Calcium & Chloride Channels Involved in Glioma Functions

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There is tight interplay between Ca2+ and Cl flux that can influence brain tumour proliferation, migration and invasion. Glioma is the predominant malignant primary brain tumour (accounts for ~80% of all cases), in which there has been an array of studies examining Cl channel expression and activity. Voltage-gated Cl channel family (ClC) proteins and Cl intracellular channel (CLIC) proteins have been largely overexpressed in glioma and are associated with increases in cell proliferation as well as enhancement of migration and invasion. Ca2+ plays fundamental roles in this phenomenon. Ca2+-activated Cl channels (CaCC) such as TMEM16A and bestrophin-1 are involved glioma formation and assist Ca2+ movement from intracellular stores to the plasma membrane. Additionally, the transient receptor protein (TRP) channel TRPC1 can induce activation of ClC-3 by increasing intracellular Ca2+ concentrations and activating Ca2+/calmodulin-dependent protein kinase II (CaMKII).  Therefore, Ca2+ and Clcurrents can concurrently mediate brain tumour cellular functions. Glioma also express volume regulated anion channels (VRACs) which is responsible for the swelling-induced chloride current, ICl,swell. This current enables glioma cells to perform regulatory volume decrease (RVD) as a survivability mechanism in response to hypoxic conditions within the tumour environment. RVD can also be exploited by glioma for invasion and migration. Effective treatment for glioma is challenging, which can be in part due to prolonged chemotherapy leading to mutations in genes associated with multi-drug resistances (MRP1, Bcl-2, and ABC family). Thus, a potential therapeutic strategy for treatment of glioma can be through the inhibition of selected chloride channels.

1. INTRODUCTION

Primary brain tumours can be classified into 120 types, divided into benign and malignant cancers. Classification is based on degree of malignancy, location, and cell type of origination. Glioma, which arise from glial cells, represent the predominant malignant primary brain tumour. In 2016, WHO made amendments to classify glioma based on diagnostic testing and treatment procedures [1]. Currently, glioma is categorized into four degrees of malignancies: low grade (Grades I-II) and high grade (Grades III-IV). These grades include astrocytoma, oligodendroglioma and ependymoma. Among high-grade glioma, 25% are anaplastic astrocytoma (Grade III) and 75% are glioblastoma (GBM) (Grade IV) [2]. GBM is exceptionally aggressive in its proliferation, migration and invasion [3] Despite heterogeneity in GBM presentation, morphological characteristics such as necrosis and vasculature defects allow for more reliable diagnosis [4]. Histological diagnosis can be further improved by the analysis of genetic mutations and biomarkers [5]. However, GBM is highly resilient to conventional cancer therapy and is typically a terminal disease [5]. Patients display a low life expectancy (~1.5 years following diagnosis) even after aggressive treatment [6]. Thus, there is urgent need for novel drug targets for the treatment of GBM [5].

In the brain, Cl channels are known to be involved in several physiological processes in the brain, including: the ClC family regulates acidification and vesicle ion homeostasis [7]; GABA- and glycine-gated Cl channels coordinate growth of neuronal stem cells via neurotransmission [3] and Ca2+-activated Cl channels are closely involved with cellular Ca2+ homeostasis. Dysregulation of these Cl channels have also been associated with a number of brain pathologies such as epilepsy, mental retardation, and hyperekplexia [7]. One trait of GBM is the involvement of aberrant Cl channel activity, which can enhance tumour proliferation, migration and invasion. Thus, channelopathy is an underlying cause, caused by overexpression and/or post-translational modifications of Clchannels [8, 9]. Moreover, the activity of Cl channels is heavily influenced by Ca2+ homeostasis via TRP and Ca2+-activated K+ (KCa) channels [8,10,11]. Ca2+ and Cl channel activity also act synergistically to induce swelling-induced chloride current (ICl,swell), which upregulates pro-survival signalling pathways and drives regulatory volume decrease (RVD) for glioma migration and invasion [12,13].

Non-glioma malignant primary brain tumours include primary cerebral lymphoma, pineal and pituitary tumours, and acoustic neuroma [2]. Nonetheless, these tumours are relatively rare compared to glioma, and there is insufficient literature documenting the involvement of Cl channels. This article overviews the mechanistic interplay between Ca2+ and Cl, noting the involvement of relevant Ca2+ and Cl channels in glioma cellular functions.  

2. CALCIUM & CHLORIDE CHANNELS INVOLVED IN GLIOMA FUNCTIONS

Ion channels have critical roles both physiologically and pathologically, including cancer. Glioma rely on migration to invade surrounding brain tissue[8]. A key proponent of invasion is cell volume change. Shrinkage can assist glioma in traversing narrow regions [2].  Osmotically-induced volume changes primarily depend on Ca2+ and Cl flux through various ion channels[2]. The key Ca2+-conducting members are transient receptor potential (TRP) and K+-activated Ca2+ (KCa) channels. In addition, the most notable Cl channels involved include Ca2+-activated Cl (CaCC) channels, as well as members of the voltage-gated Cl channel (ClC) family that contribute to the swelling-induced Cl current ICl,swell.

