Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Induces Intracellular Ca²⁺ Release through the Two-Pore Channel TPC1 in Metastatic Colorectal Cancer Cells

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca²⁺ release. NAADP-induced EL Ca²⁺ signals may be amplified by the endoplasmic reticulum (ER) through the Ca²⁺-induced Ca²⁺ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca²⁺ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca²⁺ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca²⁺ refilling of acidic organelles, caused massive Ca²⁺ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP₃)-sensitive ER Ca²⁺ store. Liposomal delivery of NAADP induced a transient Ca²⁺ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca²⁺ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP₃ receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca²⁺ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.

1. Introduction

Spatio-temporal intracellular Ca²⁺ signals regulate a multitude of functions, including proliferation, migration, differentiation, gene expression, bioenergetics and survival [1,2,3,4,5,6]. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is generated by the opening of distinct Ca²⁺-permeable channels that can be located either in the plasma membrane, such as voltage-gated Ca²⁺ channels, Transient Receptor Potential (TRP) channels, and store-operated channels (SOCs), or in endogenous organelles, such endoplasmic reticulum (ER), Golgi and the acidic vesicles of the endolysosomal (EL) system (EL) [1,5,6,7,8]. The ER represents the largest intracellular Ca²⁺ reservoir and releases Ca²⁺ in response to appropriate stimuli leading to the synthesis of the second messengers, inositol-1,4,5-trisphosphate (InsP₃) and cyclic ADP-ribose (cADPR), which respectively target InsP₃ receptors (InsP₃Rs) and ryanodine receptors (RyRs) [6,9]. InsP₃Rs and RyRs are both sensitive to cytosolic Ca²⁺ and can activate each other via a Ca²⁺-induced Ca²⁺ release (CICR) mechanism that is able to amplify local Ca²⁺ signals into a global elevation in [Ca²⁺]_i [6,9].

The ER, however, is not the sole endogenous Ca²⁺ store in mammalian cells. Acidic EL vesicles, including endosomes and lysosomes, provide an additional Ca²⁺ reservoir that is recruited by extracellular ligands through the generation of the novel Ca²⁺-releasing second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) [7,10,11]. Acidic organelles contain a relatively high amount of Ca²⁺, i.e., ≈500 µM [12], which is sequestered in a pH-dependent manner through a yet to be identified mechanism, although a Ca²⁺/H⁺ exchanger (CAX) has been recently identified in non-placental mammalian cells [13,14,15]. NAADP mediates Ca²⁺ release from acidic stores across the phylogenetic tree by activating a novel superfamily of voltage-gated ion channels, i.e., two-pore channels 1–3 (TPC1-3) [16,17,18,19]. However, only TPC1 and TPC2 are present in humans, mice and rats. NAADP does not directly target TPC1 and TPC2, but its action requires the interposition of an NAADP-binding protein, which could also mediate NAADP-dependent Ca²⁺ release from ER-embedded RyRs [20,21]. NAADP-induced mobilization of the acidic Ca²⁺ pool may be then amplified by InsP₃Rs and RyRs through the mechanism of CICR at the very narrow (<30 nm) membrane contact sites (MCSs) that occur between EL and ER vesicles [12,14,22]. TPC1 is widely distributed across the EL system, while TPC2 mainly resides at late endosomes and lysosomes [16,19,23]. Either NAADP, TPCs or a combination of both control a growing number of processes, including fertilization, insulin secretion, angiogenesis, vasculogenesis, nitric oxide (NO) release, autophagy, and neurite extension [24,25,26,27,28,29,30,31], and have been involved in the pathogenesis of several diseases [10,32].

Recent work showed that TPCs are also involved in tumorigenesis [33,34]. For instance, TPC1 transcripts were approximately three to eight folds higher than TPC2 in the SKBR3 cells, a human breast cancer cell line [18]. Subsequently, it was found that TPC1 and TPC2 transcripts were similarly expressed in a different set of human breast cancer cells, although genetic silencing of either channel did not affect proliferation in the highly aggressive MDA-MB-468 cell line [35]. More recently, TPC1 and TPC2 transcripts were identified in several cancer cell lines established from the liver, bladder and blood [36]. Notably, genetic silencing of TPC1 and TPC2 inhibited bladder cancer cell migration by impairing the formation of leading edges [36]. Moreover, pharmacological blockade of TPCs with the selective antagonist, NED-19, and genetic silencing of TPC2 reduced lung metastasis in a mammary cancer mouse model [36]. Finally, NED-19 impaired tumor growth, vascularization and metastasis in a mouse model of vascular endothelial growth factor (VEGF)-secreting melanoma and reduced VEGF-induced Ca²⁺ signals in B16 melanoma cells [37]. Therefore, TPCs are emerging as a promising molecular target to design alternative anticancer therapies [32,33,34].

Herein, we sought to investigate the role of NAADP-induced intracellular Ca²⁺ signaling in primary cultures of metastatic colorectal cancer (mCRC) cells established from liver metastasis of human patients [38,39]. We found that an EL Ca²⁺ store is present and functionally coupled to InsP₃-dependent ER Ca²⁺ release in mCRC cells. Accordingly, NAADP-induced intracellular Ca²⁺ mobilization was dampened by disrupting both the EL and ER Ca²⁺ pools. NAADP-induced Ca²⁺ release was also blocked by NED-19 and by genetic silencing of TPC1, the major TPC isoform expressed in mCRC cells. Finally, pharmacological and genetic blockade of TPC1 dramatically reduced fetal bovine serum (FBS)-induced Ca²⁺ release and proliferation in mCRC cells, thereby hinting at TPC1 as a novel therapeutic target in mCRC patients.

