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Effects of arsenic trioxide on voltage-dependent potassium channels and on cell proliferation of human multiple myeloma cells

2007; Lippincott Williams & Wilkins; Volume: 120; Issue: 14 Linguagem: Inglês

10.1097/00029330-200707020-00012

2542-5641

Jin Zhou, Wei Wang, Qingfang Wei, Tieming Feng, Lijun Tan, Baofeng Yang,

Neuroscience and Neuropharmacology Research

Arsenic trioxide (ATO) can induce cellular apoptosis and inhibit the activities of multiple myeloma (MM) cells in vitro,1 but how it works is not very clear. Recent studies showed that ATO worked on the voltage-dependent potassium channel and L-type calcium channel in myocardial cells,2–5 but the effect of ATO on ion channels of tumor cells was rarely reported. As the potassium channel plays an important role in controlling cell proliferation,6 we studied the effects of ATO on the voltage-dependent potassium current (Ikv) of the voltage-dependent potassium channel in an MM cell line, and probed into the relationship between changes of the Ikv caused by ATO and cell proliferation. METHODS Materials ATO (5 mmol/L) was produced by Harbin Yida Medicine Ltd., China. Tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), K-aspartic acid, MgCl2, EGTA, CaCl2, HEPES, KOH, NaCl, KCl, CsCl, L-glutamine, and MTT were purchased from Sigma Co., USA. Dimethyl sulphoxide (DMSO) was analytically pure and produced by Beijing Chemical Reagent Co., China. Culture medium RPMI-1640 with 10% fetal bovin serum (FBS) was the production of GIBCO Co., USA. Patch clamp recording systems (Axopatch200B, Axon, USA), flow cytometer (Becton Dickinson, USA) and enzyme immunoassay instrument (Bio-RAD Model 450, USA) were used in the study. Cell line preparation Human MM cell line RPMI-8226 was obtained from Prof. CHEN Zi-xing of Soochow Hematology Institute. RPMI-8226 cells were cultured at 37°C in humidified 5% CO2 in air, and in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mmol/L L-glutamine. The medium was changed every 2 to 3 days, and the cells were subcultured once a week. The cells were cultured and moved onto a poly-L-lysine-coated glass plate for 48 hours before whole-cell patch-clamp experiment. Cell proliferation inhibition Cell proliferation was measured by the MTT assay. The cells (1×104 cells per well) were cultured in a flat-bottomed 96-well plate for 24, 48, and 72 hours respectively with ATO (final concentration: 0.5, 1, 2, 4 and 8 mmol/L), and every group was repeated 5 times, then 20 μl MTT buffer was added to each well, and incubated for another 4 hours. The supernatant was decanted and 100 μl DMSO was added to each well. After the crystal dissolution by gently votexing, the absorbency values were read at 570 nm. Inhibition rate= (1-average absorbency in experiment group/average absorbency in control group) × 100%. Whole-cell patch clamp recording To record voltage-dependent potassium current, the standard whole-cell patch clamp technique was used. The axopatch 200 B patch clamp amplifier was connected with a computer. Tip resistances when bathed in normal Tyrode's solution ranged from 3 to 5 MΩ at the room temperature of 22°C. The pipette solution contained (mmol) 135 K-aspartic acid, 2 MgCl2, 1.1 EGTA, 0.1 CaCl2 and 10 HEPES-KOH buffer, pH 7.2. The cells were perfused with the extracellular solution containing (mmol) 136.5 NaCl, 5.4 KCl, 1.8 CaCl2, 0.53 MgCl2, 5.5 glucose, and 5 HEPES-NaCl buffer, pH 7.4. The voltage-dependent potassium current was recorded. Currents were studied using a depolarizing pulse of 1sec duration from -90 to +50 mV in 10 mV steps driven from a holding membrane potential of -80 mV. The current values were expressed in pA/pF. Cell cycle analysis RPMI-8226 cells (1.0×105 cells/ml) were washed twice with PBS, and then fixed with 70% ice-cold ethanol for 24 hours at 4°C. After being washed with PBS, the fixed cells were incubated with Tris-HCl buffer (pH 7.4) containing 10 μg/ml RNA enzyme for 30 minutes at 37°C. The cells were stained with propidium iodide (PI) and DNA content was analyzed by flow cytometry. The data were collected, saved, and analyzed by the ModFitLT software. Statistical analysis All data were expressed as mean ± standard deviation (SD). Statistical analysis was determined using independent-sample t test; all analyses were performed by SPSS v.10.0. A P value less than 0.05 was considered statistically significant. RESULTS Voltage-dependent potassium current in RPMI-8226 cell membrane (Ikv) The average resting membrane potential and capacity of RPMI-8226 cell were (-42±2) mV and (38±2) pF, respectively. Outward current was elicited when depolarization pulse reached -30 mV with the cell membrane potential maintaining at -80 mV. The current became stronger along with the increased depolarization potential. When the cell membrane was held at -80 mV and the instantaneous current was 0, with the concentration of extracellular K+ was changed from 5.4 mmol, 10 mmol, 40 mmol to 80 mmol, the measuring potential was (-54±1) mV, (-49±3) mV, (-30±1) mV and (-10±2) mV(n=11), respectively. These results demonstrated that the change of RPMI-8226 cell membrane current depended on the concentration of extracellular K+. We concluded that it was n-type voltage-dependent potassium current, because the elicited