Korean J Physiol Pharmacol.  2013 Dec;17(6):511-516. 10.4196/kjpp.2013.17.6.511.

The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

• Park KS
• Han MH
• Jang HK
• Kim KA
• Cha EJ
• Kim WJ
• Choi YH
• Kim Y

• KMID: 2285478
• DOI: http://doi.org/10.4196/kjpp.2013.17.6.511

Abstract

Bladder cancer is the seventh most common cancer in men that smoke, and the incidence of disease increases with age. The mechanism of occurrence has not yet been established. Potassium channels have been linked with cell proliferation. Some two-pore domain K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to be overexpressed in cancer cells. Here we focused on the relationship between cell growth and the mechanosensitive K2P channel, TREK2, in the human bladder cancer cell line, 253J. We confirmed that TREK2 was expressed in bladder cancer cell lines by Western blot and quantitative real-time PCR. Using the patch-clamp technique, the mechanosensitive TREK2 channel was recorded in the presence of symmetrical 150 mM KCl solutions. In 253J cells, the TREK2 channel was activated by polyunsaturated fatty acids, intracellular acidosis at -60 mV and mechanical stretch at -40 mV or 40 mV. Furthermore, small interfering RNA (siRNA)-mediated TREK2 knockdown resulted in a slight depolarization from -19.9 mV+/-0.8 (n=116) to -8.5 mV+/-1.4 (n=74) and decreased proliferation of 253J cells, compared to negative control siRNA. 253J cells treated with TREK2 siRNA showed a significant increase in the expression of cell cycle boundary proteins p21 and p53 and also a remarkable decrease in protein expression of cyclins D1 and D3. Taken together, the TREK2 channel is present in bladder cancer cell lines and may, at least in part, contribute to cell cycle-dependent growth.

Keyword

Bladder cancer; Cell cycle; Proliferation; Small interfering RNA; TREK2

MeSH Terms

Acidosis
Blotting, Western
Cell Cycle
Cell Line
Cell Proliferation
Cyclins
Fatty Acids, Unsaturated
Humans*
Incidence
Male
Patch-Clamp Techniques
Potassium Channels
Real-Time Polymerase Chain Reaction
RNA, Small Interfering
Smoke
Urinary Bladder Neoplasms
Urinary Bladder*
Cyclins
Fatty Acids, Unsaturated
Potassium Channels
RNA, Small Interfering
Smoke

Figure

• 

  Fig. 1 TREK2 channel expression in human bladder cancer cells. (A) Messenger RNA (mRNA) of ion channels related to the TREK1 (Accession No; AF129399) and TREK2 (Accession No; AF279890) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) analysis. TREK1 (355base pair [bp]) and TREK2 (291 bp) were detected. (B) Immunoblot showed presence of TREK2 channel protein in the human bladder cancer 253J cell line. TREK2 transfected CHO cells were used as a control. (C) Representative confocal microscopic analysis of TREK2 in bladder cancer cell line 253J. Cells were stained with an anti-TREK2 antibody and F-actin. Scale bar, 20 µm.

• 

  Fig. 2 The physiologic properties of TREK2 at a single channel level in bladder cancer 253J cells. (A) TREK2 in CHO cells transfected with DNA encoding TREK2 and GFP and in 253J cells was measured in the excised inside-out patch configuration at the holding potential values shown on the left. The current trace was obtained in symmetrical 150 mM KCl solutions. The letters "c" and "o" represent the "closed" and "open" states of the channels, respectively. (B) The I-V relationships showed inward rectification, and each point is the mean of 4 experiments with standard error (S.E) represented by the error bars. (C~E) Current tracing showed arachidonic acid, intracellular pH, and mechanosensitivity of the native TREK2-like channel in 253J cells at -60 mV, +40 mV, and -40 mV. Negative pressure (-10 mmHg or -20 mmHg) was applied through the pipette. The panel below each of the figures shows the single channel trace on an expanded time scale.

• 

  Fig. 3 The effect of TREK2 knockdown on the growth of 253J bladder cancer cells. (A and B) TREK2 mRNA and protein levels in 253J cell were determined after knockdown of TREK2 by siRNA through qRT-PCR and Western blot. Expression of TREK2 mRNA after transfection of TREK2 siRNA or negative control siRNA was normalized. TREK2 protein was examined by Western blot analysis after transfection of the negative control or TREK2 siRNA in 253J cells. GAPDH was used as a control. (C) The membrane potential was measured at current clamp (I=0) in a whole cell patch configuration. The 253J cells were treated with FITC-labeled negative control siRNA and FITC-labeled TREK2 siRNA for 72 hours. The data were represented as the mean±S.E. (t-test, p value <0.05). (D) The antiproliferative effect of TREK2 knockdown by siRNA in 253J cells. Cells were treated for three days with TREK2 siRNA or (-)control siRNA in 2% serum culture media. After treatment, proliferation was measured by XTT assay. Error bars represent the mean±S.E for 38 separate experiments. Asterisks indicate values that are different from the respective control (t-test, p<0.05). (E) Effect of TREK2 siRNA on 253J cell growth. Cells were captured 48 hours after transfection with TREK2 siRNA using a Nikon microscope at 10×10 magnification. Scale bar, 100 µm.

• 

  Fig. 4 Cell cycle arrest at G0/G1 after TREK2 siRNA transfection in bladder cancer 253J cells. (A) The bar graph shows that TREK2 siRNA treated cells resulted in an increased percentage of cells in G0/G1 and a decreased percentage of cells in the S phase of the cell cycle (t-test p<0.05) (B) Representative Western blot showing changes in the levels of associated proteins in cell cycle arrest of human bladder cancer 253J cells. Knockdown of TREK2 decreased the expression of cyclin D1, cyclin D3, cdk2, cdk4, and cdk6 and increased protein levels of p21 and p53.

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