Cancer Therapy Vol 2, 263-270, 2004

Department of Physiology, University of Extremadura, Cáceres, Spain

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*Correspondence: Dr. Juan A. Rosado, Faculty of Veterinary Medicine, Department of Physiology, University of Extremadura. Av. Universidad s/n, 10071 Cáceres. Spain; Phone: +34 927257154; Fax: +34 927257154; e-mail: jarosado@unex.es

Key words: calcium, tumorigenesis

Abbreviations: calcium-sensing receptors, (CaR); cytosolic calcium concentration, ([Ca²⁺]_c); calcium concentration in the stores, ([Ca²⁺]_s); mitochondrial calcium concentration, ([Ca²⁺]_m); nuclear calcium concentration, ([Ca²⁺]_n); calcium influx factor, (CIF); 1)2-diacylglicerol, (DAG); endoplasmic reticulum, (ER); G protein-coupled receptors, (GPCR); inositol trisphosphate, (IP₃); nicotinic acid—adenine dinucleotide phosphate, (NAADP); phosphatidylinositol 4)5-bisphosphate, (PIP₂); protein kinase C, (PKC); Phospholipase C, (PLC); plasma membrane Ca²⁺ ATPase, (PMCA); receptor-operated channels, (ROC); sarcoendoplasmic reticulum Ca²⁺ ATPase, (SERCA); second messenger-operated channels, (SMOC); store-operated channels, (SOC); 2)5-di(tert-butyl)-1)4-benzohydroquinone, (TBHQ); transient receptor potential channelsf, (TRPC); voltage-operated Ca²⁺ channels, (VOC)

Calcium is a chemical element widely distributed in nature, whose physiological relevance was demonstrated by Ringer in 1883. The presence of calcium in the extracellular medium was necessary for muscle contraction. One century later it was shown that in response to a signal ionised calcium (Ca²⁺) is released to the cytoplasm from an intracellular calcium store (Streb et al, 1983). In resting conditions, eukaryotic cells are surrounded by an extracellular medium containing a concentration of Ca²⁺ ([Ca²⁺]_o) of 1.2 mM and the Ca²⁺ concentration into the main intracellular stores ([Ca²⁺]_s) ranges from 0.1 to 1 mM. By contrast, the Ca²⁺ concentrations in the cytoplasm ([Ca²⁺]_c), mitochondria ([Ca²⁺]_m) and nucleus ([Ca²⁺]_n) are about 100 nM (Petersen, 2002; Berridge et al, 2003).

When an agonist binds to a specific receptor site on the outside of a cell membrane, a transduction process occurs, creating an intracellular signal. In many cases, the signal is a rise in [Ca²⁺]_c. The increase in [Ca²⁺]_c is induced by the release of compartmentalised Ca²⁺ from intracellular stores and/or the entry of extracellular Ca²⁺ across the plasma membrane. Immediately after agonist stimulation several mechanisms, mainly pumps and exchangers, remove Ca²⁺ from the cytosol, pumping this ion outside the cell or into intracellular stores, to restore the resting [Ca²⁺]_c (Petersen, 2002; Berridge et al, 2003; Schulz and Krause, 2004).

The physiological implications of Ca²⁺ range from the role in muscle (Mc Carron et al, 2004; Morales et al, 2004) or secretory tissues (Salido et al, 1999; Raraty et al, 2000) to neural transmission (Yamashita and Sugioka, 1998; Braet et al, 2004). Additionally, Ca²⁺ is an intracellular messenger for cellular growth and proliferation (Short et al, 1993).

Accumulating evidence suggests that altered cytosolic Ca²⁺ homeostasis might be involved with excessive cell proliferation, a hallmark of tumorigenesis.

Ca²⁺ release from the intracellular compartments is regulated by channels located in the membrane of the stores, such as the IP₃ receptor, the ryanodine receptor, the NAADP receptor and the sphingosine 1-phosphate receptor (Figure 1; Berridge et al, 2000).

