THE INFLUENCE OF SPERMINE AND CHLORHEXIDINE ON THE SURVIVAL, ELECTROKINETIC AND CYTOMORPHOLOGICAL CHARACTERISTICS OF HUMAN PROSTATE CANCER CELLS

Abstract

Summary. Aim: to study the effects of spermine and the spermine oxidase inhibitor chlorhexidine, applied alone or in combination, on the viability, electrokinetic and structural-functional characteristics of human prostate cancer cells in vitro. Object and methods: Studies were conducted on cell cultures: differentiated androgen-dependent LNCaP cell line and low-differentiated androgen-independent DU-145 cell line. Cell survival was determined by the method of exclusion of the vital dye trypan blue by living cells. The electrokinetic parameters of the cells (ζ-potential and total surface charge density) were determined according to the Smoluchovsky and Quincke-Helmholtz equations. Morphological changes were assessed by light microscopy of fixed preparations of cells stained with hematoxylin and eosin. Results: The fact established by previous studies that spermine at a concentration of 1.5 mM exhibits a pronounced inhibitory effect on the growth of differentiated human prostate cancer cells of the LNCaP line, which consists in reducing their survival to 48.5 ± 1.5% compared to intact control, and when this factor is used in a concentration exceeding 5.0 mM – in their death. It was shown that poorly differentiated cells of the DU-145 line were significantly more resistant to its influence: their survival decreased to 56.0 ± 1.9% only under the influence of spermine at a concentration of 5.0 mM; a concentration of 10.0 mM caused complete cell death of both lines. The trend persisted in the case of chlorhexidine: under its influence at a concentration of 15.0 μM, the survival of cells of the hormone-dependent LNCaP line was 33.0 ± 1.5%, and the hormone-independent DU-145 cell line was 79.0 ± 2.5%. The combined use of spermine and chlorhexidine (0.6 mM and 3.2 µM, respectively) caused a strong cytotoxic effect in cells of the LNCaP line (survival 19.0 ± 1.2%), while cells of the DU-145 line maintained a fairly high rate (76.0 ± 2.1%) even with a significantly higher content of spermine (3.0 mM) and chlorhexidine (5.0 μM) in the culture medium. It was established that under the influence of chlorhexidine, the cells of both studied lines show an inversion of the sign of the surface electric charge, changing it from negative to positive under physiological conditions, just as it was shown in experiments with spermine on cells of the LNCaP line. Certain quantitative differences caused by the investigated compounds in the electrokinetic indicators of cells of hormone-dependent and hormone-independent lines were revealed. Cells of the DU-145 line under the influence of spermine, or simultaneously spermine and chlorhexidine, showed a typical apoptotic morphology: they rounded and separated from each other and the substrate, chromatin condensation, blurred contours of the surface membrane, nuclear fragmentation and the presence of apoptotic bodies were noted. Conclusions: Spermine in concentrations of 1.5-10.0 mM and chlorhexidine in concentrations of 10.0-30.0 μM, applied separately, have a cytotoxic effect on human prostate cancer cells of LNCaP and DU-145 lines in culture. Cytotoxic properties of spermine, chlorhexidine or their combination are significantly more pronounced when acting on the hormone-dependent LNCaP line cells than on the hormone-independent DU-145 line. Their combined use demonstrates a significantly higher cytotoxic effect on cells of the LNCaP line, which is not observed in the DU-145 cell line. Chlorhexidine, like spermine, causes an inversion of the surface charge in the studied cell lines, changing its sign from negative to positive. The revealed differences like changes in survival and electrokinetic parameters of LNCaP and DU-145 cells can be explained by the presence or absence of androgen binding receptors on their surface membrane. Cells of the DU-145 line under the influence of spermine, or at the same time spermine and chlorhexidine, go to the state of apoptosis, as well as cells of the LNCaP line when they are exposed to spermine.