New insights into pharmacological tools to TR(i)P cancer up.

The aim of this review is to address the recent advances regarding the use of pharmacological agents to target transient receptor potential (TRP) channels in cancer and their potential application in therapeutics. Physiologically, TRP channels are responsible for cation entry (Ca(2+) , Na(+) , Mg(2+) ) in many mammalian cells and regulate a large number of cellular functions. However, dysfunction in channel expression and/or activity can be linked to human diseases like cancer. Indeed, there is growing evidence that TRP channel expression is altered in cancer tissues in comparison with normal ones. Moreover, these proteins are involved in many cancerous processes, including cell proliferation, apoptosis, migration and invasion, as well as resistance to chemotherapy. Among the TRP superfamily, TRPC, TRPV, TRPM and TRPA1 have been shown to play a role in many cancer types, including breast, digestive, gliomal, head and neck, lung and prostate cancers. Pharmacological modulators are used to characterize the functional implications of TRP channels in whole-cell membrane currents, resting membrane potential regulation and intracellular Ca(2+) signalling. Moreover, pharmacological modulation of TRP activity in cancer cells is systematically linked to the effect on cancerous processes (proliferation, survival, migration, invasion, sensitivity to chemotherapeutic drugs). Here we describe the effects of such TRP modulators on TRP activity and cancer cell phenotype. Furthermore, the potency and specificity of these agents will be discussed, as well as the development of new strategies for targeting TRP channels in cancer.

Transient receptor potential melastatin 7 is involved in oestrogen receptor-negative metastatic breast cancer cells migration through its kinase domain.

Oestrogen receptor negative (ER(-)) invasive ductal carcinoma (IDC) represents a significant clinical challenge and therefore prompts the discovery of novel biomarkers. Transient receptor potential melastatin 7 (TRPM7), a channel protein that also contains a regulatory kinase domain, is overexpressed in IDC and regulates migration. However, the molecular mechanism remains poorly defined. Here, we examined whether TRPM7 regulates migration by its channel function or by its kinase domain. A Magnesium Inhibited Cation current was recorded in two ER(-) highly metastatic breast cancer cell lines. Down-regulation of TRPM7 neither affected Ca(2+)-, nor Mg(2+)-homoeostasis but significantly reduced cell migration via a Ca(2+)-independent pathway. Notably, the overexpression of the truncated kinase domain form of TRPM7 decreased cell migration, while the overexpression of the wild-type form strongly increased it. Concomitantly, TRPM7 silencing reduced the myosin IIA heavy chain phosphorylation. Furthermore, we found higher TRPM7 expression in ER(-) IDC tissues and lymph nodes than in the non-invasive tumoural samples. In conclusion, TRPM7 plays a critical role in breast cancer cell migration through its kinase domain, and our data support the consideration of using TRPM7 as a novel biomarker and a potential therapeutic target in the treatment of human ER(-) IDC.

InsP(3)-mediated calcium release induced by heterologous expression of total chicory Leaf RNA.

A calcium dependent-chloride current (I(ni)) was recorded in Xenopus oocytes injected with total RNA from chicory leaf tissues, following depolarization from -35 to +60 mV. However, the signal transduction mechanism mediating I(ni) is unknown. The development of this current was mimicked by intracellular injection of the second messenger InsP(3) in control (non-injected) oocytes. Moreover, InsP(3) injection after I(ni) rundown did not reinitiate the current. The same phenomenon was observed following a second injection into control oocytes. Measurement of InsP(3) production in injected oocytes showed a net increase in the InsP(3) level on depolarization. Moreover, extracellular application of caffeine (5 mM) significantly reduced the number of oocytes displaying I(ni). Also, extracellular application of U-73122, a potent PLC inhibitor, clearly reduced the occurrence of I(ni). These data provide the first evidence that the calcium homeostasis mechanism induced by heterologous expression of total RNA from chicory leaves involves the InsP(3) signaling pathway.

Changes in the K+ current-density of MCF-7 cells during progression through the cell cycle: possible involvement of a h-ether.a-gogo K+ channel.

MCF-7 cells express voltage-activated K+ channels. In the present study, we used the patch-clamp and RT-PCR techniques to investigate the involvement of these channels during the cell cycle progression. The outward rectifier current (IK) recorded during depolarization was almost completely suppressed by the classical K+ channel blocker tetraethylammonium (TEA) in MCF-7 cells. TEA also inhibited cell proliferation, as measured with 3H-thymidine incorporation. Moreover, profound changes were observed in both the resting membrane potential (RMP) and IK during the release from the G0/G1 phase of the cell cycle. MCF-7 cells arrested in G0/G1 were depolarized (-26.3 +/- 10 mV, n = 30) and IK-density was small (9.4 +/- 5.6 pA/pF, n = 60) compared to cells progressing in the G1 phase (RMP = -60 +/- 7.9 mV; n = 35 and IK-density = 30.2 +/- 8.5 pA/pF; n = 76). IK was highly sensitive to Mg2+, astemizole and TEA (10 mM). Extracellular perfusion of 5 mM Mg2+ dramatically slowed the activation and perfusion of 2 microM astemizole inhibited both IK (20 +/- 3%) and cell proliferation (23%). Moreover, the h-EAG mRNA expression was modulated during the cell cycle. Thus, these data suggested that h-EAG K+ channels play a role in controlling the proliferation and/or cell cycle.

KV1.1 K(+) channels identification in human breast carcinoma cells: involvement in cell proliferation.