At least three IP₃ receptor isoforms have been described, that appear as homo- and heterotetramers (Monkawa et al, 1995; Vermassen et al, 2004). Structurally, IP₃ receptors have three different domains: a N-terminus cytoplasmic domain with an IP₃ binding site, a regulatory central domain, which binds allosteric effectors such as Ca²⁺ or ATP (Mikoshiba, 1993; Sato et al, 2004), and a transmembrane region near the C terminus composed of six-spanning domains, involved in the formation of a tetrameric complex (Sayers et al, 1997;

Figure 1. Mechanisms of calcium signalling. Cell stimulation by agonists activate the formation of second messengers that induce the release of Ca²⁺ stored in the endoplasmic/sarcoplasmic reticulum (ER/SR) through the IP₃ receptor (IP₃R), the ryanodine receptor (RyR), the NAADP receptor (NAADPR) or the sphingosine 1-phosphate binding site (S1P-R). In addition, agonists stimulate the entry of extracellular Ca²⁺ through plasma membrane channels. Most of the cytosolic Ca²⁺ is bound to buffers, and only a small percentage binds to effectors and activates cellular functions. Ca²⁺ removal is mediated by various pumps and exchangers, including the Na⁺/Ca²⁺ exchanger (NCX) and the plasma membrane Ca²⁺ ATPase (PMCA) that extrude Ca²⁺ from the cytosol and the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) that pumps Ca²⁺ back to the ER/SR. During the Ca²⁺ signal mitochondria sequester Ca²⁺ through an uniporter that might be then released slowly into the cytoplasm through the NCX or the permeability transition pore (PTP).

Hamada et al, 2003). Cytosolic Ca²⁺ exerts a dual effect on the activity of the IP₃ receptor, so that low Ca²⁺ concentrations (<300 nM) are stimulatory while higher concentrations are either inhibitory or, after IP₃ binding, do not alter IP₃ receptor activity (Bootman and Lipp, 1999; Berridge et al, 2000).

Sphingosine 1-phosphate is a second messenger that releases Ca²⁺ from the endoplasmic reticulum by possibly binding to a sphingolipid Ca²⁺-release-mediating protein on the membrane of the endoplasmic reticulum (Mao et al, 1996). Alternatively, this lipid might activate ryanodine receptors, since sphingosine 1-phosphate enhances ryanodine binding to its receptor and Ca²⁺ release by sphingosine 1-phosphate is prevented by ryanodine receptor blockers (Dettbarn et al, 1995).

In excitable cells, such as neurones and muscle cells, Ca²⁺ entry mostly occurs through voltage-operated Ca²⁺ channels (VOC); however, in non-excitable cells, where VOC are not present Ca²⁺ influx mainly occurs through receptor-operated channels (ROC), second messenger-operated channels (SMOC) or store-operated channels (SOC).

VOCs are Ca²⁺ permeable channels that are briefly activated by changes in the membrane potential (Tsien et al, 1995). These channels are mainly found in excitable cells, where they open in response to membrane depolarisations to allow Ca²⁺ to enter the cell (McCleskey, 1994).

ROC belong to a heterogeneous family of channels that are specially relevant in secretory cells and neurones. ROCs are activated by a number of cellular agonists inducing a rapid Ca²⁺ entry indicative of a direct coupling between the receptor and a Ca²⁺ permeable channel (Sage, 1992).

The conformational coupling has recently received support from studies that propose a secretion-like coupling for the activation of SMCE (Patterson et al, 1999; Rosado et al, 2000a; Redondo et al, 2003). The secretion-like coupling appears as an integrative model where messenger molecules and the actin cytoskeleton interact to facilitate a physical and reversible coupling between elements in the ER and PM. Consistent with this proteins of the Ras family and tyrosine kinases, initially considered as members of the diffusible messenger hypothesis for the activation of SMCE, are essential for actin reorganisation induced by store depletion (Rosado and Sage, 2000b; Rosado et al, 2000b) and proteins classically involved in exocytosis, such as SNAP-25 appear to be involved in Ca²⁺ entry (Yao et al, 1999; Redondo et al, 2004) . In addition, remodelling of the cytoskeletal cortical barrier has been suggested to facilitate the activation of SMCE by CIF (Xie et al, 2002).

