Consequences of activating the calcium-permeable ion channel TRPV1 in breast cancer cells with regulated TRPV1 expression

Increased expression of specific calcium channels in some cancers and the role of calcium signaling in proliferation and invasion have led to studies assessing calcium channel inhibitors as potential therapies for some cancers. The use of channel activators to promote death of cancer cells has been suggested, but the risk of activators promoting cancer cell proliferation and the importance of the degree of channel over-expression is unclear. We developed an MCF-7 breast cancer cell line with inducible TRPV1 overexpression and assessed the role of TRPV1 levels on cell death mediated by the TRPV1 activator capsaicin and the potential for submaximal activation to promote proliferation. The TRPV1 level was a determinant of cell death induced by capsaicin. A concentration response curve with varying TRPV1 expression levels identified the minimum level of TRPV1 required for capsaicin induced cell death. At no level of TRPV1 over-expression or capsaicin concentration did TRPV1 activation enhance proliferation. Cell death induced by capsaicin was necrotic and associated with up-regulation of c-Fos and RIP3. These studies suggest that activators of specific calcium channels may be an effective way to induce necrosis and that this approach may not always be associated with enhancement of cancer cell proliferation.     

Calcium channels and cancer stem cells

Cancer stem cells (CSC’s) have emerged as a key area of investigation due to associations with cancer development and treatment resistance, related to their ability to remain quiescent, self-renew and terminally differentiate. Targeting CSC’s in addition to the tumour bulk could ensure complete removal of the cancer, lessening the risk of relapse and improving patient survival. Understanding the mechanisms supporting the functions of CSC’s is essential to highlight targets for the development of therapeutic strategies. Changes in intracellular calcium through calcium channel activity is fundamental for integral cellular processes such as proliferation, migration, differentiation and survival in a range of cell types, under both normal and pathological conditions. Here in we highlight how calcium channels represent a key mechanism involved in CSC function. It is clear that expression and or function of a number of channels involved in calcium entry and intracellular store release are altered in CSC’s. Correlating with aberrant proliferation, self-renewal and differentiation, which in turn promoted cancer progression and treatment resistance. Research outlined has demonstrated that targeting altered calcium channels in CSC populations can reduce their stem properties and induce terminal differentiation, sensitising them to existing cancer treatments. Overall this highlights calcium channels as emerging novel targets for CSC therapies.     

Ion Channels and Cancer

Membrane ion channels are essential for cell proliferation and appear to have a role in the development of cancer. This has initially been demonstrated for potassium channels and is meanwhile also suggested for other cation channels and Cl⁻ channels. For some of these channels, like voltage-gated ether à go-go and Ca²⁺-dependent potassium channels as well as calcium and chloride channels, a cell cycle-dependent function has been demonstrated. Along with other membrane conductances, these channels control the membrane voltage and Ca²⁺ signaling in proliferating cells. Homeostatic parameters, such as the intracellular ion concentration, cytosolic pH and cell volume, are also governed by the activity of ion channels. Thus it will be an essential task for future studies to unravel cell cycle-specific effects of ion channels and non-specific homeostatic functions. When studying the role of ion channels in cancer cells, it is indispensable to choose experimental conditions that come close to the in vivo situation. Thus, environmental parameters, such as low oxygen pressure, acidosis and exposure to serum proteins, have to be taken into account. In order to achieve clinical application, more studies on the original cancer tissue are required, and improved animal models. Finally, it will be essential to generate more potent and specific inhibitors of ion channels to overcome the shortcomings of some of the current approaches.     

A role for voltage gated T-type calcium channels in mediating "capacitative" calcium entry?

Calcium entry through plasma membrane calcium channels is one of the most important cell signaling mechanism involved in such diverse functions as secretion, contraction and cell growth by regulating gene expression, proliferation and apoptosis. The identity of plasma membrane calcium channels, the main regulators of calcium entry, involved in cell proliferation has been thus extensively sought. Among these, a calcium entry pathway called capacitative calcium entry (CCE), activated by calcium store depletion, is particularly important in non-excitable cells. Though this capacitative calcium entry is generally supposed to occur through TRP channels there is some evidence that voltage-dependent T-type calcium channels may contribute to calcium entry after store depletion. Here we show that though mibefradil, a T-type calcium channel blocker, is able to reduce capacitative calcium entry induced by either thapsigargin or ATP, this was not mimicked by any other T-type calcium channel inhibitors even in cells overexpressing alpha(1H) T-type calcium channels, leading us to conclude that T-type calcium channels are not responsible for the capacitative calcium entry observed in different cancer cell lines. On the contrary, we show that the action of mibefradil on capacitative calcium entry is due to an action on store-operated calcium channels.     