2.1. CALCIUM CHANNELS AND CALCIUM HOMESTASIS

Voltage-Gated Calcium Channels (VGCCs). VGCC includes low-voltage activated (T-type) and high-voltage activated (L, N, P/Q, and R-types) calcium channels, among which P/Q-type, N-type, and T-type channels are abundant of expression in brain cells. VGCCs mediate Ca2+ oscillations in primary brain tumours are involved in glioma aggressiveness and malignancy [14].  For example, T-type VGCC blocker mibefradil decreased GBM cell proliferation and migration, induced GBM cell apoptosis in vitro [15], and suppressed GBM growth in a murine xenograft model [16], providing that recovery of intracellular calcium balance by suppressing VGCCs by inhibitors or other regulators is effective approach for the treatment of primary brain tumours.

 

Transient Potential Receptor (TRP) channels. Within this superfamily of non-selective cation channels, one of the members in the TRPM (melastatin) family, TRPM7, has been strongly suggested to influence glioma cellular Ca2+ concentrations, and promote survival and motility [17,18,19]. Our lab and others have reported that TRPM7 is upregulated in the U87 and U251 GBM cell lines [17,18,19].Inhibition of TRPM7 by carvacrol [17] and xyloketal B [19] induced apoptosis, prevent migration and invasion, and downregulate RAS/MEK/MAPK and PI3K/ATK pathways. In contrary, potentiation of TRPM7 by naltriben increased GBM migration and invasion, and upregulated the ERK pathway [19]. These studies strongly indicate that TRPM7 is involved in GBM functions, as they were suppressed with TRPM7 inhibition, and vice versa.  TRPM7 is speculated to act upstream of the aforementioned pro-survival pathways through activation of receptor threonine kinases (RTKs) and the PLC pathway [17,18,19]. In A172 glioma cells, elevated TRPM7 expression promotes cell proliferation and migration through activation of the Notch and JAK2/STAT3 pathways [20]. TRPM7 also contributes to glioma migration by mediating Ca2+ entry, which can activate proteins involved in the modulation of cell adhesion dynamics [21]. In addition, TRPM7 triggered Ca2+ sparks that promoted the formation of invadosomes [21]. TRPM7 activity, and consequently intracellular [Ca2+], decreased in response to hypertonic conditions, which reduced Cl influx [22] (Fig. 1). This suggests that TRPM7 can directly and indirectly mediate Ca2+ and Cl flux, respectively.

 

Calcium-Activated Potassium (KCa) Channels. Glial cell proliferation is partly mediated by membrane conductance variations via Ca2+ and K+ flux [23] Moreover, Ca2+-activated K+-channels (KCa) can influence glioma cell migration and metastasis [24]. Large conductance KCa channels (BK) are upregulated in GBM due to a mutation in the hslo gene and show an increase in Ca2+ conductance [25] The overexpressed BK channel KCa1.1 was shown to mediate growth in Muller glial cells and I321N astrocytoma cells [25]. KCa1.1 co-localized with a member of the Cl channel (ClC) family (ClC-3), and this interaction was associated with invadopodium formation and enhanced glioma invasiveness [26]. Upregulated expression of the intermediate conductance KCa3.1 (IK) was reported in U87, which promote increased Ca2+ entry through TRP channel [27] This subsequently enhanced Ca2+ oscillations, which underlies the motility mechanisms responsible for glioma migration [28].

Role of Ca2+ homeostasis in glioma. Maintaining Ca2+homeostasis involves Ca2+ influx from extracellular space, and the exchange of Ca2+ between the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR), the cytosol, and extracellular compartments [29,30].  Ca2+ dysregulation can affect many signalling pathways in glioma, including proliferation, migration, invasion, apoptosis, and promote cancer killing immune cells [30,23].

Ca2+ signalling patterns can be conducted via a series of Ca2+ storage discharges, known as Ca2+ oscillations [31], which are coordinated by store-operated Ca2+entry (SOCE) via Ca2+ release-activated channels (CRACs) [27]. Ca2+ oscillations can be induced by Cl channel activity. Ca2+ oscillations initiate the depolymerization of actin which assists tumour proliferation, migration and invasion by promoting a counteracting cell volume increase via activation of Na+/H+ exchangers and K+/Na+/Cl transporters [27]. CRAC is made up of Orai channels and STIM sensors, which respond to Ca2+ levels in the ER and SR [24,30]. Ca2+ ions are released from the intercellular Ca2+ store mainly via inositol 1,4,5-trisphosphate receptor (IP3R), and ryanodine receptor (RyR), and excluded to the extracellular domain by ATP-driven Ca2+ pumps and Ca2+-related exchangers [30]. Glioma exhibits an increase in cytosolic Ca2+ through activation of the PLC/IP3 pathway [28] ( Fig 2).

