Blockage of Intermediate-Conductance Ca -Activated K Channels Inhibit Human Pancreatic Cancer Cell Growth in Vitro

Ion channels are important in controlling cell cycle progression and proliferation in a variety of cell types. Using the whole-cell recording mode of the patch-clamp technique, functional ion channels were electrophysiologically characterized in PANC-1 (Kras G12D ( / ), p53 R273C, p16), BxPC-3 (smad4 , p53 Y220C, p16), and MiaPaCa-2 [transforming growth factorreceptor type II defect, K-ras G12C( / ), p53 R248W, p16] human pancreatic cancer cell lines. In BxPC-3 and the MiaPaCa-2 cells, we could identify 600 or 1200 functional Ca -activated K channels (IK) per cell, respectively, whereas PANC-1 cells expressed 200 functional IK channels per cell. These channels were observed by using pipette solutions buffering [Ca ]i to 1 M. The channels were voltage-independent, blocked by charybdotoxin, clotrimazole, 1-[(2-chlorophenyl) diphenylmethyl]-1Hpyrazole (TRAM-34), and blocked by Ba in a voltage-dependent manner. In the presence of 10 M clotrimazole or TRAM-34, proliferation of the BxPC-3 as well as the MiaPaCa-2 cells was completely stopped. In contrast, proliferation of PANC-1 cells was hardly affected by clotrimazole or TRAM-34. Proliferation in all three cell lines could be inhibited in the presence of the Ca channel antagonists verapamil, diltiazem, and nifedipine. By quantitative RT-PCR, we could show that MiaPaCa-2 cells exhibit a 2.8-fold and BxPC3 cells a more than 8-fold elevated level of IK mRNA level compared with PANC-1 cells. Interestingly, in primary pancreatic tumors we found a tremendous up-regulation of IK mRNA. In eight of nine (or 89%) primary pancreatic tumor tissues, we found a 6to 66-fold increase in IK mRNA. Our findings suggest that a certain amount of functional IK channels is crucial for the proliferation of some pancreatic cancer types. The blockade of IK channels may ultimately prove useful as a therapeutic option for some patients with ductal adenocarcinoma of the pancreas with an up-regulated IK channel expression. There are a lot of indications that ion channels are important for cell cycle progression and proliferation of cells. An excellently documented example for the importance of potassium channels in proliferation and differentiation derived from studies with lymphocytes (DeCoursey et al., 1984; Matteson and Deutsch, 1984). However, this relation between potassium channels and cell proliferation has also been shown for other cell systems, for example in melanoma cells (Nilius and Wohlrab, 1992), breast carcinoma cells (Strobl et al., 1995), fibroblasts (Rane, 1999), and Schwann cells (Pappas and Ritchie, 1998). The role of potassium channels in these cellular events is usually attributed to a regulation in the intracellular calcium concentration. Alternatively, potassium channels could also control cell volume and might therefore modulate cell proliferation (Rouzaire-Dubois and Dubois, 1998). In addition, there are reports that potassium channels are involved in tumorigenesis (Pardo et al., 1999). IK channels seem to play an important role for epithelial Cl because it has been demonstrated that in human intestinal T84 cells, blockage of IK channels by clotrimazole inhibited repressed Cl secretion (Rufo et al., 1997). In addition, activation of IK channels stimulated Cl secretion in a variety of epithelial tissues (Devor et al., 1996; Singh et al., 2001). The IK channel in erythrocytes, called the “Gardos” channel (Gardos, 1958), is activated in sickle cells because of deoxygenation/sickling and is a major cause of salt loss and dehydration. It has been shown that blocking the Gardos channel with clotrimazole can improve the erythrocyte state of hydration in patients with sickle-cell disease (Brugnara et al., 1996). In addition to the influence of functional IK chanThis work was support by grants from the Deutsche Forschungsgemeinschaft (to S.G. and to K.G.), Interdisziplinäres Zentrum für Klinische Forschung B7 (to H.J. and S.G.), SFB518 (to K.G. and T.G.), and 4SC AG (Martinsried, Germany) (to S.G.). ABBREVIATIONS: IK channel, Ca -activated K channel with intermediate conductance; TGF, transforming growth factor; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; DMSO, dimethyl sulfoxide; CTX, charybdotoxin; RT-PCR, reverse transcription-polymerase chain reaction; CT, cycle threshold; CRAC, calcium release-activated Ca 2 channel; ERK, extracellular signal-regulated kinase. 0026-895X/04/6503-630–638$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 3 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2580/1129141 Mol Pharmacol 65:630–638, 2004 Printed in U.S.A. 630 at A PE T Jornals on Jne 6, 2017 m oharm .aspeurnals.org D ow nladed from nels in erythrocyte function, these channels also seem to play a role in erythroid differentiation (Vandorpe et al., 1998). In pancreatic ductal epithelial cells, Ca -activated potassium channels have been shown to mediate pancreatic secretion of fluid and electrolytes (Nguyen and Moody, 1998). In combination with the Na /H antiport and the Na /K ATPase, these channels mediate serosal H secretion, thus balancing luminal HCO3 secretion (Nguyen and Moody, 1998). To determine whether potassium channels might also play an important role in the proliferation control of pancreatic carcinoma cells, we first characterized the functionally expressed ion channels in these cells using electrophysiological techniques. In a second step, we measured the increase in cell number of these pancreatic carcinoma cell cultures under conditions in which most of the expressed potassium channels were blocked. We used three different pancreatic carcinoma cell lines that all harbor mutations in p53 and deletions of p16 (Berrozpe et al., 1994; Fujimoto et al., 2000; Moore et al., 2001). In addition to these common mutations and deletions, PANC-1 cells are characterized by a monoallelic activating K-ras mutation (Moore et al., 2001), BxPC-3 cells have a smad4 deletion (Berrozpe et al., 1994; Fujimoto et al., 2000), and MiaPaCa-2 cells lack a functional transforming growth factor(TGF) receptor type II in combination with an activating K-ras mutation on both alleles (Moore et al., 2001). Although mutations of the K-ras gene occur in more than 90% of pancreatic carcinomas (Johnson et al., 2001; Moore et al., 2001), they are not the only alterations that occur (Hruban et al., 2001). Usually, inactivation of tumor-suppressor genes, such as p16 and p53, coupled with activation of oncogenes such as K-ras, are a few of the mutations that trigger the growth of cancerous cells (Hruban et