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Knockdown of the small conductance Ca(2+) -activated K(+) channels is potently cytotoxic in breast cancer cell lines.

ABSTRACT:

Background and purpose

Small conductance calcium-activated potassium (KCa 2.x) channels have a widely accepted canonical function in regulating cellular excitability. In this study, we address a potential non-canonical function of KCa 2.x channels in breast cancer cell survival, using in vitro models.

Experimental approach

The expression of all KCa 2.x channel isoforms was initially probed using RT-PCR, Western blotting and microarray analysis in five widely studied breast cancer cell lines. In order to assess the effect of pharmacological blockade and siRNA-mediated knockdown of KCa 2.x channels on these cell lines, we utilized MTS proliferation assays and also followed the corresponding expression of apoptotic markers.

Key results

All of the breast cancer cell lines, regardless of their lineage or endocrine responsiveness, were highly sensitive to KCa 2.x channel blockade. UCL1684 caused cytotoxicity, with LD50 values in the low nanomolar range, in all cell lines. The role of KCa 2.x channels was confirmed using pharmacological inhibition and siRNA-mediated knockdown. This reduced cell viability and also reduced expression of Bcl-2 but increased expression of active caspase-7 and caspase-9. Complementary to these results, a variety of cell lines can be protected from apoptosis induced by staurosporine using the KCa 2.x channel activator CyPPA.

Conclusions and implications

In addition to a well-established role for KCa 2.x channels in migration, blockade of these channels was potently cytotoxic in breast cancer cell lines, pointing to modulation of KCa 2.x channels as a potential therapeutic approach to breast cancer.

SUBMITTER: Abdulkareem ZA

PROVIDER: S-EPMC4737296 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium-4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities.

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