Health Implications of Dietary Amines 391 Potassium channel blockers quinidine and caesium halt cell proliferation in C6 glioma cells via a polyamine-dependent mechanism T.M. Weiger* 1,2 , S. Colombatto† 2 , V. Kainz* 2 , W. Heidegger‡, M.A. Grillo† and A. Hermann* *Division of Animal Physiology, Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria, †Department of Medicina e Oncologia Sperimentale, University of Torino, Torino, Italy, and ‡Department of Molecular Biology, University of Salzburg, Salzburg, Austria Abstract Potassium channels are ubiquitous in cells and serve essential functions in physiology and pathophysio- logy. Potassium channel blockers have been shown to block tumour growth by arresting cells at the G 0 /G 1 checkpoint of the cell cycle. We investigated the effect of quinidine and caesium (Cs + ) on cell proliferation, LDH (lactate dehydrogenase) release, free internal calcium, membrane potential, polyamine concentration, ODC (ornithine decarboxylase) activity and polyamine uptake in C6 glioma cells. The EC 50 for reducing cell pro- liferation was 112 μM for quinidine, whereas Cs + was less effective with an EC 50 of 4.75 mM. KCl or sucrose did not affect proliferation. LDH release was augmented by quinidine. Quinidine caused a transient increase in free internal calcium but decreased calcium after a 48 h incubation period. Further 300 μM quinidine de- polarized the cell membrane in a similar range as did 30 mM KCl. Quinidine decreased cellular putrescine beyond detection levels while spermidine and spermine remained unaffected. ODC activity was reduced. Addition of putrescine could not override the antiproliferative effect owing to a reduced activity of the polyamine transporter. Our study indicates that the antiproliferative effect of quinidine is not due to a simple membrane depolarization but is caused by a block of ODC activity. Introduction Potassium channels are found ubiquitously in animal and plant cells. They control the membrane potential as well as electrical signalling and are known to be involved in cell proliferation. Potassium channel blockers such as quinidine, 4-AP (4-aminopyridine), Cs + or TEA (tetraethyl-ammon- ium) have been shown to stop cell proliferation and tumour growth in a variety of cell types via an arrest in the G 0 /G 1 transition during the cell cycle (for reviews, see [1,2]). How- ever, the cellular mechanism by which potassium channel activity contributes to cell division is not clear. Polyamines not only modulate potassium channels [3,4], but also are crucial for cells to pass through the cell cycle [5]. We invest- igated the effects of Cs + , which is used in cancer therapy [6,7], and quinidine, a drug used in the clinic for treatment of malaria [8] and heart arrhythmias [9], on polyamine homoeostasis, membrane potential, mitochondrial activity, calcium levels and cytotoxicity in C6 glioma cells. Key words: cell cycle, cell proliferation, mitochondrion, ornithine decarboxylase (ODC), potassium channel, quinidine. Abbreviations used: 4-AP, 4-aminopyridine; BrdU, 5-bromo-2 ′ -deoxyuridine; FCS, fetal calf serum; HS, horse serum; LDH, lactate dehydrogenase; NF2, neurofibromatosis type 2; ODC, ornithine decarboxylase; TEA, tetraethyl-ammonium; XTT, sodium 3,3 ′ -[1-(phenylamino- carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate. 1 To whom correspondence should be addressed (email thomas.weiger@sbg.ac.at). 2 These authors contributed equally to this work. Materials and methods Cell culture C6-glioma cells were kept at 37 ◦ C, 5% CO 2 and 90% humidity in Ham’s F-10 medium supplemented with 9% FCS (fetal calf serum) and 1% penicillin/streptomycin. In the case of polyamine substitution experiments, FCS was replaced by HS (horse serum). For experiments cells were plated on poly(D-lysine)- or fibronectin-coated 96-well microtitre plates, flasks or coverslips. At 5 h after seeding, control medium was changed to experimental medium containing either quinidine or Cs + . Cell proliferation assay Cell numbers were measured with a Crystal Violet assay as described previously [10,11]. Net proliferation was deter- mined by subtracting the number of initially seeded cells. Controls were set to 100% unless mentioned otherwise. Analysis of dose–response relationships Data were fitted according to the equation: Y = 1 −  [c h ]  c h + EC h 50  where Y is normalized response (values from control experiments were set to 1), c is concentration and h is Hill coefficient. C  2007 Biochemical Society