Substance P immunoreactive nerve fibres are related to gastric cancer differentiation status and could promote proliferation and migration of gastric cancer cells

Tachykinins such as SP (substance P) may be involved in the progression of gastric adenocarcinoma through binding to NK-1 receptor. However, the existence and relationship between SP and gastric cancer progression and differentiation remained unknown. We have studied the NK-1 receptor in human gastric cancer tissue and MKN45 cell line and found SP-containing nerve fibres in human gastric cancer and found that the amounts of SP-positive nerves were related to gastric cancer differentiation. SP could promote proliferation, adhesion, migration and invasion of MKN45 cells in vitro. In addition, the intracellular calcium level of MKN45 cells was elevated after SP stimulation, and administration of CRACs (calcium release-activated calcium channels) inhibitor SKF-96365 could partially abolish these effects induced by SP. These results demonstrated that NK-1 receptor and SP-containing nerves existed in human gastric cancer; SP positive nerves may play an important role in human gastric cancer progression, and calcium is critically significant among SP-induced biological effects.     

Calcium signaling regulates fundamental processes involved in Neuroblastoma progression

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor in children. Despite NB’s relative rarity, high-risk NB patients have a poor prognosis with survival rate less than 50%. This is even worse for patients with relapsed or refractory NB. Finding effective alternative treatment strategies is therefore a must.Calcium is an intracellular messenger that is unequivocally present in normal physiology mediating proliferation, growth, migration, cell division, angiogenesis and cell death, as well as pathophysiological processes such as those included in Weinberg’s hallmarks of cancer. Within the past 20 years, the molecular identity of most calcium channels has been revealed, however for some of these channels the precise gating mechanism and their role in normal physiology is still elusive.Here we review the recent findings of components of calcium signaling that are deregulating in the malignant progression of NB. We further integrate critical calcium signaling pathways using patient-derived expression analysis.Revealing the roles of these calcium pathways in tumor development, progression, microenvironment and importantly - protection against antineoplastic drugs may hopefully lead to novel treatment strategies in the future.     

Old proteins,developing roles: The regulation of calcium channels by synaptic proteins

Coupling of presynaptic voltage-gated calcium channels to synaptic release machinery is critical for neurotransmission. It was traditionally believed that anchoring calcium channels close to the calcium micro-domain dependent release machinery was the main reason for the physical interactions between channels and synaptic proteins, however in recent years, it is becoming clear that these proteins additionally regulate channel activity, and such processes as channel targeting and alternative splicing, to orchestrate a much broader regulatory role in controlling calcium channel function, calcium influx, and hence neurotransmission. Calcium signalling serves a multitude of cellular functions and therefore requires tight regulation. Specific, often calcium-dependent interactions between synaptic proteins and calcium channels appear to play a significant role in fine-tuning of the synaptic response over development. While it is clear that investigation of a few of the multitude of synaptic proteins will not provide a complete understanding of calcium channel regulation, consideration of the emerging mechanisms by which synaptic protein interactions might regulate calcium channel function is important in order to understand their possible contributions to synaptic transmission. Here, we review the current state of knowledge of the molecular mechanisms by which synaptic proteins regulate presynaptic calcium channel activity.     

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

Coupling of presynaptic voltage-gated calcium channels to the synaptic release machinery is critical for neurotransmission. It was traditionally believed that anchoring calcium channels close to the calcium microdomain dependent release machinery was the main reason for the physical interactions between channels and synaptic proteins, however in recent years, it is becoming clear that these proteins additionally regulate channel activity, and such processes as channel targeting and alternative splicing, to orchestrate a much broader regulatory role in controlling calcium channel function, calcium influx and hence neurotransmission. Calcium signalling serves a multitude of cellular functions and therefore requires tight regulation. Specific, often calcium-dependent interactions between synaptic proteins and calcium channels appear to play a significant role in fine-tuning of the synaptic response over development. While it is clear that investigation of a few of the multitude of synaptic proteins will not provide a complete understanding of calcium channel regulation, consideration of the emerging mechanisms by which synaptic protein interactions might regulate calcium channel function is important in order to understand their possible contributions to synaptic transmission. Here, we review the current state of knowledge of the molecular mechanisms by which synaptic proteins regulate presynaptic calcium channel activity.     

