450 nm, 550 nm and 650 nm (50nm FWHM) inserted between sample and photomultiplier tube. Improvement in experimental equipment as well as mathematical analysis of data have shown the possibility to use the DL in cancer research as tool to investigate the status of human cells and tissues, so showing the possibility to discriminate between normal and tumor conditions [2, 3]. The study performed on human leukemia Jurkat T-cells [4-5], on follicular and anaplastic human Thyroid cancer cells [6], on glioblastoma multiforme [7] gave the possibility to correlate DL to apoptosis and oxidative stress. The results pointed out the mitochondrial origin of this ultraweak luminescence and in particular to its correlation with the electron flow in the complex I of the mitochondrial respiratory chain [7, 8], whose natural biomarkers, NADH, flavins and singlet oxygen, emit respectively in blue, green-yellow and red regions. Worth to note that in addition to their established role in generating energy for the cell, mitochondria represent an essential component of many apoptotic pathways, and features of DL have been correlated to apoptosis and oxidative stress. More recently in the framework of the research project ETHICS “Pre-clinical experimental and theoretical studies to improve treatment and protection by charged particles” funded by INFN, we studied DL emitted by non-tumorigenic breast epithelial cell line and metastatic breast cancer cell line in order to found possible correlations between Delayed Luminescence and in vitro damaging induced by ion irradiation. The experimental results showed not only DL dependence on cell line but also its ability to give early information of the effects induced by proton dose [9]. References [1] Berberan-Santos M.N. et al., Mathematical functions for the analysis of luminescence decays with underlying distributions 1. Kohlrausch decay function (stretched exponential), Chem. Phys. 315, 171–182 (2005), doi.org/10.1016/j.chemphys.2005.04.006. [2] Niggli H.J. et al., Laser-Ultraviolet-A induced ultraweak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts, Indian J. Exp. Biol. 46, 358-363 (2008). [3] Musumeci F. et al., Discrimination between normal and cancer cells by using spectral analysis of delayed luminescence, Appl. Phys. Lett. 86, 153902 (2005), doi.org/10.1063/1.1900317 . [4] Baran I. et al., Effects of menadione, hydrogen peroxide and quercetin on apoptosis and delayed luminescence of human leukemia Jurkat T-cells, Cell Biochem. Biophys., 58, 169-179 (2010), doi: 10.1007/s12013-010-9104-1. [5] Baran I. et al., Detailed analysis of apoptosis and delayed luminescence of human leukemia Jurkat Tcells after proton-irradiation and treatments with oxidant agents and flavonoids, Oxidative Med. Cell. Longev., 2012, 498914 (2012), doi:10.1155/2012/498914. [6] Scordino A. et al., Delayed luminescence to monitor programmed cell death induced by Berberine on thyroid cancer cells, J. Biomed. Opt., 19, 117005 (2014), doi: 10.1117/1.JBO.19.11.117005. [7] Grasso R. et al., The delayed luminescence spectroscopy as tool to investigate the cytotoxic effect on human cancer cells of drug-loaded nanostructured lipid carrier, Proc. SPIE 9887, Biophotonics: Photonic Solutions for Better Health Care V, 988723, (2016), doi: 10.1117/12.2227514. [8] Baran I. et al., Mitochondrial respiratory Complex I probed by delayed luminescence spectroscopy, J. Biomed. Opt., 18, 127006 (2013), doi: 10.1117/1.JBO.18.12.127006. [9] Grasso R. et al., Delayed luminescence in a multiparameter approach to evaluation and reduction of radiobiological risks. Novel Biophotonics Techniques and Applications IV, A. Amelink, ed., Vol. 10413 of SPIE Proceedings (Optical Society of America, 2017), paper 104130L (2017); doi: 10.1117/12.2285851.