Gliomas are highly invasive tumor cells which synthesize numbers of proteins/enzymes performing different functions. Some of these enzymes affect the cells survival, proliferation and migration, the others force the cells to commit suicide known under the term apoptosis – “programmed cell death”. One of the “Janus faced” regulatory enzymes synthesized by glioma cells is protein kinase C delta (PKCδ). Its inhibition or activation significantly influences apoptosis.

Fig. 1.

The primary question in this study was how to estimate the PKCδ expression in U87 MG glioma cells by using common biological techniques and advanced computational approaches. We have used hypericin, a well-known PKC inhibitor, to study PKCδ distribution and expression in U87 MG cells. Hypericin is a hydrophobic molecule that possess suitable properties for its application in the field of photodynamic therapy (PDT) of cancerous and non-cancerous diseases. The principle of PDT lies in a selective activation of photo-sensitive molecule localized at specific area in cancer cell. Hypericin was demonstrated to be localized at different places within cancer cells, depending on its administered concentration, incubation time and transport system.

In this study, the subcellular distribution of hypericin was observed to be similar to the distribution of the fluorescent labeled ceramide, a probe for Golgi apparatus staining. We have shown the analogy in cellular uptake pathway of hypericin and ceramide by using fluorescence lifetime imaging technique. Previously, ceramide has been demonstrated to induce PKCδ intracellular relocalization with the Golgi apparatus as the final target. Here we have found the increased Golgi apparatus accumulation with PKCδ phosphorylated at Ser645 site after hypericin administration. The Western blot analysis demonstrated the increased expression of this phosphorylated form of PKCδ. Furthermore, 3D image reconstructions and numerical analysis of the achieved images gave additional information useful for better understanding of PKCδ role in U87 MG cells and its involvement in apoptotic pathways.

We have developed an empirical model characterizing with the PKCδ spatial redistribution and its localization in U87 MG cells. The model we have proposed takes into account the structure of the available experimental data that are used to determine model parameters. The approach we applied is generally known as “divide and conquer” strategy. The visualized volumes of the PKCδ droplets located within the intra/extra – nuclear compartments represent inputs for quantification, description, modeling, that finally lead to better understanding of the cell death mechanisms. We studied volume dynamics in non-equilibrium but also in local equilibrium regime with small fluctuations. A new perspective for the theory comes from empirical finding that the fluctuations during equilibration can be modeled by the stochastic process of the Ornstein-Uhlenbeck type.

Veronika Huntosova
Center for Interdisciplinary Biosciences, Faculty of Science, P.J. Safarik University, Kosice, Slovakia

Publication

Estimation of PKCδ autophosphorylation in U87 MG glioma cells: combination of experimental, conceptual and numerical approaches.
Misuth M, Joniova J, Belej D, Hrivnak S, Horvath D, Huntosova V
J Biophotonics. 2016 May 9

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