SOC and now also SIC: Store-operated and store-inhibited channels

There is a specialized form of calcium influx that involves a close communication between endoplasmic reticulum and the channels at the plasma membrane. In one side store depletion activates channels known as store-operated channels (SOC), which are responsible of the well-studied store-operated calcium entry (SOCE). SOC comprises two different types of channels. Orai, which is exclusively activated by store depletion being the channel responsible of the calcium release-activated calcium current, and transient receptor potential canonical channel, which in contrast, is activated by store depletion only under specific conditions and carries nonselective cationic currents. On the other hand, it has been recently shown that store depletion also inhibits calcium channels. The first member identified, of what we named as store-inhibited channels (SIC), is the L-type voltage-gated calcium channel. Stores control both SOC and SIC by means of the multifunctional protein STIM1. The identification of SOC and SIC opens a new scenario for the role of store depletion in the modulation of different calcium entry pathways, which may satisfy different cellular processes.     

Arachidonic acid, ARC channels, and Orai proteins

A critical role for arachidonic acid in the regulation of calcium entry during agonist activation of calcium signals has become increasingly apparent in numerous studies over the past 10 years or so. In particular, low concentrations of this fatty acid, generated as a result of physiologically relevant activation of appropriate receptors, induces the activation of a unique, highly calcium-selective conductance now known as the ARC channel. Activation of this channel is specifically dependent on arachidonic acid acting at the intracellular surface of the membrane, and is entirely independent of any depletion of internal calcium stores. Importantly, a specific role of this channel in modulating the frequency of oscillatory calcium signals in various cell types has been described. Recent studies, subsequent to the discovery of STIM1 and the Orai proteins and their role in the store-operated CRAC channels, have revealed that these same proteins are also integral components of the ARC channels and their activation. However, unlike the CRAC channels, activation of the ARC channels depends on the pool of STIM1 that is constitutively resident in the plasma membrane, and the pore of these channels is comprised of both Orai1 and Orai3 subunits. The clear implication is that CRAC channels and ARC channels are closely related, but have evolved to play unique roles in the modulation of calcium signals—largely as a result of their entirely distinct modes of activation. Given this, although the precise details of how arachidonic acid acts to activate the channels remain unclear, it seems likely that the specific molecular features of these channels that distinguish them from the CRAC channels – namely Orai3 and/or plasma membrane STIM1 – will be involved.     

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.     

Physiopathology of neuronal voltage-operated calcium channels.

Voltage-operated calcium channels are multimeric transmembrane proteins crucially involved in control of calcium homeostasis. Multiple types of voltage-operated calcium channels have been described in both the nervous system and peripheral tissues. Different channels can be classified according to either their biophysical properties or their pharmacology, biochemical and molecular structure, and localization and functional role. Concentrating on neuronal cells, this paper reviews the different properties of low- and high-voltage activated channels, as well as various attempts to subdivide high-voltage activated channels into different subtypes (L, N, omega, P, etc.). The availability of selective drugs (such as dihydropyridines) and natural toxins (such as omega-Conotoxin, omega-agatoxin, and funnel-web spider toxins), which bind to specific channel subtypes, has greatly helped in channel classification. The emerging view is that there are many members of the family of voltage-operated calcium channels, each with its own molecular structure, a different pharmacology, a different localization, and possibly a different physiological role. Different calcium subtypes are selectively affected in human and animal diseases. The use of omega-Conotoxin has led to identification of the channel subtype (omega) specifically affected in Lambert-Eaton myasthenic syndrome (a human disease of neurotransmission), and has permitted development of new diagnostic approaches to the disease.     