 

2.2. CHLORIDE CHANNELS

Cl channels, the primary anion channels in human cells, are classified based on properties such as voltage-gating, ligand-gating, intracellular phosphorylation, ATP hydrolysis, and cell-volume swelling [32]. They play a vital role in cell volume regulation. In response to hypotonic conditions, swelling-activated Cl channels induce ion efflux to reduce swelling by coordinating with other ion channels, pumps, and co-transporters [32] . However, this cell shrinkage assists in glioma invasion throughout the brain [33]. Moreover, Cl channel activity is strongly correlation with glioma cell cycle progression [34].

2.2.1. Ca2+-Activated ClChannels (CaCC)

The molecular identity is still under controversial, candidates of which include TMEM16A, CLCA, ClC-3 and bestrophins. Among these candidates, TMEM16A is currently considered as a strong one coding for CACC [35].

Ano-1 (TMEM16A). This CaCC channel family is ubiquitously expressed in all tissue types and consists of 10 members. TMEM16A can assist in the activation of otherCa2+-activated channels via linkingCa2+ stores from IP3 and ER to the plasma membrane. In addition, it exhibits an outwardly rectifying, time-dependent current, and suggested to regulate cell volume [36]. Upregulation of TMEM16A has been reported in other cancers to promote cell proliferation through increased activity of the ERK pathway, which is also prominent in glioma.

TMEM16A has been reported to have both Ca2+ and voltage-dependence. At low intracellular [Ca2+], TMEM16A exhibits an outward Cl current when the cell is depolarized. Interestingly, TMEM16A can be activated even at hyperpolarized potential if Ca2+ is present. However, in the absence of Ca2+, the channel is inactivated [37].

 

Bestrophin-1 (Best1). This CaCC family is comprised of 4 members, and in humans, found in the retina and brain [36]. The current of Best1 exhibits an outward rectification in response to cell volume increase [38]. Best1 is typically localized to the ER and interact with the Ca2+ sensor, STIM1. When Ca2+ is released from the ER, Best1 acts as a counterion channel and assists in activation of TMEM16A [39]. Retinal pigment epithelium (RPE) cells expressing Best1 mutants had reduced performance in volume regulation decrease [40]. In glial cells, Best1 is responsible for astrocytic GABA and glutamate release. Best1 is also expressed in glioma, and has been speculated to play a role in tumour volume regulation [41].

 

Chloride channel regulator (CLCA). In humans, CLCA exists in three forms: hCLCA1, hCLCA2, hCLCA3. Whereas CLCA1 directly interacts with TMEM16A, CLCA2 interacts with STIM1 and Orai1 to activate Ca2+ store release [42]. Additionally, CLCA1 and CLCA2 can increase the activity of TMEM16A [42].  CLCA mediates Cl conductance, and can affect cell-cell adhesion, cell cycle progression and apoptosis [43]. Expression of CLCA has been associated with asthma and cystic fibrosis. Moreover, it plays a key anti-cancer role by activating the tumour suppressor p53, and thus can be exploited as a potential therapeutic target for treatment of glioma. However, reports of CLCA expression in the human brain have been elusive [43].

 

2.2.2. Voltage-gated Cl (ClC) Superfamily

ClCs consist of 18 segments assembled in a tiled configuration interweaved into the cell membrane [44]. ClCs are divided into three groups: ClC-1, ClC-2, hClC-Ka and hClC-Kb; ClC-3 to ClC-5, and ClC-6 and -7 [45]. The involvement of ClC has been reported in many cancer types [46]. Expression of CLCs in tumours, including glioma, is upregulated, although downregulation of CLC has been reported in other types of cancers [30,42]. Specifically, ClC-1 [47], ClC-2, ClC-3 and ClC-5 [33] were expressed in glioma. Within these, ClC-2 and ClC-3 were overexpressed and enhanced glioma cell viability, proliferation, migration and invasion [33].

In glioma, Cl conductance contributes to a large portion of the overall conductance of the cell. Thus, the overall cellular membrane potential lies close to the Clreversal potential [48]. In glioma, applying a hyperpolarizing voltage between -80to -120 mV (relative to a resting potential of -40mV) induces a voltage- and time- dependent inward Cl current [33]. Glioma cells also experience a voltage-dependent outward Cl current from -60 to +100mV [33]. Inwardly- and outwardly-rectifying currents (following hyperpolarization and depolarization, respectively) were reduced with knockdown of ClC-2 and ClC-3, respectively. Additionally, the rate at which the ClC-3 outward current increases along the voltage steps can be enhanced by addition of intracellular Ca2+[39], thus indicating that Ca2+ plays a role in regulating Cl channel conductance. Cl conductance in glioma changes cyclically through cell cycle progression, where it is highest in early G1 phase and lowest in S phase [49]. Ca2+ has also been observed to change during the cell cycle [50], thereby indicating a possible relationship between Ca2+ and Cl currents in glioma cell cycle progression.

