Quercetin-induced Growth Inhibition in Human Bladder Cancer Cells Is Associated with an Increase in Ca-activated K Channels

Abstract

Quercetin (3,3',4',5,7-pentahydroxyflavone) is an attractive therapeutic flavonoid for cancer treatment because of its beneficial properties including apoptotic, antioxidant, and antiproliferative effects on cancer cells. However, the exact mechanism of action of quercetin on ion channel modulation is poorly understood in bladder cancer 253J cells. In this study, we demonstrated that large conductance Ca(2+)-activated K(+) (BK(Ca)) or MaxiK channels were functionally expressed in 253J cells, and quercetin increased BK(Ca) current in a concentration dependent and reversible manner using a whole cell patch configuration. The half maximal activation concentration (IC(50)) of quercetin was 45.5±7.2 µM. The quercetin-evoked BK(Ca) current was inhibited by tetraethylammonium (TEA; 5 mM) a non-specific BK(Ca) blocker and iberiotoxin (IBX; 100 nM) a BK(Ca)-specific blocker. Quercetin-induced membrane hyperpolarization was measured by fluorescence-activated cell sorting (FACS) with voltage sensitive dye, bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3); 100 nM). Quercetin-evoked hyperpolarization was prevented by TEA. Quercetin produced an antiproliferative effect (30.3±13.5%) which was recovered to 53.3±10.5% and 72.9±3.7% by TEA and IBX, respectively. Taken together our results indicate that activation of BK(Ca) channels may be considered an important target related to the action of quercetin on human bladder cancer cells.

Keywords: BKCa; Bladder cancer cell; Proliferation; Quercetin; Voltage sensitive dye.

Fig. 1

Functional expression of Ca²⁺-activated K current in bladder cancer cells. (A) Messenger RNA (mRNA) of ion channels related to the K_Ca currents was amplified by reverse transcription-polymerase chain reaction (RT-PCR) analysis. BK_Ca (310 base pair [bp]), SK1 (282 bp), SK2 (214 bp), and SK3 (311 bp) were detected and GAPDH (258 bp) was used as a positive control. (B) Characterization of Ca²⁺ activated K⁺ channels (K_Ca) using whole cell recording in 253J cells. Current held at -60 mV was applied by step pulse from -120 mV to 100 mV in increments of 20 mV for 150 ms. Pipette solutions contained 140 mM KCl, 1 mM MgCl₂, 5 mM Mg-ATP, and 2 mM EGTA, and bath solutions contained 143 mM NaCl, 5.4 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, and 10 mM glucose. Representative outward current was blocked by 5 mM tetraethylammonium (TEA) and 100 nM iberiotoxin (IBX), a MaxiK specific blocker. Arrow indicates zero current.

Fig. 2

Inhibition of quercetin-evoked outward current by a K channel blocker in 253J cells. Voltage steps of 150 ms duration were applied from -120 mV to 100 mV in 20 mV increments every 10 sec. (A) Outward current activated by 50 µM quercetin was inhibited by TEA and recovered after TEA washout. (B) The specific BK_Ca channel inhibitor, IBX, inhibited ~97% of the quercetin-evoked whole cell outward current and the current was almost irreversible with IBX-free solution including quercetin in bladder cancer cells. (C) Dosedependent effects of quercetin on bladder cancer cells at 60 mV were plotted (n=5 cells per concentration). Quercetin substantially activated the outward current dose dependently. Quercetin-evoked current continually recorded for 150 ms ramp pulse from -100 to 100 mV at a holding potential of -60 mV. The bath solution contained normal Tyrode's (NT) and 0 µM, 1 µM, 10 µM, 30 µM, 50 µM, and 100 µM quercetin. Each point represents the mean ± standard deviation of the mean (SD) (n=5).

Fig. 3

The changes of membrane potential using voltage sensitive dye, bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3)) with various K⁺ concentrations, K⁺ channel blockers, and quercetin. Fluorescence-activated cell sorting (FACS) assay results from 253J cells loaded with DiBAC₄(3) (100 nM) are shown. (A) As graded increase of KCl concentration mean fluorescence intensity (MFI) was increased (the peak shifted right) (B) Treatment with quercetin (30 µM) decreased DiBAC₄(3) MFI (the peak shifted left). The application of TEA (5 mM, dotted line) in quercetin-free solution augmented MFI more than control (shaded bar), whereas co-treatment with TEA (5 mM) and quercetin (thick solid line) prevented quercetin-induced hyperpolarization (thin solid line).

Fig. 4

The effect of proliferation by quercetin and K channel blockers in 253J cells (A) Cells were treated for three days with quercetin (50 µM) or various K channel blockers in the presence or absence of quercetin. After treatment, proliferation was detected by XTT assay. Error bars represent mean±standard error [SE] for 32 separate experiments. Dimethyl sulfoxide (DMSO) was used with the same volume of quercetin. Asterisks indicate values which are different from the respective control (t-test, p<0.05). (B) Effect of quercetin and K channel blocker on 253J cell growth and viability. Cells were grown in 2% serum culture media and captured 24 hrs after addition of channel blockers and quercetin using a Nikon microscope at 10×20 magnification. Scale bar, 200 µm