current was voltage-dependent and could be deactivated by repeated depolarization. Effect of ATO on Ikv in RPMI-8226 cell line Effect of ATO on the relation between current and voltage of Ikv in RPMI-8226 cell line The current extent of Ikv was partially inhibited after administration of 2 μmol ATO during all the gradients of the voltage being held, while Ikv almost recovered to the control level after ATO was washed away (Fig. 1A). The relation between current and voltage of cell membrane Ikv in RPMI-8226 cell line was determined, and depolarization was switched from -90 mV to +50 mV. When the depolarization pulse reached +50 mV, the peak amplitude was reduced by 2 μmol of ATO by 38% (Fig. 1B). Under the condition of currents being clamped, the resting membrane potential was depolarized from -42±2 mV to (-35±4) mV, and there was a difference between them (n=6, P<0.01).Fig. 1.: Effects of ATO on Ikv. A: Relationship between the current and potential after stimulation with first time-course depolarization at every 10 mV pace, the baseline potential at —80 mV. A1: The current of the control; A2: Current of RPMI-8226 after incubation in 2 μmol ATO for 5 minutes; B: Relationship between the current and potential of Ikv in RPMI-8226 after incubation in ATO; C: Effect of ATO on the resting membrane potential in RPMI-8226. *P<0.01, compared with control group.ATO inhibited Ikv in a concentration-dependent manner In a whole-cell voltage-clamp mode, Ikv was measured before and after 5-minute exposure to various concentrations of ATO (0.5, 1, 2, 4, and 8 μmol/L). The current amplitudes of Ikv were all depressed at these concentrations during the whole voltage-clamp period. The inhibition of currents was dose-dependent, and half-maximal inhibitory concentration (IC50) value for ATO was 4.1 μmol/L. Ikv almost recovered to the control level after ATO being washed away. Comparing the effect of ATO with other drugs on Ikv TEA and 4-AP are voltage-dependent potassium channel blockers. The effects of 10 mmol TEA, 1 mmol 4-AP, 10 mmol 4-AP, and various concentrations of ATO on Ikv were compared. Ikv was significantly depressed by 10 mmol 4-AP and 8 μmol ATO by 80%, less depressed by 1 mmol 4-AP and 1 mmol ATO by 20%, and most gently depressed by 10 mmol TEA by 15%. Effects of ATO on cell proliferation in RPMI-8226 cell line ATO inhibited RPMI-8226 cell proliferation in a time-and dose-dependent manner and its IC50 was 3.4 mmol. Cell proliferation was almost completely inhibited by 8 mmol ATO (Fig. 2). The concentration of ATO, which effectively depressed Ikv, ranged from 1 mmol to 8 mmol, similar to that for inhibiting cell proliferation (P<0.01).Fig. 2.: Effects of ATO on cell proliferation and Ikv of MM cell line.ATO effectively depressed Ikv, ranged from 1 mmol to 8 mmol, similar to inhibiting cell proliferation.Effect of ATO on cell cycle of RPMI-8226 cell line DNA content of RPMI-8226 cells was analyzed by flow cytometry after co-cultured with 2 mmol ATO for 6, 12, 24 and 48 hours. Cell cycle analysis showed a time-dependent increase in accumulation of G0/G1 phase cells and a gradually decrease of S-phase cells. These data suggested a significant negative correlation between apoptosis ratio and number of S-phase cells (Table, P<0.01).Table: Effects of ATO on cell cycle of RPMI-8226DISCUSSION Recent study has shown that the activation of potassium channels in non-excitable cells and changes of membrane potential can affect the mitogen-induced cell activation process, and therefore interfere cell proliferation.6 Potassium blockers such as 4-AP and TEA can inhibit proliferation of malignant cells or induce apoptosis of the cells.7 MM is a kind of B-cell malignancy, which is rich in voltage-dependent potassium channels in the cellular membrane.8–10 There are two basic biophysical characteristics of these channels, voltage-dependent activity and high selectivity to K+. In this study, the voltage-dependent potassium channels in the membrane of MM were verified by K+ specific blockers: Cs+, TEA and 4-AP. We found ATO can inhibit Ikv in a dose-dependent manner. During K+ channel blockade, the inhibition extent of 1 mmol ATO to Ikv was similar to that of 1 mmol 4-AP and much stronger than that of 10 mmol TEA. The effect of 8 mmol ATO was similar to that of 10 mmol 4-AP. These data suggested that ATO inhibited Ikv of potassium channels to a large extent, which is similar to the effect of voltage-dependent potassium channel blockers. At various concentrations, a positive correlation could be seen between decreased current and cell proliferation inhibition, suggesting that inhibitory effect of ATO on cell proliferation was related to the decreased activity of potassium channels and the resultant cell depolarization. Flow cytometry showed that the ratio of RPMI-8226 cells in early G1 phase increased gradually along with prolonged action of ATO in contrast to that decreased in S phase. This indicated that proliferation of RPMI-8226 cells was inhibited by ATO in early G1 phase. Besides, apoptosis of RPMI-8226 cells induced by ATO was also observed. According to our findings, we speculated that ATO may trigger apoptosis by the intrinsic signal-pathway through Ikv inhibition and depolarization of cell membrane, but further studies are needed to find out the exact mechanism.