Early detection of glioma is difficult due to their invasion into the brain’s white matter, and not apparent until patients exhibit pain or impaired motor activity [51]. Early signs of glioma include brain swelling, which causes intracellular pH deregulation, and changes in signalling substrates and enzyme concentration [52]. Cl channels can induce decrease in cell volume by initiating passive Cl efflux, which is accompanied by increase in K+ conductance [13]. This results in surrounding aquaporins to efflux waters which leads to RVD [48]. In order to maintain constant Cl and salt efflux, Cl transporters are required to actively bring Cl back into the cell to maintain membrane potential and the electrochemical gradient [48]. RVD has been shown to assist glioma invasion throughout the tight brain parenchyma by changing cell volume and shape [13] (Fig 3).

 

Swelling-induced Cl currents (ICl,swell). An increase in anionic membrane permeability following cell-swelling was first reported ~40 years ago [53]. Further research showed that the increase in permeability caused ICl,swell, which facilitated subsequent regulatory volume decrease (RVD) via volume regulated anion channels (VRAC) [54]. However, volume sensitive outward rectifying (VSOR) channels and volume sensitive organic osmolyte/anion channels (VSOAC) have also been attributed to RVD [36]. It is unclear whether VRAC, VSOR, and VSOAC are composed of related proteins or separate distinct entities, and the identity of the Cl channel(s) responsible for ICl,swell remains ill-defined. One of the early candidates for VRAC was the leucine-rich repeat containing 8A (LRRC8A), which is a widely expressed Cl channel regulated by cell volume and Ca2+[55]. The action of mechanism of LRRC8A in volume regulation is unknown, but it is strongly suggested that LRRC8A must form a heterodimer with an additional LRRC8 protein to form functional VRAC [51]. Impairment of LRRC8A alone was detrimental to cell volume regulation. Specifically, suppression of LRRC8A in HEK cell lines, HeLa and T-lymphocyte cell lines prevented RVD through inactivation of ICl,swell [55]. Moreover, glioma was unable to perform RVD after silencing of LRRC8A, which resulted in reduced cell viability and proliferation, as well as increased sensitivity to chemotherapeutic drugs temozolomide and carmustine [56].

Importantly, certain members of the ClC family can also be activated by cell swelling to generate ICl,swell, notably ClC-2 and ClC-3 [57,58]. However, there is controversy in the literature whether ClC-2 or ClC-3 is the predominant channel in generating ICl,swell, with multiple studies supporting and refuting their importance. ClC-2 expressed in Xenopus oocytes activated in response to hypotonic solution-induced cell-swelling [57] In SF9 cells, CLC-2 promoted RVD [59]. Consistent with this, knockdown of ClC-2 decreased volume regulation in hepatoma cells [60]. However, ClC-2 knockout mice did not experience a change in RVD compared to controls [61], and ClC-2 current in T84 cells was only modulated by cell-swelling when exposed to hyperpolarization [48]. For ClC-3, early studies demonstrated that its downregulation suppresses RVD in HeLa cells [62] and inhibits ICl,swell in rat brain epithelial cells [63]. However, overexpression of ClC-3 in HEK293 cells did not affect ICl,swell and had no role in RVD [64]. Furthermore, in human pulmonary artery smooth muscle cells, ClC-3 knockdown did not affect ICl,swell [65], and ICl,swell was not affected in ClC-3 knockout mice [66]. These contrasting results suggest that whether CLC-2 or CLC-3 is the dominant player in generating ICl,swell may be cell type dependent.

Nonetheless, the most recent and strongest candidate for ICl,swell in glioma is ClC-3. It was reported that ClC-3 coordinates with other Cl channels and co-transporters to maintain cell volume, and inhibition of ClC-3 alone is insufficient to mitigate glioma invasion [46]. ClC-3 is a membrane delimited channel that is activated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation [67] (Fig 2) . Expression of ClC-3 is 10 folds higher in glioma than non-malignant brain tumours [67] ClC-3 activation by CaMKII indicates that changes in intracellular Ca2+ concentrations can play a role in conducting ICl,swell, further suggesting synergy between Ca2+ and Cl currents in promoting glioma malignancy.