Voltage gated calcium channels in molluscs: classification,Ca2+ dependent inactivation,modulation and functional roles

Molluscan neurons and muscle cells express transient (T-type like) and sustained LVA calcium channels, as well as transient and sustained HVA channels. In addition weakly voltage sensitive calcium channels are observed. In a number of cases toxin or dihydropyridine sensitivity justifies classification of the HVA currents in L, N or P-type categories. In many cases, however, pharmacological characterization is still preliminary. Characterization of novel toxins from molluscivorousConus snails may facilitate classification of molluscan calcium channels. Molluscan preparations have been very useful to study calcium dependent inactivation of calcium channels. Proposed mechanisms explain calcium dependent inactivation through direct interaction of Ca²⁺ with the channel, through dephosphorylation by calcium dependent phosphatases or through calcium dependent disruption of connections with the cytoskeleton. Transmitter modulation operating through various second messenger mediated pathways is well documented. In general, phosphorylation through PKA, cGMP dependent PK or PKC facilitates the calcium channels, while putative direct G-protein action inhibits the channels. Ca²⁺ and cGMP may inhibit the channels through activation of phosphodiesterases or phosphatases. Detailed evidence has been provided on the role of sustained LVA channels in pacemaking and the generation of firing patterns, and on the role of HVA channels in the dynamic changes in action potentials during spiking, the regulation of the release of transmitters and hormones, and the regulation of growth cone behavior and neurite outgrowth. The accessibility of molluscan preparations (e.g. the squid giant synapse for excitation release studies,Helisoma B5 neuron for neurite and synapse formation) and the large body of knowledge on electrophysiological properties and functional connections of identified molluscan neurons (e.g. sensory neurons, R15, egg laying hormone producing cells, etc.) creates valuable opportunities to increase the insight into the functional roles of calcium channels.     

Regulation of Ca2+ influx in myocardial cells by beta adrenergic receptors,cyclic nucleotides,and phosphorylation

Calcium channels in the heart play a major role in cardiac function. These channels are modulated in a variety of ways, including protein phosphorylation. Cyclic AMP-mediated phosphorylation is the best understood phosphorylation mechanism which regulates calcium influx into cardiac cells. Binding of an agonist (e.g., a catecholamine) to the appropriate receptor stimulates production of cyclic AMP by adenylate cyclase. The cyclic AMP may subsequently bind to and activate a cyclic AMP-dependent protein kinase, which then can phosphorylate a number of substrates, including the calcium channel (or a closely-associated regulatory protein). This results in stimulation of the calcium channels, greater calcium influx, and increased contractility. The cyclic AMP system is not the only protein kinase system in the heart. Thus, the possibility exists that other protein kinases may also regulate the calcium channels and, hence, cardiac function. Recent evidence suggests that cyclic GMP-mediated phosphorylation may play a role opposite to cyclic AMP-mediated phosphorylation, i.e., inhibition of the calcium current rather than stimulation. Other recent evidence also suggests that a calcium/calmodulin-dependent protein kinase and calcium/phospholipid-dependent protein kinase (protein kinase C) may also regulate the myocardial calcium channels. Thus, protein phosphorylation may be a general mechanism whereby calcium channels and cardiac function are modulated under a variety of conditions.     

The pharmacology and regulation of T type calcium channels: new opportunities for unique therapeutics for cancer

T type calcium channels are implicated in a fascinating array of physiological and pathophysiological phenomena, as reviewed extensively in this issue of Cell Calcium. It is also evident from this timely collection of papers that knowledge about T channels is nascent. This creates the exciting potential to explore an uncharted territory for new insights into important physiological processes and associated diseases. Equally exciting and perhaps more important is the opportunity to exploit the developing understanding of T channels to design new therapeutic approaches to diseases that are now intractable to varying degrees. This review will focus on the controversy surrounding the proposed novel functions for and means of regulation of T type Ca2+ channels, particularly with regard to the potential role of pharmacologic modulation of T type Ca2+ channels in cytostatic cancer chemotherapy. In addition, we will concentrate on recent studies of the molecular pharmacology and physiology of T type channels, since these areas (molecular pharmacology [T.N. Heady, J.C. Gomora, T.L. Macdonald, E. Perez-Reyes, Molecular pharmacology of T-type Ca2+ channels, Jpn. J. Pharmacol. 85 (2001) 339-350.]; physiology [E. Perez-Reyes, Molecular physiology of low-voltage-activated t-type calcium channels, Physiol. Rev. 83 (2003) 117-161.]) have been very well-reviewed and the reader is referred to these authoritative sources.     

