Philippe Delcourt

Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation

Store‐operated calcium entry (SOCE) is the main Ca2+ influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh‐7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh‐7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6‐overexpressing or TRPC6‐knockdown Huh‐7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6‐overexpressing Huh‐7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6‐knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)‐induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh‐7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh‐7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. Conclusion: TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis. (HEPATOLOGY 2008;47:2068–2077.)

Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells.

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (Icold/menthol) that shows inward rectification and high Ca2+ selectivity, which are dramatically different properties from “classical” TRPM8-mediated Icold/menthol. Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both Icold/menthol and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca2+ release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca2+ release channel, potentially involved in a number of Ca2+- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

Role of Cationic Channel TRPV2 in Promoting Prostate Cancer Migration and Progression to Androgen Resistance

Castration resistance in prostate cancer (PCa) constitutes an advanced, aggressive disease with poor prognosis, associated with uncontrolled cell proliferation, resistance to apoptosis, and enhanced invasive potential. The molecular mechanisms involved in the transition of PCa to castration resistance are obscure. Here, we report that the nonselective cationic channel transient receptor potential vanilloid 2 (TRPV2) is a distinctive feature of castration-resistant PCa. TRPV2 transcript levels were higher in patients with metastatic cancer (stage M1) compared with primary solid tumors (stages T2a and T2b). Previous studies of the TRPV2 channel indicated that it is primarily involved in cancer cell migration and not in cell growth. Introducing TRPV2 into androgen-dependent LNCaP cells enhanced cell migration along with expression of invasion markers matrix metalloproteinase (MMP) 9 and cathepsin B. Consistent with the likelihood that TRPV2 may affect cancer cell aggressiveness by influencing basal intracellular calcium levels, small interfering RNA-mediated silencing of TRPV2 reduced the growth and invasive properties of PC3 prostate tumors established in nude mice xenografts, and diminished expression of invasive enzymes MMP2, MMP9, and cathepsin B. Our findings establish a role for TRPV2 in PCa progression to the aggressive castration-resistant stage, prompting evaluation of TRPV2 as a potential prognostic marker and therapeutic target in the setting of advanced PCa.

Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78

The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca2+ homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca2+ homeostasis, and the ER Ca2+ store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca2+ leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca2+ depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca2+ leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca2+ permeability and subsequent ER stress can lead to development of a novel therapeutic approach.—Hammadi, M., Oulidi, A., Gackière, F., Katsogiannou, M., Slomianny, C., Roudbaraki, M., Dewailly, E., Delcourt, P., Lepage, G., Lotteau, S., Ducreux, S., Prevarskaya, N., Van Coppenolle, F. Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78. FASEB J. 27, 1600–1609 (2013). www.fasebj.org

Intermediate-conductance Ca 2 -activated K channels (IK Ca1) regulate human prostate cancer cell proliferation through a close control of calcium entry

Accumulating data point to K+ channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K+ channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IKCa1) in human PCa as well as their involvement in cell proliferation. We showed that IKCa1 mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IKCa1 potassium channel suppressed PCa cell proliferation. The activation of IKCa1 hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IKCa1 channel is likely to promote carcinogenesis in human prostate tissue.

Novel metallocenic compounds as antimalarial agents. Study of the position of ferrocene in chloroquine

Abstract The synthesis, characterization and antimalarial activity of two new ferrocene–chloroquine compounds are reported. One of them, 7-chloro-4- N -[(4- N′ -ethyl- N′ -ferrocenylmethyl)ammonio-1-methylbutylamino]quinolin-1-ium bi-tartrate ( 2 ) showed very promising antimalarial activity in vivo on mice infected with Plasmodium berghei N. and Plasmodium yoelii NS. and in vitro against chloroquine resistant-strains of Plasmodium falciparum .

Synthesis of ferroquine enantiomers: first investigation of effects of metallocenic chirality upon antimalarial activity and cytotoxicity.

Ferroquine (FQ) is a new antimalarial agent with a high blood schizotoncidal activity. Previous studies on this compound were done with racemate mixtures. As FQ possesses planar chirality, pure enantiomers were obtained by enzymatic resolution in order to compare their antimalarial activities and cytotoxicities. (+)‐FQ and (−)‐FQ were equally active in vitro, at nanomolar concentrations. Both enantiomers were slightly less active than the racemate in vivo; cytotoxicities were similar. Actually, the racemate represents the optimal formulation. To the best of our knowledge, this is the first investigation of biological activities of compounds with metallocenic chirality.

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

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.

Bisphenol A stimulates human prostate cancer cell migration via remodelling of calcium signalling

Bisphenol A (BPA), the principal constituent of reusable water bottles, metal cans, and plastic food containers, has been shown to be involved in human prostate cancer (PCa) cell proliferation. The aim of the present study was to explore the effect of BPA on PCa cell migration and the pathways involved in these processes. Using the transwell technique, we clearly show for the first time that the pre-treatment of the cells with BPA (1–10 nM) induces human PCa cell migration. Using a calcium imaging technique, we show that BPA pre-treatment induces an amplification of Store-Operated Calcium Entry (SOCE) in LNCaP cells. RT-PCR and Western blot experiments allowed the identification of the ion channel proteins which are up-regulated by BPA pre-treatments. These include the Orai1 protein, which is known as an important SOCE actor in various cell systems, including human PCa cells. Using a siRNA strategy, we observed that BPA-induced amplification of SOCE was Orai1-dependent. Interestingly, the BPA-induced PCa cell migration was suppressed when the calcium entry was impaired by the use of SOCE inhibitors (SKF96365, BTP2), or when the extracellular calcium was chelated. Taken together, the results presented here show that BPA induces PCa cells migration via a modulation of the ion channel protein expression involved in calcium entry and in cancer cell migration. The present data provide novel insights into the molecular mechanisms involved in the effects of an environmental factor on cancer cells and suggest both the necessity of preventive measures and the possibility of targeting ion channels in the treatment of PCa cell metastasis.

Differential roles of STIM1, STIM2 and Orai1 in the control of cell proliferation and SOCE amplitude in HEK293 cells

Orai1, together with STIM1 and STIM2, constitutes the molecular basis for store-operated calcium entry (SOCE) and we have investigated their role in cell proliferation and cell cycle progression in HEK293 cells. 48-h serum deprival, and a 24-h treatment with 1 mM hydroxyurea or with 10 microM RO-3306--a cyclin-dependent kinase 1 inhibitor--induced cell cycle block in G1, S and G2/M, respectively. SOCE amplitude, monitored in whole-cell voltage clamped cells, was markedly reduced (60-70%) in all conditions, with full reversibility within 4h. Silencing of Orai and STIM1 using siRNA resulted in a large inhibition of SOCE (70-80%) whereas siSTIM2 had a smaller but significant effect (30%). However, the cell population doubling time was not affected in siSTIM1 cells (18 h, the same as in control cells) but was increased in both siOrai1 cells (29 h) and in siSTIM2 (23 h) even when combined with siSTIM1. This suggests that STIM1 plays no role in cell proliferation in HEK293 cells while STIM2 is involved in both SOCE and cell proliferation in these cells. Finally, the cell cycle block induced SOCE inhibition was associated with reduced Orai1 expression with full recovery within 4h, whereas the expression of STIM1 and STIM2 remained unaltered. These observations reveal a tight relation between cell proliferation, calcium entry and Orai1 expression in HEK293 cells.