Effects of ICl,swell on glioma signalling and cellular functions. Activity of ion channels is regulated by various signalling proteins and their associated pathways [8]. Signalling pathways that act via phosphorylation of tyrosine, threonine, and serine residues regulate most voltage gated ion channels, including those in the ClC family, by altering their voltage sensitivity [26] or by promoting flux of signalling molecules between organelles within the cell [68]. Ion channels also associate with cell-adhesion molecules such as integrin, which create cell signalling complexes with various other receptors. Activity of these cell-adhesion molecules can affect cell differentiation and neuron outgrowth [8]

We recently investigated the pharmacological effects of DCPIB, a selective inhibitor of ICl,swell, on U87 and U251 GBM cell viability, proliferation, migration, and invasion. We found that glioma exhibited ICl,swell following hypotonic solution-induced cell swelling, and this current was abolished with application of DCPIB. Moreover, inhibiting ICl,swell suppressed glioma cellular functions, as DCPIB treatment of U87 and U251 cells dose-dependently reduced cell viability and colony formation. Glioma migration and invasion were also significantly inhibited by DCPIB [12]. In GBM, the JAK/STAT3 and PI3K/ATK pathways are hyperactivated in GBM and are found to be essential for cell viability and migration [69]. We observed that DCPIB suppressed  JAK2/STAT3 and Akt/PI3K signaling and thus  speculate that these pathways are involved in the underlying mechanism by which ICl,swell suppression inhibits GBM cellular functions [12]. Specifically, we propose that ICl,swell could activate these pathways via phosphorylation by receptor threonine kinases (RTKs) (Fig 4).

Our findings are consistent to the reported by  Sforna et al. [70], who demonstrated in GBM the mechanism by which Cl channels induced ICl,swell in response to acute and chronic hypoxia. Similarly, they observed that cell swelling generated ICl,swell  resulted in RVD. This was hypothesized to prevent cell necrosis following hypoxic stress. However, following chronic hypoxia, GBM cells had suppressed ICl,swell and slower RVD. In the GL-15 human GBM cell line, Catacuzzeno et al. [13] reported that ICl,swell required the activity of U73122-sensitive phospholipase C (PLC), membrane permeable diacylglycerol (DAG), and EHT1864-sensitive Rac1 small GTPase [13] They also found that ICl,swell can be activated by fetal calf serum (FCS), which suggested that leakage of the blood brain barrier potentially plays a role in tumour cell invasiveness [13].

2.2.3. Other Types of Cl Channels

Cystic fibrosis transmembrane conductance regulator (CFTR). Consisting of 12 membrane spanning segments and two nucleotide binding domains (NBD1 and 2) plus a regulatory R domain, CFTR is activated via cAMP-dependent phosphorylation and binding of ATP at the nucleotide binding domains [44]. CFTR acts as a conductance regulator of several channels such as suppression of CaCC and the outwardly rectifying Cl channel (ORCC) [71]. It also acts as a regulator of TRPV4, which was shown to deliver the Ca2+ signal for RVD in epithelia [36]. CFTR modulates inflammation and apoptotic pathway, and CFTR gene mutation has been associated with cervical and pancreatic cancer [46]. Thus, CFTR can potentially also play a role in glioma, although its role here has yet to be reported.

Cl intracellular channel protein (CLIC). Also known as the p64 family, CLIC consists of six members: CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, and CLIC6 [72]. Their main roles include regulation of endosomal trafficking, actin-dependent membrane remodelling, and tubulogenesis [72]. Additionally, both overexpression and underexpression of CLIC1 and CLIC4 have been observed in several tumour cell lines and have been considered to be therapeutic targets for chemotherapy [46] . CLIC4 is regulated by p53 and tumor necrosis factor α (TNFα), and has been observed to translocate from the plasma membrane to the nucleus in response to stressful conditions to assist in apoptosis, thus highlighting its role in tumour suppression [46]. Specifically, in glioma, suppression of CLIC4 has been reported to increase apoptosis induced by hydrogen peroxide [50]. Furthermore, CLIC1 expression is upregulated in GBM at higher levels than low grade glioma [46]. Suppression of CLIC1 in GBM decreases cell proliferation and hinders the self-renewal ability of cancer stem cells [73].

Maxi Cl channel. Found in multiple cell types including glia, neurons, lymphocytes, macula densa, cardiac muscle cells, maxi Cl channels are involved in controlling cell membrane potential, apoptotic pathway and cell volume regulation. Swelling, hypoxia and ischemia activate this channel [74], which suggests the potential of its role in glioma cellular functions.

