Tetraethylammonium (TEA), 4-aminopyridine (4-AP), glibenclamide, phloretin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), diazoxide, iberiotoxin, tetrodotoxin (TTX) and propidium iodide (PI) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). The Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) apoptosis detection kit was procured from Antgene Biotech (Wuhan, China). RNase A was purchased from Fermentas International Inc. (Burlington, Ontario, Canada). All other chemicals were of standard analytical grade.

The human glioma cell line U87-MG was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in RPMI-1640 supplemented with 10% FBS and 100 units of penicillin/streptomycin in 5% CO₂ at 37°C. The cells were passaged every 3 days and maintained in exponential growth to ~80% confluency for later experiments.

A modified MTT assay was used to examine the effect on cell proliferation. Briefly, cells were seeded in a 96-well plate at 5,000 cells/well and incubated overnight. After drug treatment for 48 h, 20 μl of MTT solution (5 mg/ml) was added to each well, and the samples were incubated for an additional 4 h. Subsequently, the supernatant was removed, and the cells were dissolved in 150 μl of DMSO. Finally, absorbance was measured at 570 nm using a 96-well microplate reader (Thermo Scientific, USA).

Cells were double stained using an Annexin V-FITC/PI apoptosis detection kit. Briefly, after exposure to different drugs for 48 h, the cells were harvested, washed twice with cold PBS and resuspended in Annexin V binding buffer. Then, 5 μl of FITC-labeled Annexin V and 5 μl of PI were added. The cells were incubated for 15 min at room temperature in the dark with gentle oscillation. After the addition of 200 μl of binding buffer to each tube, the cells were analyzed within 1 h using a flow cytometer (Becton-Dickinson, USA).

Cells were detached by trypsinization, seeded at 5×10⁵ cells/well in a 6-well plate and incubated overnight. The cells were then treated with various drugs at different concentrations. Subsequently, the cells were harvested into cold PBS at different time points, fixed in ice-cold 70% ethanol and stored at 4°C overnight for subsequent cell cycle analysis. Fixed cells were washed twice with PBS and resuspended in 1 ml of the staining reagents (100 μg/ml RNase A and 50 μg/ml PI). The samples were incubated in the dark for 30 min, and the distribution of cells in the various phases of the cell cycle was measured by flow cytometry.

Cells were grown overnight, washed twice with Hank’s balanced salt solution (HBSS) and loaded with 1 μmol/l Fluo3-AM for 30 min in the dark at room temperature. Then cells were washed twice with Ca²⁺-free HBSS, re-suspended in Ca²⁺-free HBSS and incubated at room temperature for 20 min in the dark. When appropriate, the cells were pretreated with K⁺ channel blockers or openers for 5 min before the initiation of Ca²⁺ influx. Ca²⁺ influx was measured as changes in fluorescent signals, which were recorded using a fluorescence microscope (Olympus, Japan) and analyzed using Matlab software.

To further verify the antiproliferative efficacies of K⁺ channel blockers, an in vivo experiment was performed using 6- to 8-week old nude mice. The nude mice were randomly divided into control and treated groups. The treated groups were subcutaneously injected with a suspension of 5×10⁶ U87-MG cells with 4-AP (5 mmol/l), TEA (20 mmol/l) or glibenclamide (200 μmol/l), and mice in the control group were injected with a suspension of 5×10⁶ cells with 0.01 mol/l PBS (8). Tumor size was measured every 3 days and tumor volume was determined by calculating (length × width)²/2. At the end of the experiment, tumors were excised and weighed. A tumor growth curve was plotted according to the tumor volume, and the inhibitory rates of tumor growth were calculated according to the tumor weight. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Membrane currents were recorded using a whole-cell voltage clamp and borosilicate glass pipettes (outer diameter, 1.5 mm; direct current resistance, 3–6 MΩ) constructed using a two-step puller (P-97; Sutter, USA). To investigate the K_V currents, the pipette solution consisted of (in mmol/l) KCl 140, MgCl₂ 2.5, HEPES 10, EGTA 11 and ATP 5 and the pH was adjusted to 7.2 (11). To investigate the K_ATP currents, the micropipettes were filled with (in mmol/l) KCl 140, MgCl₂ 1, ATP 0.01, EGTA-KOH 5 and HEPES-KOH 5 and the pH was adjusted to 7.3 (12). The cells were bathed in an extracellular solution containing (in mmol/l) NaCl 135, KCl 5.4, MgCl₂ 1.0, NaH₂PO₄ 0.33, CaCl₂ 1.8, HEPES 10 and D-glucose 10 with 1 μmol/l TTX. The osmolarity was adjusted to 330 mOsm/l with sucrose and the pH was adjusted to 7.4. Whole-cell patch clamp recordings were performed at room temperature using a patch clamp amplifier (Axon-200B) (13). Adjustments of capacitance compensation and series resistance compensation were performed before the membrane currents were recorded. The membrane currents were filtered at 10 kHz (−3 dB) and the data were processed using Clampfit (Axon, USA).

