CaV3.2 T-type Calcium Channels Are Involved in Calcium-dependent Secretion of Neuroendocrine Prostate Cancer Cells

Florian Gackière,Gabriel Bidaux,Philippe Delcourt,Fabien Van Coppenolle,Maria Katsogiannou,Etienne Dewailly,Alexis Bavencoffe,Myriam Tran Van Chuoï-Mariot,Brigitte Mauroy,Natalia Prevarskaya,Pascal Mariot

doi:10.1074/jbc.m707159200

Abstract

Because prostate cancer is, in its early stages, an androgen-dependent pathology, treatments aiming at decreasing testosterone plasma concentration have been developed for many years now. However, a significant proportion of patients suffer a relapse after a few years of hormone therapy. The androgen-independent stage of prostate cancer has been shown to be associated with the development of neuroendocrine differentiation. We previously demonstrated that neuroendocrine prostate cancer cells derived from LNCaP cells overexpress CaV3.2 T-type voltage-dependent calcium channels. We demonstrate here using prostatic acid phosphatase as a marker of prostate secretion and FM1-43 fluorescence imaging of membrane trafficking that neuroendocrine differentiation is associated with an increase in calcium-dependent secretion which critically relies on CaV3.2 T-type calcium channel activity. In addition, we show that these channels are expressed by neuroendocrine cells in prostate cancer tissues obtained from patients after surgery. We propose that CaV3.2 T-type calcium channel up-regulation may account for the alteration of secretion during prostate cancer development and that these channels, by promoting the secretion of potential mitogenic factors, could participate in the progression of the disease toward an androgen-independent stage.

Highlights

Hormone refractory disease, deprivation of androgens has no further incidence on the growth of the prostate cancer, and no curative therapy is currently effective
We show in this article that LNCaP cells display a calcium-dependent pathway of regulated secretion and that neuroendocrine differentiation is associated with an increase in prostatic acid phosphatase (PAP)2 secretion
We show that T-type calcium channels could promote secretion upon membrane depolarization and that PAP secretion by LNCaP cells is dependent on T-type (␣1H) calcium channel activity

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Treatments—LNCaP cells were cultured as previously described [11]. To induce neuroendocrine differentiation, LNCaP cells were cultured with 1 mM dibutyryl cyclic AMP and 100 ␮M isobutylmethylxanthine for 3– 6 days. Electrophysiological Recordings—Patch clamp recordings were performed in the whole-cell configuration as previously described [24] using a RK-300 patch clamp amplifier (Biologic, Grenoble, France). The second method is a sensitive fluorimetric assay (Molecular Probes, mIU1⁄7mlϪ1) based on the cleavage of 6,8difluoro-4-methylumbellyferyl phosphate (DIFMUP) by phosphatases [26], generating DIFMU, whose fluorescence was excited at 360 nm and measured at 450 nm. SiRNAs were incubated in culture medium without serum for 5–10 min at room temperature to form the transfection complexes and were added dropwise onto the cells. Analysis of the ␣1H Subunit Gene Expression of a Voltage-dependent T-type Calcium Channel (Reverse Transcription-PCR)— Reverse transcription-PCR was carried out as previously described [16]. Differences were considered significant where p Ͻ 0.05 (*), p Ͻ 0.01 (**), and p Ͻ 0.001 (***)

RESULTS

We then carried out a set of experiments to assess whether

This shows that the expression of

ADDITIONS AND CORRECTIONS