Glycine-gated Cl and GABA channels. These heteropentameric proteins are comprised of three subunits (α, β and γ), which in turn constitute four different membrane-integrated segments, building up the core side of the channel from the C-terminal side and are extended to the extracellular surface through the N-terminal side. This creates the ligand binding site of the channel [44]. GABAA is a Cl channel comprised of eight subfamilies, which differ in functionality and pharmacologically based on their subunit structure [3]. Physiologically, GABAA receptor channels enable Clflux into neurons to counteract depolarization and suppress action potentials [3]. Nevertheless, GABA signalling is also involved in growth regulation of neuronal stem cells, neuroblasts and neuronal tumour cells. In glioma, the decrease in GABA receptors has been correlated with the tumour’s degree of malignancy. GABA receptors are absent or misfolded in GBM which could attribute to its enhanced malignancy [3].

2.2.4. Role of Cl Channels in Glioma Cell Proliferation, Apoptosis, Migration and Invasion

Proliferation. When the cell experiences adverse conditions, CLIC-1 activity enables Cl influx during the G1/S phase of the cell cycle to induce proliferation [75,76]. Inhibition of CLIC-1 can prolong the duration of the cell cycle [77]. GBM cell lines have been shown to overexpress CLIC-1 proteins in order to promote proliferation [78]. Pharmacological inhibition or knockdown of CLIC-1 decreases GBM proliferation and tumorigenesis [26]. For mitosis to be successful, cell volume decrease for pre-mitotic condensation (PMC) is required prior to the M phase [26]. During PMC, ClC-3 membrane expression is elevated. Suppressing ClC-3 halts PMC and thus cell cycle progression. Furthermore, inhibiting GABA-gated Cl channels in the U3013 GBM cell line stunted cell growth. This strongly suggests that Cl current is required for cell proliferation and differentiation [5]

 

Apoptosis. Glioma requires Cl channel activity to initiate an apoptotic volume decrease (AVD). Inhibition of Cl channels prevents AVD in addition to caspase activation and DNA fragmentation  [26]. Knockdown of CLIC4 in glioma results in an elevation of apoptosis induced by hydrogen peroxide. Although Bax is associated with CLIC4 in apoptosis, Bax/Bcl2 expression did not change even with CLIC4 suppression [79].  However, when glioma was exposed to hypoxia, CLIC4 was upregulated along with an increase in Bax/Blc2 and caspase-3 expression [79]. However, Bax-induced apoptosis was not prevented by CLIC4 suppression, which suggested different signalling pathways [76]. CLIC4 is also involved in p53 and cMyc activated apoptosis [46]. When U251 cells were starved, CLIC4 inhibition and subsequent upregulation of beclin 1 resulted in enhanced autophagy. CLIC4 inhibition also induced mitochondrial and ER apoptosis as a result of Bax/Bcl-2 and cytochrome c release, and increase in caspase e and CHOP, respectively [80]. In C6 cells, silencing the ClC-4 gene resulted in hydrogen-induced and TNFα-mediated apoptosis [79].

 

Migration and Invasion. To traverse the narrow parenchyma space within the brain, glioma undergoes cell volume decrease, which is primarily regulated by Cl flux. Inhibition of Clchannels reduces glioma migration [26]. When treated with the Cl inhibitor NPPB, glioma crossed the Transwell membrane at a slower rate [24]. Replacing Cl with Ior Br allowed glioma to retain its original ability to migrate [24] .

Invasion of glioma is mediated by Cl and K+ efflux, which results in RVD [24] . GABA-gated Cl channels can induce Cl accumulation and thus regulate RVD [24]. Na-K-Cl cotransporter (NKCC) is the main transporter in glioma responsible for Claccumulation. The uptake of Clis balanced by efflux of Cl via KCC1 and KCC3a transporters, which can also regulate migration [24] . ClC-3 forms protein complexes with matrix metalloproteinase-2 (MMP-2), and has been observed to co-localize with KCa channels in the lipid raft domain of invadipodia [76] .

3. INTERACTION BETWEEN CALCIUM AND CHLORIDE

Effects of Ca2+ on Cl signalling. Ca2+ can influence glioma migration and invasion via modulation of Cl channel activity [39]. Elevated Ca2+ influx through TRP channel, TRPC1, is essential for epidermal growth factor (EGF)-induced invasion in several glioma cell lines [8]. Furthermore, this Ca2+ influx also activates CAMKII-activated ClC-3 activity, which results in an efflux of Cl ions and RVD for cell migration. Similarly, in GBM cell lines, potentiated KCa3.1 and ClC-3 activity by bradykinin was necessary for chemotactic cell migration [10]. Bradykinin was found to interact with GPCR receptors which led to increased intracellular Ca2+. This resulted in upregulated KCa3.1 as well as CAMKII-activated ClC-3 activity, causing Cl and K+ efflux that resulted in cell volume decrease required for GBM migration and invasion [10].  In U87 cells, FCS was shown to increase activity of KCa3.1 through Ca2+ oscillations, and subsequently increased Cl channel activity. This decreased cell volume and assisted glioma migration [11].