NAADP receptors

Of the established Ca(2+) mobilizing messengers, NAADP is arguably the most tantalizing. It is the most potent, often efficacious at low nanomolar concentrations. Recent studies have identified a new class of calcium release channel, the two-pore channels (TPCs), as the likely targets for NAADP. These channels are endolysosomal in localization where they mediate local Ca(2+) release, and have highlighted a new role of acidic organelles as targets for messenger-evoked Ca(2+) mobilization. Three distinct roles of TPCs have been identified. The first is to effect local Ca(2+) release that may play a role in endolysosomal function including vesicular fusion and trafficking. The second is to trigger global calcium release by recruiting Ca(2+)-induced Ca(2+) release (CICR) channels at lysosomal-ER junctions. The third is to regulate plasma membrane excitability by the targeting of Ca(2+) release from appropriately positioned subplasma membrane stores to regulate plasma membrane Ca(2+)-activated channels. In this review, I discuss the role of NAADP-mediated Ca(2+) release from endolysosomal stores as a widespread trigger for intracellular calcium signaling mechanisms, and how studies of TPCs are beginning to enhance our understanding of the central role of lysosomes in Ca(2+) signaling.     

Functional interaction between mouse spermatogenic LVA and thapsigargin-modulated calcium channels

The acrosome reaction in mouse is triggered by a long-lasting calcium signaling produced by a chain of openings of several calcium channels, a low-voltage-activated (LVA) calcium channel, an inositol trisphosphate receptor (IP(3)R), and the store-operated calcium channel TRP2. Since mature sperm cells are refractory to patch clamp experiments, we study the functional interactions among those sperm calcium channels in spermatogenic cells. We have studied the role of cytosolic calcium in voltage-dependent facilitation of low voltage-activated calcium channels. Calcium concentration was modified through the inclusion of the calcium buffers, EGTA and BAPTA, in the recording pipette solution, and by addition of calcium modulators like thapsigargin and the calcium ionophore A23187. We demonstrate that lowering calcium concentration below resting level allows to evidence a voltage-dependent facilitation. We also show that LVA calcium channels present strong voltage-dependent inhibition by thapsigargin. This effect is independent of cytosolic calcium elevation secondary to calcium store depletion and to the activation of TRP channels. Our data evidence an interesting functional relationship, in this cell type, between LVA channels and proteins whose activity is related to calcium filling state of the endoplasmic reticulum (presumably TRP channels and inositol triphosphate receptor). These relationships may contribute to the regulation of calcium signaling during acrosome reaction of mature sperm cell.     

Receptor-Operated Calcium Influx Mediated by Protein Tyrosine Kinase Pathways

Abstract

Calcium influx from the extracellular space elicited by activation of heterotrimeric G protein-coupled and heptahelical receptors plays a critical role in transmembrane signal transduction in a wide variety of cell systems. In nonexcitable cells, the precise voltage-independent mechanism by which calcium enters the cell remains unknown. Multiple mechanisms appear to be operating in different cell types (1–3): 1. G protein-operated calcium influx, 2. second messenger-operated calcium influx, 3. capacitative calcium influx, and 4. phosphorylation of calcium channels. Receptor-operated calcium channels have a fundamental role in stimulus-secretion coupling in many different cells, but these channels remain to be purified and cloned. This review proposes that receptor-operated calcium influx is mediated by protein tyrosine kinase pathways. The function of protein tyrosine kinase pathways and their interactions with other receptor-operated calcium influx mechanisms are described.     

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).     

Establishing life is a calcium-dependent TRiP: Transient receptor potential channels in reproduction

Calcium plays a key role in many different steps of the reproduction process, from germ cell maturation to placental development. However, the exact function and regulation of calcium throughout subsequent reproductive events remains rather enigmatic. Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. On the one hand, endometrial cell will undergo massive changes to support an implanting embryo, including stromal cell decidualization. On the other hand, trophoblast cells from the trophectoderm surrounding the inner cell mass will differentiate and acquire new functions such as hormone secretion, invasion and migration. The need for calcium in the different gestational processes implicates the presence of specialized ion channels to regulate calcium homeostasis. The superfamily of transient receptor potential (TRP) channels is a class of calcium permeable ion channels that is involved in the transformation of extracellular stimuli into the influx of calcium, inducing and coordinating underlying signaling pathways. Although the necessity of calcium throughout reproduction cannot be negated, the expression and functionality of TRP channels throughout gestation remains elusive. This review provides an overview of the current evidence regarding the expression and function of TRP channels in reproduction.