Functional expression of BK channel α and γ subunits in the LNCaP prostate cancer cell line, in normoxic and hypoxic conditions

B. Evans, C. Breen, R. Kennedy, K. McCloskey. CCRCB, Queen's University Belfast, Belfast, United Kingdom.

Introduction: Prostate cancer (PCa) affects 1 in 8 men in their lifetime and is the 5^th most common cancer death in men (1). PCa have a hypoxic tumour environment, moreover, treatment with androgen deprivation therapy further drives hypoxia (2). Ion channels present a unique opportunity for novel therapeutic targets given their biophysical and pharmacological diversity. BK channels are associated with several cancer hallmarks, including proliferation (3). BKγ is an modulatory subunit that complexes with pore forming BKα allowing BK to be active in androgen sensitive PCa LNCaP cells at resting membrane potentials in the absence of calcium (4). This study aimed to characterise the functional expression of BKα and BKγ in LNCaP under normoxia and hypoxia and elucidate whether BK activity was linked with cell proliferation/viability.

Methods: PCR, Western blot and immunofluorescence, cell viability and patch-clamp assays were performed. BK modulators mallotoxin (0.5-10μM) and paxilline (0.3-1μM) were used, (vehicle DMSO <0.1%). Hypoxia was achieved with a hypoxic chamber, 37°, 0.1%O₂ (5%CO₂) for 48hs. Data are expressed as mean±SEM; ANOVA/unpaired Student’s t-tests were performed; P<0.05 considered as significant.

Results: BKα (KCNMA1) gene expression was greater in hypoxic vs normoxic cells (1.47±0.14 fold n=4, P<0.05) although protein expression was similar. Conversely, BKγ (LRRC26) expression was reduced (0.58±0.13 fold, n=4, P<0.01) at gene and also protein level. LNCaP had paxilline-sensitive (300nM) BK currents in normoxia and hypoxia (n=6; n=5 respectively) with similar activation curves. BK current density was similar in hypoxia and normoxia (n=5). BK activator, mallotoxin (5μM) leftward-shifted the activation curve; V₅₀ from 35mV to 17.7mV (n=3). The mallotoxin-effect was attenuated by paxilline co-treatment. Mallotoxin significantly decreased proliferation/viability in both conditions in a concentration-dependent manner (1μM-10μM, n=3). Mallotoxin was apparently less effective in hypoxia. Paxilline partially rescued the mallotoxin-effect (0.5μM) on proliferation/viability by 21±7.3%) in normoxia.

Conclusion: Differential gene expression of BKα/BKγ under hypoxia did not significantly alter channel activity. Mallotoxin was apparently more effective in normoxia than hypoxia. In normoxia, the mallotoxin-reduction in cell viability was partially-rescued by paxilline suggesting that BK channel activation reduced viability. Any non-BK effects of mallotoxin require further study.

References:

(1) Cancer Research UK, http://www.cancerresearchuk.org/

(2) Byrne NM et al. (2016) Br J Cancer 114:659-668.

(3) Bloch M et al. (2007) Oncogene 26: 2525-2534.

(4) Yan J and Aldrich RW (2010) Nature 466: 513-516