Several CaCC channel genes are upregulated, including anoctamin, Cl channel regulator (ClCA) and bestrophin [39] . Moreover, TMEM16A is found to be overexpressed in glioma and helps promote cell proliferation, migration and invasion [81]. The proposed underlying mechanism is that upregulated TMEM16A activates the NF‑κB signalling pathway which increases expression of oncogenes (e.g. cyclin D1, cyclin E, and c‑myc). The NF-κB pathway also promotes activity of MMP-2 and MMP-9, which regulate the structural integrity of the ECM in the brain and promote glioma migration and invasion [81] . GABA-gated Cl channels also increase intracellular Ca2+ concentration in glial cells, since hyperpolarization from Cl influx induces activation of voltage-gated Ca2+ channels, resulting in Ca2+ influx [18].

 

Effects of Cl on Ca2+signalling.Inversely, Cl channels can also regulate cellular Ca2+and its associated signalling pathways. As previously mentioned, GABAA receptors mediate Cl current-which can hyperpolarize the membrane potential, resulting cell inhibition in adult neurons. However, in astrocytoma and oligodendroglioma, GABAA receptor channels can also depolarize the cell while intracellular Cl concentrations are high. Depolarization causes an increase in intracellular Ca2+ likely via the activation of voltage activated Ca2+ channels (VGCC) [82,18]. Elevation of intracellular Ca2+ by VGCCs have led to uncontrolled proliferation and migration in cancer [14]. In neurons, activation of CaCC channels following Ca2+ influx caused an efflux of Cl, which depolarized the cell and activated VGCCs for even more Ca2+ influx and thus further depolarization [83] Although this continuous depolarization has not been reported in glioma, inhibition of VGCC to reduce Ca2+ entry decreased glioma cell proliferation and induced apoptosis [14]. Thus, this suggests that intracellular Ca2+elevation induced by hyperpolarization is important for the activation of Ca2+-dependent proliferative and survival pathways.

4. PHARMACOLOGY

There is growing evidence of the role of Cl channels in cancer pathology, thus illustrating the potential of exploiting them as therapeutic targets for chemotherapy [32, 8].Volume-activated Clcurrent can be inhibited by an array of compounds, which are divided into the following classes: Class I compounds are transported into the cell via phosphoglycoprotein (P-gp) and stop channel activation without pore blocking. These compounds react inside the cell following ATP hydrolysis; Class II compounds are transported into the cells vial P-gp and physically block the channel by binding to its extracellular area. Note that some class II compounds (e.g.tamoxifen) are P-gp independent and bind to other receptors; and Class III compounds (e.g. DIDS and NPPB) are unable to suppress P-gp- dependent drug transport, but can block volume-activated Cl current [84, 85].

DIDS, a classical VSOR and RVD inhibitor [52] (Fig. 5), acts as an anti-apoptotic compound by increasing the cell’s water content. Additionally, in epidermoid cancer cells, DIDS prevented decrease in cell viability when trichostatin (TSA) and cisplatin were also added simultaneously [86]. Treatment of TSA and cisplatin increased caspase-3 activity, which induced apoptosis and decreased cell viability [86] . Hypoxia-induced swelling was blocked by DIDS and NPPB [68]. In astrocytoma, DIDS, DNS and Zn2+ decreased cell proliferation [34].

Additionally, RVD is also inhibited by NPPB and Cd2+ [52] and to a lesser extent by the K-Cl cotransporter inhibitor dihydroindenyloxyacetic (DIOA). Treatment of glioma with these inhibitors combined resulted in greater inhibition of RVD than when used separately [52]. NPPB was reported to completely suppress glioma cell invasion [87] (Fig. 5). However, GBM cells were potentially overdosed and cell invasion was halted due to cytotoxic levels of NPPB [88]. Note that NPPB is non-specific and can inhibit the function of multiple Cl channels (e.g. ClC-2, ClC-3, and CFTR) [33] , as well as KCa channels [89, 88].

The alkylating agent temozolomide (TMZ) is the current standard chemotherapeutic drug for GBM treatment. TMZ can conjugate with NPPB, creating TMZ-NPPB, which blocks Cl currents (with similar efficacy as NPPB alone) in glioma and suppresses cell viability, migration and invasion [9]. TMZ-NPPB is more stable than TMZ, thus having greater therapeutic potential for treating glioma [9]. Furthermore, sensitivity to TMZ can be restored by inhibiting PI3K [90].

DCPIB is a selective ICl,swell inhibitor which can also impede RVD [68]. After DCPIB treatment, we observed reduction of intracellular Cl. In addition, DCPIB abolished ICl,swell in GBM, and suppressed glioma viability, proliferation, colony formation, migration and invasion [12].