The data are expressed as the means ± standard error (± SE). Statistical significance was assessed using analysis of variance (ANOVA). P-values of <0.05 were considered to indicate statistically significant results.

To identify the K⁺ channels that affect U87-MG cell proliferation, we used a variety of K⁺ channel blockers and openers and an MTT assay was used to determine the number of live cells remaining after the drug treatment. Among the blockers, 4-AP and TEA are transient outward and delayed rectifier K_V channel blockers, respectively (14), whereas glibenclamide is a specific blocker of K_ATP channels, and iberiotoxin is a specific K_Ca channel blocker. As shown in Fig. 1, cell proliferation was significantly inhibited by 4-AP and TEA in a dose-dependent manner and glibenclamide also significantly decreased the U87-MG cell number. However, 100 nmol/l iberiotoxin, a concentration that completely blocks K_Ca channels (15), only moderately inhibited U87-MG cell proliferation. Meanwhile, diazoxide, an opener of K_ATP channels and phloretin, an opener of K_Ca channels, had no significant effects on cell proliferation. Taken together, these data reveal that K_V and K_ATP channels but not K_Ca channels have important roles in the proliferation of U87-MG cells.

To elucidate the mechanisms through which K⁺ channels influence the proliferation of glioma cells, flow cytometry was performed to investigate whether the U87-MG cell cycle may be influenced by various K⁺ channel blockers or openers. As shown in Fig. 2A and B, the percentage of U87-MG cells in the G₀/G₁ phase was significantly enhanced after exposure to 4-AP, TEA and glibenclamide for 48 h, whereas the percentage of cells in the S phase was markedly reduced. Iberiotoxin had no obvious effect on the U87-MG cell cycle at a concentration of 25 nmol/l. Meanwhile, activating K_ATP channels with diazoxide and activating K_Ca channels with phloretin increased the percentage of U87-MG cells in the S phase. These results were consistent with previous reports (16) that K⁺ channels are believed to facilitate progression through G₁/S checkpoint.

To determine whether the reduction in cell viability caused by K⁺ channel blockers was related to apoptotic cell death, the effects of different K⁺ channel blockers and openers on U87-MG cell apoptosis were studied using Annexin V-FITC/PI staining. As illustrated in Fig. 2C, the percentage of Annexin V-FITC-positive apoptotic cells was increased by adding 4-AP or glibenclamide to the U87-MG cells (5.1±0.8% of control, 44.7±3.8% of 5 mmol/l 4-AP and 18.3±1.32% of 200 μmol/l glibenclamide). However, 4-AP only slightly increased necrosis, whereas glibenclamide induced noticeable necrosis (1.8±0.26% of control, 3.2±0.26% of 5 mmol/l 4-AP and 24.8±3.08% of 200 μmol/l glibenclamide). In contrast, at some of the tested concentrations, treatment with TEA, iberiotoxin, phloretin or diazoxide for 48 h had no apparent effect on cell apoptosis.