The secretion-like coupling model for the activation of SMCE proposes a direct interaction between the IP₃Rs in the ER and a Ca²⁺ channel in the PM. The involvement of IP₃Rs in SMCE has widely been confirmed (Irvine, 1990; Mikoshiba, 1997), and the role of mammalian homologues of the Drosophila TRPC has received support from studies that demonstrate coupling between TRPCs and IP₃Rs in transfected cells (Kiselyov et al, 1998; Vazquez et al, 2001) and in cells naturally expressing TRPCs (Rosado and Sage, 2000c; Rosado et al, 2002). In this model, the actin cytoskeleton plays a dual role as for secretion, both as a cortical barrier preventing constitutive coupling and also supporting the transport of portions of the ER towards the PM (Rosado and Sage, 2001).

Ca²⁺ removal from the cytosol is carried out by several Ca²⁺ pumps and exchangers which reintroduce Ca²⁺ into the internal stores or extrude it out of the cell (Meldolesi and Pozzan, 1998).

Ca²⁺ uptake into the intracellular stores mostly occurs against a concentration gradient, since [Ca²⁺]_c is lower than [Ca²⁺]_s which at rest is in a high-micromolar to low-millimolar concentration range. Released Ca²⁺ is returned to the stores by the sarcoendoplasmic reticulum Ca²⁺ ATPase (Figure 1; SERCA). There are three different SERCA genes, and additional isoform subtypes are generated by alternative splicing (Exton, 1997). SERCA has a high affinity for Ca²⁺ (0.1-0.4 m M), which suggests that SERCA is likely to be activated by an increase in [Ca²⁺]_c and inhibited by an increase in [Ca²⁺]_s (Carafoli, 1992).

Several pharmacological tools, such as thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBHQ) and cyclopiazonic acid, have been developed to investigate the role of SERCA in Ca²⁺ signalling. Among them, the most widely used is thapsigargin, which binds to all SERCAs although with different affinity (Cavallini et al, 1995) and causes an irreversible inhibition of their activity by blocking the ATPase in the Ca²⁺-free state (Wictome et al, 1992). A similar effect is induced with TBHQ, although with lower potency, and some isoforms seems to be insensitive to this inhibitor, which has been used to identify distinct intracellular Ca²⁺ stores (Cavallini et al, 1995) that, in turn, activate different Ca²⁺ entry mechanisms (Rosado et al, 2004).

Perhaps the major mechanism for the removal of cytosolic Ca²⁺ is the extrusion of Ca²⁺ to the extracellular medium against a concentration gradient. Ca²⁺ efflux is mainly carried out by two different transporters, the plasma membrane Ca²⁺ ATPase (PMCA) and the Na⁺/Ca²⁺ exchanger (Figure 1).

The PMCA is an ATPase highly sensitive to vanadate and lanthanum (Pedersen and Carafolli, 1987; Pariente et al, 1999; Lajas et al, 2001). Molecular biology studies revealed the expression of at least four PMCA isoforms in humans: PMCA1-4, although its number is increased by the existence of alternative splice variants (Strehler and Zacharias, 2001). The structure of the PMCA consists of ten transmembrane domains and five extracellular regions, with the NH2 and COOH termini located in the cytosolic site of the membrane (Guerini, 1998; Strehler and Zacharias, 2001).

PMCA activity is regulated by several messenger molecules including Ca²⁺/calmodulin, protein tyrosine kinases, PIP₂, protein serine/threonine kinases and by proteases like calpain (Strehler and Zacharias, 2001; Pariente et al, 2003) and agonists might either increase or inhibit the PMCA activity by activating these intracellular pathways (Rosado and Sage, 2000d; Pariente et al, 2001).

On the other hand, the Na⁺/Ca²⁺ exchanger is a bi-directional electrogenic ion transporter that couples the movement of Na⁺ in one direction with the transport of Ca²⁺ in the opposite direction. The Na⁺/Ca²⁺ exchanger modulates [Ca²⁺]_c by either removing Ca²⁺ from the cytosol (forward mode) or by transporting Ca²⁺ inside the cell (reverse mode). Three different Na⁺/Ca²⁺ exchangers have been described. Two of them are electrogenic, the K⁺-independent Na⁺/Ca²⁺ exchanger, which catalyses the countertransport of either 3 or 4 Na⁺ for 1 Ca²⁺ (Blaustein and Lederer, 1999) and the K⁺-dependent Na⁺/Ca²⁺ exchanger, which catalyses the exchange of 4 Na⁺ by 1 Ca²⁺ and 1 K⁺ (Dong et al, 2001). In addition, an electroneutral Na⁺/Ca²⁺ exchanger has been described in mitochondria (Matsuda et al, 1997).

