Monitoring of the prostate tumour cells redox state and real-time proliferation by novel biophysical techniques and fluorescent staining

Abstract

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 IC₅₀ for zinc(II) is 55.5 and 150.8 μM for PC-3 and PNT1A, respectively. MTT-determined IC₅₀ are >1.3-fold higher. Impedance-based viability correlates with viable count (r > 0.92; p < 0.03), not with MTT. Two-fold lower intracellular zinc(II) in the tumour PC-3 cell line was found. After zinc(II) treatment >2.6-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 ratio > 1), when exposed to zinc(II) compared to PC3, where predominance of oxidised forms remains at all concentrations. IC₅₀ 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.