To further explore the mechanism of K⁺ channel involvement in U87-MG cell proliferation, we examined the effects of different K⁺ channel blockers and openers on Ca²⁺ influx. Ca²⁺ influx was induced by the addition of 0.5 mmol/l CaCl₂ to the bathing medium (Ca²⁺-free HBSS), which caused a rapid increase in cytosolic Ca²⁺ to a level that was sustained for several minutes. As shown in Fig. 3, 4-AP (5 mmol/l) and glibenclamide (200 μmol/l) nearly abolished the increase in [Ca²⁺]_i induced by exogenously applied Ca²⁺ (P<0.01), and TEA (20 mmol/l) reduced the peak Ca²⁺ response by 8.5±0.5% (P<0.05), indicating that K_V and K_ATP channels may modulate Ca²⁺ influx into glioma cells and subsequently modulate the proliferation and apoptosis of these cells. Iberiotoxin (25 nmol/l) decreased the peak Ca²⁺ response by 2.1±0.1%, which was not significant when compared with the control cells (P>0.05).

To provide direct evidence that K_V or K_ATP channels are responsible for tumor development, the antitumor activities of K_V or K_ATP channel blockers were investigated in nude mice with established human glioma U87-MG xenografts. As shown in Fig. 4A, U87 xenografts grew rapidly in mice, and the tumor size in the control group reached 1189.2±203.3 mm³ over the duration of the experiment (27 days). Statistically, there were significant differences in both tumor volume and weight between the control and the treated groups. Mice treated with 4-AP at 5 mmol/l and glibenclamide at 200 μmol/l showed inhibition rates of 46.2 and 43.8%, respectively, which were significant (P<0.01) when compared with the control. TEA at 20 mmol/l suppressed tumor growth by 33.7% (P<0.05). The actual tumor weights at the time of sacrifice are shown in Fig. 4B. No obvious toxic effects were observed in any of the groups during the experiment. These results suggest that K_V and K_ATP channels play important roles in glioma development in vivo.

To determine whether K⁺ channel blockers inhibits cell proliferation and tumorigenesis via blockage of whole-cell K⁺ currents, we next studied the dose-dependent effects of these three blockers on K_V/K_ATP currents in U87-MG cells. Representative recordings of the K_V currents (transient and persistent K⁺ currents) were evoked with a step-up depolarization protocol. Briefly, the membrane potential was pre-hyperpolarized from −50 mV to −110 mV for 100 msec, depolarized from −50 to +60 mV (10 mV increment/step, duration 200 msec) and subsequently restored to the original depolarizing potential of −50 mV (Fig. 5A). Depolarizing the voltage from −110 to −50 mV and maintaining the level of −50 mV for 100 msec inactivated transient K⁺ currents and a further depolarization voltage step from −50 to +60 mV evoked persistent K⁺ currents (Fig. 5B). The transient component (Fig. 5C) was then visualized in isolation using point-by-point subtraction of the persistent component (as shown in Fig. 5B) from the total outward current (as shown in Fig. 5A). As shown in Fig. 5D, 5 mmol/l TEA remarkably inhibited the persistent outward currents and the persistent component was reduced completely after the application of TEA (40 mmol/l). Meanwhile, 4-AP (1 mmol/l) markedly suppressed the transient outward current and 4-AP (10 mmol/l) completely and reversibly blocked the transient components in each cell examined (Fig. 5E).

To examine the effect of glibenclamide on K_ATP currents, K_ATP currents were activated by using standard whole cell recording with a pipette solution containing only 10 mmol/l ATP (12). The membrane potential was normally held at −40 mV and the currents were evoked by a series of 400 msec depolarizing and hyperpolarizing current steps (−100 mV to +80 mV in 20 mV steps, Fig. 5F). As shown in Fig. 5G, the whole-cell K⁺ currents observed with low intracellular ATP were inhibited by extracellular glibenclamide (300 μmol/l). These data suggest that these three K⁺ channel blockers have the same working concentration range for inhibiting K_V/K_ATP currents, cell proliferation and tumor growth, indicating that the effects of 4-AP, TEA and glibenclamide are indeed mediated by blockage of K⁺ channels.