Chlorotoxin (Ctx), an inhibitor of small conductance Cl channels (Fig. 5), is currently under phase I/II clinical trials for glioma treatment [46]. Action of mechanism of Ctx is via internalization of a complex formed by MMPs and ClC-3 at the cell membrane [26]. Ctx selectively inhibits ClC-3 expression [8], and suppresses glioma migration both in vitro cell cultures and in vivo mouse models, [26,87] Knockdown of ClC-3 with siRNA in addition to Ctx treatment only had slight increase in efficacy compared to Ctx alone, thus suggesting that Ctx is a potent ClC-3 inhibitor [76].  Ctx prevented glioma invasion into fetal rat brain aggregates and was able to irreversibly block RVD [91]. The Ctx derivative, TM-601, has been used in clinical trials to treat high grade glioma [26].

Metformin, traditionally used as a drug for type-2 diabetes, has also been reported to have anti-tumorigenic properties. Specifically, metformin suppresses cell proliferation in GBM cell lines. Metformin acts by directly binding to ClC-1 and preventing Clconductance, which consequently suppresses cell proliferation [92,75]. Related biguanides such as phenformin, moroxydine, and cycloguanil show similar mechanisms of ClC-1 inhibition, and also suppress cell proliferation to varying efficacy [75] .

Ani9 is a selective inhibitor of TMEM16A. In glioma cells, Ani9 almost completely suppressed TMEM16A activity. Due to its high specificity, Ani9’s suppression of overall VRAC function was lower than that of less selective CaCC channel inhibitors such as T16Ainh-A01 and NPPB. Additionally, increasing the concentration of Ani9 did not contribute to higher impedance of VRAC [93] (Fig. 5).

Finally, tamoxifen, a commonly used medication for breast cancer prevention [94], and the K+ channel blocker tetraethylammonium chloride (TEA), have been reported to reduce Cl current resulting in a decrease in cell invasion and migration in glioma [91]. They also reduced RVD in glioma cells that were subjected to osmotic swelling [91] (Fig. 5), suggesting that they may be potent inhibitors of VRAC.

 

Multidrug resistance (MDR). Tumour cell resilience to chemotherapy is a major challenge in glioma treatment. MDR is characterized as resistance to an array of anti-cancer drugs with differing structure and mechanism of action [95]. MDRs expel anti-cancer drugs by ATP- dependent proteins within the ATP-binding cassette (ABC) transporter superfamily, which includes P-gp, multidrug resistance protein (MRP), and major vault protein (MVP) [96,97].

P-gp plays important roles in translocating substrate molecule. Malignant tumours express P-gp in order to protect themselves during chemotherapy. Increase in P-gp expression before and after therapy indicated that P-gp is potentially involved in intrinsic and acquired MDR in glioma. Increase in Cl- current in response to cell swelling has been correlated with upregulation of P-gp [98]. It was initially debated whether P-gp was a Cl- channel itself, or a Cl- channel regulator. It was later demonstrated that suppression of P-gp did not abolish swelling-induced currents, thus  suggesting that P-gp does not have intrinsic Cl- channel properties [99]

GBM showed higher MDR expression than low grade astrocytoma [96], Overexpression of MRP1 has been associated with MDR [96]. Expression of MRP1 is higher in Grade III and IV glioma compared to lower grade glioma [100]. Increase in MRP1 expression is suggested to correlate with intrinsic or extrinsic drug resistant [101]. Overexpression of MVP is also seen in many tumours including glioma[96,102]. Increase in MVP expression is observed after chemotherapy [96].

Therapeutic potential for glioma. Combination drug therapy can improve glioma treatment outcomes. Uptake of doxorubicin in U87 cells was increased upon treatment with the P-gp inhibitor, antisense oligodeoxynucleotides [103]. The MRP inhibitor indomethacin enhanced the cytotoxic effects of etoposide and vincristine on multiple glioma cell lines [104]. Silencing MRP1 allowed etoposide to recover its cytotoxic effects against etoposide-resistant T98G-VP and Gli36-VP cell lines [96]. siRNA can also be used to silence MDR1 to induce tumour apoptosis [104] .

5. CONCLUSION

Cl channels play a significant role in glioma pathology. The regulation of Cl flux is essential for glioma proliferation, migration and invasion. When expressed aberrantly, Cl channels can suppress glioma apoptosis and improve viability. Specifically, the swelling-induced Cl current ICl,swell plays a key role in the regulation of cell volume to sustain cell viability, promote invasion and migration, and initiate proliferation signalling pathways. ICl,swell is strongly speculated to be generated by ClC-3 in glioma, which is activated by CaMKII. Furthermore, there is strong evidence in the literature supporting the important relationship between Cland Ca2+ signalling in contributing to tumour malignancy. Therapeutic drugs targeting Cl channels can exploit this Cl and Ca2+ interplay, and potentially be pivotal for glioma treatment.

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