Monitoring of the prostate tumour cells redox state and real-time proliferation by novel biophysical techniques and fluorescent staining
| Autor: | Monika Pavkova-Goldbergova, Martina Raudenská, Sona Krizkova, Markéta Sztalmachová, René Kizek, Jiri Sochor, Branislav Ruttkay-Nedecky, Petr Babula, Vojtech Adam, Michal Masarik, Marián Hlavna, Natalia Cernei, Ondrej Zitka, Jaromír Gumulec |
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| Rok vydání: | 2012 |
| Předmět: |
Male
Cell Survival Blotting Western Biophysics Tetrazolium Salts chemistry.chemical_element Cell Growth Processes Zinc Cell morphology Biochemistry Redox 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Cell Line Tumor Humans Metallothionein MTT assay Fluorescent Dyes 030304 developmental biology 0303 health sciences Formazans Reverse Transcriptase Polymerase Chain Reaction Acridine orange Prostatic Neoplasms Glutathione Molecular biology Zinc Sulfate Microscopy Fluorescence chemistry Dielectric Spectroscopy 030220 oncology & carcinogenesis Cancer cell Linear Models RNA Oxidation-Reduction |
| Zdroj: | Integrative Biology. 4:672-684 |
| ISSN: | 1757-9708 |
| DOI: | 10.1039/c2ib00157h |
| Popis: | The present paper is focused on zinc(ii) treatment effects on prostatic cell lines PC-3 (tumour) and PNT1A (non-tumour). Oxidative status of cells was monitored by evaluation of expression of metallothionein (MT) isoforms 1A and 2A at the mRNA and protein level, glutathione (oxidised and reduced), and intracellular zinc(ii) after exposition to zinc(ii) treatment at concentrations of 0-150 μM using electrochemical methods, western blotting and fluorescent microscopy. A novel real-time impedance-based growth monitoring system was compared with widely used end-point MTT assay. Impedance-based IC(50) for zinc(ii) is 55.5 and 150.8 μM for PC-3 and PNT1A, respectively. MTT-determined IC(50) are1.3-fold higher. Impedance-based viability correlates with viable count (r0.92; p0.03), not with MTT. Two-fold lower intracellular zinc(ii) in the tumour PC-3 cell line was found. After zinc(ii) treatment2.6-fold increase of intracellular zinc(ii) was observed in non-tumour PNT1A and in tumour PC-3 cells. In PC-3 cells, free and bound zinc(ii) levels were enhanced more markedly as compared to PNT1A. PNT1A produced 4.2-fold less MT compared to PC3. PNT1A cells showed a 4.8-fold increase trend (r = 0.94; p = 0.005); PC-3 did show a significant trend at MT1 and MT2 protein levels (r = 0.93; p = 0.02) with nearly ten-fold increase after 100 μM zinc(ii) treatment. In terms of redox state, PNT1A had a predominance of reduced GSH forms (GSH : GSSG ratio1), when exposed to zinc(ii) compared to PC3, where predominance of oxidised forms remains at all concentrations. IC(50) differs significantly when determined by MTT and real-time impedance-based assays due to dependence of impedance on cell morphology and adhesion. When real-time growth monitoring, precise electrochemical methods and fluorescent microscopy are performed together, accurate information for metal fluxes, their buffering by thiol compounds and monitoring of the redox state become a powerful tool for understanding the role of oxidative stress in carcinogenesis. |
| Databáze: | OpenAIRE |
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