Electrophysiological, immunocytochemical, and RT-PCR methods were used to identify a K(+) conductance not yet described in MCF-7 human breast cancer cells. A voltage-dependent and TEA-sensitive K(+) current was the most commonly observed in these cells. The noninactivating K(+) current (I(K)) was insensitive to iberiotoxin (100 nM) and charybdotoxin (100 nM) but reduced by alpha-dendrotoxin (alpha-DTX). Perfusion of alpha-DTX reduced a fraction of I(K) amplitude in a dose-dependent manner (IC(50) = 0.6 +/- 0.3 nM). This DTX sensitive I(K) exhibited a voltage threshold at -20 mV and was not inactivated. The time constant of activation was 5.3 +/- 2.2 ms measured at +60 mV. The averaged half-activation potential and slope factor values were 14 +/- 1.6 mV and 10 +/- 1.4, respectively. Immunocytochemical analysis demonstrated that plasma membrane was labeled by anti-Kv1.1 but not by anti-Kv1.2 nor anti-Kv1.3 antibodies. Furthermore, only Kv1.1 mRNA was detected in MCF-7 cells. Incubation in 1 and 10 nM alpha-DTX reduced cell proliferation by 20 and 30%, respectively. These data provide the first evidence of Kv1.1 K(+) channels expression in MCF-7 cells and indicate that these channels are implicated in cell proliferation.

Potassium channels in rat prostate epithelial cells.

Voltage-dependent K(+) channels were identified and characterized in primary culture of rat prostate epithelial cells. A voltage-dependent, inactivating K(+) channel was the most commonly observed ion channel in both lateral and dorsal cells. The K(+) current exhibited a voltage threshold at -40 mV. Averaged half-inactivation potential (V(1/2)) and the slope factor (k) values were -26 mV and 6, respectively. It showed a monoexponential decay with an inactivation time constant of about 600 ms at +60 mV. The deactivation time constant at -60 mV was 30 ms and the reversal potential was estimated at -80 mV, suggesting that current was carried by potassium ions. The scorpion venom peptides charybdotoxin (5 nM) and margatoxin (1 nM), inhibited K(+) current at all membrane potentials with a rapid and a slow reversibility respectively. Both tetraethylammonium (10 mM) and 4-aminopyridine (50 microM) reduced K(+) current by approximately 40%. We conclude that plasma membranes of lateral and dorsal rat prostate epithelial cells contain Kv K(+) channels that have biophysical and pharmacological properties consistent with those of the Kv1.3 family.

A calcium homeostasis mechanism induced by heterologous expression of total RNA from chicory leaves in Xenopus oocytes.

Xenopus oocytes were injected with total RNA from chicory leaf tissues and then examined by the voltage-clamp technique. A double-step voltage protocol was used, with an initial hyperpolarization step from the holding potential of -35 to -140 mV followed by a second depolarization step to +60 mV. Two different outward currents were observed, one noninactivating (Ini), and one inactivating (Ii). Only the noninactivating outward current (Ini) could be induced by depolarization from -35 to +60 mV. The mean amplitude of Ini was 2915 +/- 848 nA (n = 11). This current, carried by chloride ions, declined nearly to the baseline in 153 +/- 64 sec (n = 13), and was highly dependent on intracellular calcium. After the rundown of Ini, the same oocyte was depolarized from -140 to +60 mV. This protocol induced an inactivating outward current (Ii) with a mean amplitude of 4461 +/- 1605 nA (n = 13). Ii was also carried by chloride ions and dependent on extracellular calcium. Ii was strongly inhibited by 100 micron extracellular La3+. These two types of chloride currents were also observed after IP3 injection in control oocytes. Ini and Ii were not observed in noninjected oocytes or water-injected oocytes. We suggest that the expression of total chicory leaf tissue RNA in Xenopus oocytes reveals a calcium homeostasis mechanism responsible for calcium mobilization from internal stores and subsequent calcium entry.

Seasonal influences on the functional expression of the endogenous L-type Ca2+ channels in Pleurodeles oocytes: role of cAMP?

We have investigated the seasonal regulation of the Ba current (IBa) through the L-type Ca2+ channels in Pleurodeles oocytes and its relation with the intracellular cAMP level. IBa-density remained relatively constant from January to February, increased from March to April (with a maximum increase in March) and decreased from May to July. While the permeable cAMP analogue 8Br-cAMP reduced IBa during the breeding season, it was without effect during the resting season. However, IBa recorded during the two seasons was increased by a dihydropyridine agonist Bay K 8644 and blocked by cadmium. The main conclusion is that the seasonal reduction in the L-type Ca2+ current amplitude may be correlated with a high intracellular cAMP level and may account for the inability of oocytes to mature during the resting season.

Voltage-gated calcium channels in Pleurodeles oocytes: classification, modulation and functional roles.

In unfertilised Pleurodeles oocytes, two distinct types of high voltage-activated Ca2+ channels are expressed: a slowly inactivating Ca2+ channel and a transient one. The first is dihydropyridine-sensitive and is referred to as the L-type Ca2+ channel. The transient channel is highly sensitive to Ni2+. Phosphorylation through protein kinases G and A facilitates and inhibits the L-type Ca2+ channel respectively. The transient type channel is insensitive to stimulation by protein kinases (A and G). The functional expression of L-type and transient Ca2+ channels is modulated by the two maturation seasons. The transient Ca2+ currents are only observed during the resting season, while the L-type current is observed either alone during the breeding season or in association with the transient current during the resting season. Moreover, the current density of the L-type Ca2+ channel is much greater during the breeding season than the resting season. Thus, the wide distribution of L-type Ca2+ channels in Pleurodeles oocytes during the two seasons suggests that the roles of these channels may be important in the regulation of the maturation process.