Ultrasound and cisplatin combined treatment of human melanoma cells A375--the study of sonodynamic therapy.

Sonodynamic therapy, an effect of low-power ultrasound field and the anticancer drug cisplatin, was studied in vitro on human melanoma cells A375. The viability of cells has been studied by standard 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide viability assay according to different modes of treatment: application of cisplatin alone, exposure of ultrasound field alone, exposure to ultrasound followed by cisplatin and application of cisplatin followed by exposure to ultrasound. Ultrasound was used at a therapeutic intensity of 1 W∙cm(-2) for 10 min. Concentration of cisplatin in the cell suspension was always 2.3 µM. The results show that sonodynamic therapy is one of the possibilities of how to intensify standard cytostatic therapy. This conclusion is supported by reducing the viability of studied cells, especially 72 h after the treatment. The time sequence of application of ultrasonic field and cytostatics appears to be a significant factor affecting the changes in cell viability. Maximum suppression of viability has been found when applying the experimental design involving application of cisplatin followed by exposure to ultrasound; the final value of viability of combined affected cells was more than 10% lower than for cisplatin treatment alone.

Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture.

The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 W x cm(-2) in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were--1 s drop time, 5 mV x s(-1) voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag/AgCl/3 M KCl as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy.

Biological effects of combined ultrasound and cisplatin treatment on ovarian carcinoma cells.

The effects of low-power ultrasound, the anti-cancer drug cisplatin, and their combined application were studied in two lines of human ovarian carcinoma cells, A2780 and A2780cis. Four modes of treatment were used: exposure to ultrasonic field, application of cisplatin, exposure to ultrasound followed by cisplatin, and presence of cisplatin followed by exposure to application ultrasound. Ultrasound was used at intensities of 0.5 W/cm(2) and 1.0 W/cm(2) for 10 min, cisplatin was applied at concentrations of 1 microM and 6 microM per cell suspension treated in A2780 and cisplatin-resistant A2780cis cells, respectively. The results of each experimental treatment were assessed by the resultant cell viability related to the viability of control cells, using a standard MTT test. It was shown that a combined effect of ultrasound and cisplatin was more effective than that of ultrasound or cisplatin alone. It also appeared that the order of application played a role, with the cisplatin-ultrasound treatment lowering cell viability more than the ultrasound-cisplatin treatment. It can be assumed that the exposure of cells to a low-power ultrasonic field has an immediate effect on the structure of cell surfaces and, consequently, on entry of cisplatin into the cell. The study also included observations on changes in the cell cycle associated with the treatments used in both cell lines and their evaluation by flow cytometry.

Photochemical properties of a potential photosensitiser indocyanine green in vitro.

The present study investigates the photochemical properties of a potential photosensitiser, indocyanine green (ICG), in an in vitro HeLa cell system. Cell proliferation was studied after a combined effect of ICG, at a concentration range of 24-94 microM, and therapeutic laser irradiation at several different energy densities. In addition, ICG cytotoxicity was evaluated in HeLa cells and V79 Chinese hamster by the MTT assay. Phototoxicity was evaluated at 1, 24, and 48 h after irradiation. No phototoxic effect was detected 1h after irradiation. The maximum phototoxic effect of ICG on HeLa cells was detected for an ICG concentration of 94 microM, a laser output of 360 mW, and an energy density of 99 J/cm(2) at 24h after irradiation. Potentiation of the ICG phototoxic effect was achieved by adding 20 microM H(2)O(2), which was a non-toxic concentration for HeLa cells in this experimental design. At 48 h after laser irradiation a statistically significant difference was found between the toxicity of ICG plus peroxide, as compared to ICG alone. The addition of H(2)O(2) at a concentration of 20 microM caused a significant increase in phototoxicity of ICG for HeLa cells. Our results confirm that ICG could be a perspective agent for use in photodynamic therapy and that its phototoxic effect can be potentiated by addition of an oxidative agent.

Discrete Fourier transform-based analysis of HeLa cell microtubules after ultrasonic exposure.

Cytoskeletal structures can be affected by external factors including ultrasound. Our task was to develop a structure analysis method to evaluate these changes quantitatively. We exposed HeLa cells to continuous ultrasound (1 MHz, 1 and 2 W/cm2, 10 min at 37 degrees C). The microtubules were detected by the monoclonal antibody TU-01/SwAM/FITC, observed in a fluorescence microscope and photographed digitally. The images were processed by "FFT magic" software. The structure analysis is based on frequency domain filtering using discrete Fourier transform. The basic idea is to design filters to extract information describing best the structural changes. The properties of the filter can be enhanced by direction filtering, i.e., extraction of a symmetric angular segment in the frequency domain centered on a zero frequency. The final image is a normalized sum of inverse FFT's of such segmented spectra. We needed a method yielding a single number assigned to the structure, e.g., the ratio of the area of microtubules to the total cell area. Assuming that the image background intensity is constant, we can use thresholding to detect areas occupied by the cells. The information about the area of the microtubules is contained in a wide range of higher intensities. Therefore, we use a gamma correction. The area occupied by microtubules is then considered an area with intensities above the selected threshold. There were tested three different filters to extract information about microtubules. The mathematical method chosen seems sensitive enough for quantitative assessment of changes of the microtubular network.