Downregulation of Ca²⁺-Activated Cl⁻ Channel TMEM16A by the Inhibition of Histone Deacetylase in TMEM16A-Expressing Cancer Cells

Abstract

The Ca 21 -activated Cl 2 channel transmembrane proteins with unknown function 16 A (TMEM16A; also known as anoctamin 1 or discovered on gastrointestinal stromal tumor 1) plays an important role in facilitating the cell growth and metastasis of TMEM16A-expressing cancer cells. Histone deacetylase (HDAC) inhibitors (HDACi) are useful agents for cancer therapy, but it remains unclear whether ion channels are epigenetically regulated by them. Using real-time polymerase chain reaction, Western blot analysis, and whole-cell patch-clamp assays, we found a significant decrease in TMEM16A expression and its functional activity was induced by the vorinostat, a pan-HDACi in TMEM16A-expressing human cancer cell lines, the prostatic cancer cell line PC-3, and the breast cancer cell line YMB-1. TMEM16A downregulation was not induced by the chemotherapy drug paclitaxel in either cell

Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

“…It has reported that AATB (4-(acetylamino)- N -[2-amino-5-(2-thienyl)phenyl]-benzamide) is a selective HDACi for HDAC1 and HDAC2 (half maximal (50%) inhibitory concentration (IC 50 ) = 7 and 49 nM for HDAC1 and HDAC2, respectively and IC 50 ≥ 10 μM for the other HDAC isoforms) [35]. In addition, in our previous study, inhibitory effects of target gene transcription by 30 nM and 300 nM AATB were very similar to those by HDAC1 and HDAC2 siRNAs, respectively [36]. The inhibitory effects of K Ca 1.1 transcription were demonstrated through the pharmacological blockade of HDAC1 and HDAC2 by 300 nM AATB ( n = 4, p < 0.01), whereas no significant changes were found by the respective inhibition of HDAC1, HDAC3, and HDAC6 by treatments with 30 nM AATB, 1 μM T247 [ N -(2-aminophenyl)-4-[1-(2-thiophen-3-ylethyl)-1 H -(1),(2),(3)triazol-4-yl]benzamide], and 1 μM NCT-14b [( S )- S -7-(adamant-1-ylamino)-6-(tert-butoxycarbonyl)-7-oxoheptyl-2-methylpropanethioate] for 48 h ( n = 4 for each, p > 0.05).…”

“…They were maintained at 37 °C in 5% CO 2 with RPMI 1640 medium, Dulbecco’s modified Eagle’s (DMEM) medium, or Leibovitz’s L-15 medium containing 10% fetal bovine serum (Sigma, St. Louis, MO, USA) and a penicillin (100 units/mL)-streptomycin (0.1 mg/mL) mixture [36]. A cell viability assay using WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H -tetrazolium, monosodium salt), which is a colorimetric assay to measure the viable cell numbers by the cleavage of tetrazolium salts was performed according to our previous study [36]. Briefly, using a density of 10 5 cells/mL, cells were cultured in duplicate in 96-well plates for 0–4 days.…”

“…Four hours after the addition of WST-1 reagent (Dojindo, Kumamoto, Japan) into each well, the absorbance was measured in a microplate reader Multiskan FC (Thermo Fisher Scientific, Yokohama, Japan) at a test wavelength of 450 nm as a reference wavelength of 620 nm. In the siRNA-mediated blockade of K Ca 1.1, HDAC2, and HDAC3, Lipofectamine ® RNAiMAX reagent (Thermo Fisher Scientific) was used [36]. Commercially available siRNA oligonucleotides against human K Ca 1.1/HDAC2/HDAC3 and control siRNA (type A) were purchased from Santa Cruz Biotechnology.…”

“…Total RNA extraction from cell lines and reverse-transcription were performed as previously reported [36]. cDNA products were amplified with gene-specific PCR primers, designated using Primer Express TM software (Ver 3.0.1, Life Technologies, Carlsbad, CA, USA).…”

HDACs and HDAC Inhibitors in Cancer Development and Therapy

“…T247 and T326 inhibited the growth of colon and prostate cancer cells (Suzuki et al 2013). In TMEM16A–expressing cancer cells, T247 also exerts a suppressive effect on cancer cell viability via downregulatingTMEM16A (Matsuba et al 2014). …”