Mitochondria are a relevant component of the Ca²⁺ signalling machinery. Localised in the vicinity of the Ca²⁺ channels, mitochondria sequesters Ca²⁺ coming from intracellular stores or the extracellular medium modulating the Ca²⁺ signals (Figure 1; Gonzalez and Salido, 2001; Parekh, 2003).

Ca²⁺ entry into mitochondria is regulated by a high capacity and low affinity uniporter that transports Ca²⁺ down a electrochemical gradient. This uniporter requires local high [Ca²⁺]_c to function suggesting that mitochondria should be close to the Ca²⁺ channels (Berridge et al, 2000; Pariente et al, 2003). Efflux of Ca²⁺ occurs by two different exchangers that countertransport Ca²⁺ for either Na⁺ or H⁺, or through a permeability transition pore that have two different states, a reversible low conductance state, that allow mitochondria to participate in Ca²⁺ signalling and an irreversible high conductance state that collapses the mitochondrial membrane potential leading to the activation of apoptosis (Ichas et al, 1997; Berridge et al, 2000).

Mitochondrial Ca²⁺ accumulation has a dual role in cell function, an universal role that consist in the activation of mitochondrial enzymes involved in the generation of ATP, and a more specific role involved in the modulation of the spatio-temporal aspects of Ca²⁺ signalling (Camello-Almaraz et al, 2002; Brini, 2003; Gonzalez et al, 2003; Parekh, 2003).

It is well known that approximately 98-99 % of Ca²⁺ ions in the cytosol are bound by buffer molecules, including Ca²⁺ binding proteins (Neher and Augustine, 1992; Mogami et al, 1999). There is a variety of Ca²⁺-binding proteins involved in Ca²⁺ signalling either acting as Ca²⁺ sensors, effectors or buffers that either initiate, execute or terminate Ca²⁺-dependent functions. Most of Ca²⁺-binding molecules inside the cell are Ca²⁺ buffer proteins that maintain a low [Ca²⁺]_c at rest, including calretinin, calbindin and parvalbumin in the cytosol and calsequestrin and calreticulin in the intracellular stores.

Cancer is a disease characterised by a complete deregulation of the cell proliferation cycle. It is well known that cell exposure to external agents, such as some heavy metals (Krantz and Dorevicht, 2004), virus infection (Shah et al, 2004) or radiations, increases the number of cancer incident. In addition, intracellular signals can also induce tumorigenesis. Among the different cancer types and locations, lung, skin or prostate cancer in men and breast cancer in women appear as the more common neoplasia.

To understand the mechanism involved in gene deregulation, we have to investigate the signalling mechanisms that induce the activation of transcription factors (Arbiser, 2004), where Ca²⁺ signals are among the most important pathways involved in tumorigenesis (Lipskaia and Lompré, 2004). A number of studies have reported alterations in Ca²⁺ homeostasis in tumoral cells, ranging from those that affect the [Ca²⁺]_o to those that involve dysfunction of one or several intracellular Ca²⁺ mobilising mechanism.

Since cancer is predominantly a disease of disordered balance between proliferation, differentiation and apoptosis, an altered function of the extracellular calcium-sensing receptors (CaR) might contribute to the progression of the neoplastic disease. Parathyroid hyperplasias as well as colon carcinoma have been shown to be correlated with an altered expression of CaR (Martín-Salvago et al, 2003; Rodland, 2004), leading to loss of the growth suppressing effects of elevated extracellular Ca²⁺. CaR dysfunction has been reported to be involved in cancer progression and its activation has been linked to an increased expression and secretion of parathyroid hormone-related peptide, a promoter of the metastatic progress in bone as well as a causal factor involved in hypercalcemia of malignancy (Rodland, 2004).

