Computerized detection of morphological changes to glioma cells during estramustine and ion-channel blocker perifusion

Abstract

Summary A perifusion technique for microscopy with computerized detection of early changes in cell morphology during continuous perifusion was used to show that the geometry of cultured glioma cells (MG-251) changes rapidly when they are exposed to estramustine phosphate (EMP). When the cells were exposed to 20 or 40 mg 1-' EMP, cell volume projected cell area (PCA) rapidly increased. When the Na+, K+-ATPase blocker ouabain (100 ,umol 1-') was added to the EMP (40 mg 1-') perifusion, the acute EMP response was eradicated. When the PCA curve for ouabain alone was subtracted from the curve of combined ouabain and EMP perifusion, the resulting curve showed that ouabain completely blocked the EMP-induced increase in PCA. When the Na+, K+, Cl-co-transport inhibitors bumetanide (10 lmol 1-'), or furosemide (100 ,umol 1-'), were added to EMP (40 mg 1-'), the acute increase in PCA seen for EMP alone was also completely blocked. This study shows that inhibitors of ion transmembrane transport can modify EMP-induced cell volume increases. This may be of particular importance since the blockers have been found to interfere also with the cytotoxic function of EMP during cell culture. Thus, it is possible that cell volume changes could serve as a rapid technique for predicting the cytotoxic activity of antineoplastic drugs.Keywords: cell volume; cytotoxicity; estramustine; microperifusion; potassium flux An intact cell volume is known to be of critical importance for the preservation of cell functions, including growth and proliferation (Hoffman and Simonsen, 1989). Extensive studies on cell volume regulation in the last few years indicate that a wide variety of cells share common regulatory capacities, although a pronounced diversity exists between different cell types in the nature of the ion transport systems involved. It is well known that transmembrane fluxes of cations are part of important cell functions, such as maintenance of the membrane potential regulation, intracellular pH (DeWeer, 1985), and volume regulation in anisotonic media (Hoffman and Simonsen, 1989). Predictive tumour sensitivity tests have received increasing attention, and several different predictive cellular and animal systems have been developed with varying degree of success (Chapman et al, 1989). There is, thus, still a strong need for more effective predictive tests of drug sensitivity in clinical oncology.Estramustine phosphate (EMP), a cytotoxic nor-nitrogen mustard derivative of oestradiol 1 7p-phosphate, is generally accepted in the treatment of advanced prostatic carcinoma (Madajewics et al, 1980), and recently it has been shown to exert considerable cytotoxic effects on several glioma cell lines von Schoultz et al, 1990). In the present study, we have investigated the effect of bumetanide and furosemide, inhibitors of Na+, K+, Cl-co-transport, and ouabain, an inhibitor of Na+, K+-ATPase, on the cytotoxic effect of EMP. This was done by means of a new microscopic technique that provides the ability to follow early alterations in cell size an...

Diverging Effects of 5-HT3 Receptor Antagonists Ondansetron and Granisetron on Estramustine-Inhibited Cellular Potassium Transport

“…In previous reports we have shown that very early changes in cell size and shape correlated with the cytotoxicity to glioma cells of the anticancer drug estramustine phosphate (Engström et al 1991;Behnam-Motlagh et al 1997). The increase in cell volume indicated that estramustine phosphate caused a reduction of cellular ion efflux reflected in a decreased 86 Rb π influx with a total net accumulation of ions and water into the cell (Engström et al 1991).…”

“…Thus, there exist a possibility of interactions between drugs that influence transmembrane fluxes of cations and anti-neoplastic drugs. The Na π , K π , 2Cl ª -cotransport inhibitors furosemide and bumetanide reduced the cytotoxicity of estramustine phosphate to pulmonary mesothelioma (P 31) cells (Sandström et al 1994), and also completely blocked estramustine phosphate-induced cell volume changes of glioma cells (Behnam-Motlagh et al 1997). Grankvist/klinkemi.umu.se). formed fibroblasts (Grankvist et al 1992).…”

Ondansetron but not granisetron affect cell volume regulation and potassium ion transport of glioma cells treated with estramustine phosphate

“…Changes in cell size and shape has been shown to correlate with EMP cytotoxicity to glioma cells (Engstrom et al 1991;Behnam-Motlagh et al 1997). At least three different potassium ion flux pathways seem to exist in the tumour cell line used (malignant glioma Mg251), i.e., Na + , K + , ATPase, Na + , K + , 2Cl --cotransport system, and K channels with high conductance (Sandstrom et al 1994).…”

“…The technique has been used in previous reports regarding EMP cytotoxicity (Engstrom et al 1991;Behnam-Motlagh et al 1997;Behnam-Motlagh et al 2000). In short, we used a special type of microperifusion chamber consisting of glass slides forming a compartment in which cancer cells could be perifused.…”

“…Thus, there exists a potential possibility of interactions between drugs that influence transmembrane fluxes of cations and antineoplastic drugs. Indeed, the Na + , K + , 2Cl --cotransport inhibitors furosemide and bumetanide were found to reduce the cytotoxicity of EMP to pulmonary mesothelioma (P31) cells (Sandstro¨m et al 1994), and also to completely block EMP-induced cell volume changes of glioma cells (Behnam-Motlagh et al 1997). Moreover, ondansetron possesses a distinct ability to reverse K + influx of mesothelioma cells (P31) exposed to EMP, whereas granisetron does not.…”