Abstract
Neuroendocrine differentiation of prostate epithelial cells is usually associated with an increased aggressivity and invasiveness of prostate tumors and a poor prognosis. However, the molecular mechanisms involved in this process remain poorly understood. We have investigated the possible expression of voltage-gated calcium channels in human prostate cancer epithelial LNCaP cells and their modulation during neuroendocrine differentiation. A small proportion of undifferentiated LNCaP cells displayed a voltage-dependent calcium current. This proportion and the calcium current density were significantly increased during neuroendocrine differentiation induced by long-term treatments with cyclic AMP permeant analogs or with a steroid-reduced culture medium. Biophysical and pharmacological properties of this calcium current suggest that it is carried by low-voltage activated T-type calcium channels. Reverse transcriptase-PCR experiments demonstrated that only a single type of LVA calcium channel mRNA, an alpha(1H) calcium channel mRNA, is expressed in LNCaP cells. Quantitative real-time reverse transcriptase-PCR revealed that alpha(1H) mRNA was overexpressed during neuroendocrine differentiation. Finally, we show that this calcium channel promotes basal calcium entry at resting membrane potential and may facilitate neurite lengthening. This voltage-dependent calcium channel could be involved in the stimulation of mitogenic factor secretion and could therefore be a target for future therapeutic strategies.
Highlights
Neuroendocrine differentiation of prostate epithelial cells is usually associated with an increased aggressivity and invasiveness of prostate tumors and a poor prognosis
We have investigated in this study the possible expression of voltage-gated calcium channels in human prostate cancer LNCaP cells and their modulation during neuroendocrine differentiation induced by increasing cytosolic cAMP or by reducing steroids in the culture medium
Neuroendocrine Differentiation Induced by cAMP Permeant Analogs Is Associated with an Increased Inward Current—We first assessed by morphometric assays and Western blotting that culturing LNCaP cells for 3–5 days with cAMP permeant analogs (dibutyryl cAMP (Bt2cAMP), 8-bromo-cAMP (8-Br-cAMP), 1 mM) or a phosphodiesterase inhibitor induced neuroendocrine differentiation
Summary
LNCaP cells were purchased from the American Type Culture Collection and grown as recommended in RPMI 1640 (Biowhittaker, Fontenay sous Bois, France) supplemented with 10% fetal bovine serum (Seromed, Poly-Labo, Strasbourg, France) and 5 mM L-glutamine (Sigma, L’Isle d’Abeau, France). Cells were routinely grown in 50-ml flasks (Nunc, Poly-Labo) in a humidified atmosphere at 37 °C (95% air, 5% CO2). To study the role of steroids in the expression of voltage-dependent calcium channels, cells were grown in phenol red-free RPMI 1640 supplemented with 10% charcoal-stripped fetal bovine serum (culture medium thereafter referred to as steroid-reduced medium). Cells were subcultured in Petri dishes (Nunc) using trypsin. The culture medium was changed every 3 days. The treatment was initiated and the culture medium containing the treatments (Bt2cAMP, IBMX) was changed every day.
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