The mechanisms involved in the regulation of [Ca²⁺]_c are also either involved or altered in tumoral cells. In the last years a number of studies have provided information about the transduction mechanisms involved in Ca²⁺ entry induced by mitogenic factors, binding either to tyrosine kinase receptors or to G protein coupled receptors. Among the early events induced by mitogens Ca²⁺ mobilisation and specially Ca²⁺ entry are widespread signals (Munaron, 2002), which supports the involvement of Ca²⁺ signalling in the development of neoplastic transformation.

In preneoplastic mouse JB6 epidermal cells extracellular Ca²⁺ entry has been shown to be necessary for the induction of neoplastic transformation and cell treatment with calcium channel blockers, such as lanthanum or nifedipine, reduce their transformation (Smith et al, 1986). In addition, the sustained Ca²⁺ store depletion and prevention of the refilling via SMCE influences the rate of cellular protein synthesis and reduces cell proliferation in experimental cancer (Palakurthi et al, 2000). These results indicate that cell transformation requires the influx of external Ca²⁺ and in most cases it has been shown to accelerate Ca²⁺ entry; therefore, reduction of this mechanism might reduce the progression of the tumour and might be one of the targets of cancer therapy. This is the case of the human prostate cancer cells, where blockade of the Ca²⁺ entry by using TH-1177, extend the mean life span in mice inoculated with these cells by 38 % without obvious cytotoxicity (Haverstic et al, 2000). In addition, a potential antitumor action for another Ca²⁺ channel blocker, the dihydropyridine amlodipine, has recently been described (Yoshida et al, 2004). Amlodipine has been reported to reduce the tumour growth and increase the survival of mice inoculated with human epidermoid carcinoma A431 cells (Yoshida et al, 2004).

Several alterations have also been observed in the function of the mechanisms involved in Ca²⁺ removal from the cytosol, which indicate that this transporters might be involved in the development of neoplasia. Recent studies have reported that an altered expression of the PMCA might occur in tumorigenesis. The expression of PMCA 1 in MCF-7 and MDA-MB-231 breast cancer cells is significantly increased compared to that in normal cells (Lee et al, 2002). This effect might be a cause of the cell transformation or a response to this stimuli since PMCA expression has been shown to be lower in skin and lung fibroblast transformed by treatment with SV40 (Reisner et al, 1997).

An interesting issue that is still unclear is how cancer cells avoid apoptosis. It has been reported that early preneoplastic cells are highly susceptible to apoptosis, whereas later preneoplastic cells are quite resistant (Preston et al, 1997). Although several hypothesis have been proposed to explain the resistance to apoptosis, a plausible mechanism involving Ca²⁺ signalling has been presented, in which reduced Ca²⁺ levels in the ER were observed in early preneoplastic cells that undergo apoptosis compared to a higher level of stored Ca²⁺ in the ER in late preneoplastic cells. The mechanism by which these cells accumulated different amount of Ca²⁺ in the ER lies on the different activation of SMCE. In late preneoplastic cells, less susceptible to apoptosis and therefore with a great potential to become transformed, Ca²⁺ entry is increased compared to that found in cells in the early steps of neoplastic transformation (Preston et al, 1997). The role of Ca²⁺ entry in apoptosis is supported by a more recent report suggesting that a reduced SMCE correlates with the development of apoptosis (Jayadev et al, 1999).

In the last decade, the scientists have considerably advanced in the cancer therapy, trying to apply genetic therapy to stop the development of several cancer types. At present, there is not treatment that can totally sure the cancer elimination, and quite often they found another problem, cancer cells can leave the main focus, travel around the body and proliferate in other substrate, the so called metastatic cells. The current therapies usually have severe side effects in the patients. However, the recent observations provide reasons to be optimistic and raise the possibility that directed synthesis of novel and specific Ca²⁺ channel blockers or inhibitors of Ca²⁺ mobilising mechanisms could be useful in the therapy of cancer by minimising effects on normal organs and cells and by maximising the cytostatic efficacy for the neoplastic tissue of clinical concern.

Juan A. Rosado