2. Results

2.1. A Functional Lysosomal Ca²⁺ Store Is Present in Metastatic Colorectal Cancer Cells

In order to assess the presence of a functional lysosomal Ca²⁺ store, mCRC cells were loaded with the Ca²⁺-sensitive fluorophore, Fura-2/AM, as shown elsewhere [39]. Subsequently, mCRC cells were challenged with the lysosomotropic agent, glycyl-l-phenylalanine-2-naphthylamide (GPN), a substrate of the exopeptidase cathepsin C that results in osmotic lysis and loss of lysosomal membrane integrity [40]. GPN has been widely employed to investigate EL Ca²⁺ signalling in a variety of cell types with the notable exception of cancer cells [14,41,42]. The acute addition of GPN (200 μM) caused a rapid and transient increase in [Ca²⁺]_i in the absence of extracellular Ca²⁺ (0Ca²⁺) (Figure 1A), which reflects intracellular Ca²⁺ release from the endogenous lysosomal store [41,43].

To further support this observation, mCRC cells were stained with Lysotracker Red, a fluorescent weak base that accumulates in acidic organelles [43]. As shown in Figure S1, GPN (200 μM, 30 min) caused the loss of Lysotracker Red fluorescence, which is consistent with labelling of lysosomal vesicles. We further probed the effects of the ionophore nigericin, which dissipates the proton gradient sustaining lysosomal Ca²⁺ refilling [15,40]. Similar to GPN, nigericin (50 μM) caused a transient increase in [Ca²⁺]_i under 0Ca²⁺ conditions (Figure 1B) and erased Lysotracker Red fluorescence (Figure S1), thereby confirming the EL origin of the intracellular Ca²⁺ signal. As summarized in Figure 1C,D, respectively, there was no significant difference between GPN and nigericin in the percentage of responding cells and in the magnitude of the peak Ca²⁺ response. Taken together, these findings show for the first time that a functional lysosomal Ca²⁺ store is present in mCRC cells and is able to increase the [Ca²⁺]_i.

2.2. InsP₃-Induced ER Ca²⁺ Release Sustains the Ca²⁺ Response to GPN in Metastatic Colorectal Cancer Cells

As mentioned earlier, lysosomal Ca²⁺ release may be amplified by ER-dependent Ca²⁺ mobilization through the CICR mechanism, which results in the appearance of regenerative Ca²⁺ waves [10,44]. For instance, GPN induces long-lasting intracellular Ca²⁺ oscillations in human fibroblasts that are curtailed by previous depletion of the EL Ca²⁺ store or pharmacological blockade of InsP₃Rs [41,45]. To challenge this model in mCRC cells, we exploited cyclopiazonic acid (CPA), an established inhibitor of Sarco-Endoplasmic Reticulum Ca²⁺-ATPase (SERCA) activity, which prevents Ca²⁺ sequestration into ER lumen and results in depletion of the ER Ca²⁺ pool. We have recently shown that pretreating mCRC cells with 30 μM CPA for 30 min under 0Ca²⁺ conditions is sufficient to deplete the InsP₃-sensitive ER Ca²⁺ store in mCRC cells [39]. As shown in Figure 2, this protocol significantly (p < 0.05) reduced the percentage of cells responding to GPN (200 μM) (Figure 2A,C) and the peak magnitude of endogenous Ca²⁺ release (Figure 2A,D).

Likewise, CPA (30 μM, 30 min) significantly (p < 0.05) reduced the percentage of responding cells and the peak Ca²⁺ response to nigericin (50 μM) (Figure 2B–D). In agreement with these observations, 2-Aminoethoxydiphenyl borate (2-APB) (50 μM, 30 min), which selectively blocks InsP₃Rs in the absence of extracellular Ca²⁺ [46,47], significantly (p < 0.05) reduced the percentage of responding cells (Figure 3B) and the peak Ca²⁺ response to GPN (Figure 3A,C). RyRs could also amplify EL Ca²⁺ release [14]. Caffeine (5 mM), a membrane-permeant agonist of RyRs, did not increase the [Ca²⁺]_i in mCRC cells (Figure S2A). Moreover, qRT-PCR could not find RyR1-3 transcripts in these cells (Figure S2B). These data, therefore, indicate that GPN-induced lysosomal Ca²⁺ mobilization is amplified by InsP₃-dependent ER Ca²⁺ release through the CICR mechanism, as described elsewhere [41].

2.3. NAADP Gates TPC1 to Induce Lysosomal Ca²⁺ Release in Metastatic Colorectal Cancer Cells

In order to assess whether and how NAADP mobilizes lysosomal Ca²⁺, we exploited the NAADP-containing liposomal preparation that we have recently described [48]. As illustrated in Figure 4A, liposomal delivery of NAADP (at 1:20 dilution) caused a rapid Ca²⁺ transient under 0Ca²⁺ conditions, while restoration of extracellular Ca²⁺ did not cause any discernible increase in [Ca²⁺]_i. NAADP is therefore unlikely to evoke extracellular Ca²⁺ entry in mCRC cells. Control experiments revealed that liposomal formulations devoid of NAADP did not cause any Ca²⁺ signal (Figure 4A). The Ca²⁺ response to NAADP (1:20) was impaired by disruption of acidic stores with GPN (200 μM, 30 min) (Figure 4B), which significantly (p < 0.05) reduced both the percentage of responding cells (Figure 4C) and the peak Ca²⁺ response (Figure 4D).

qRT-PCR showed that TPC1 transcripts were ≈50-folds (50.2 ± 7.4, n = 4) higher than TPC2 (Figure 5A), as also found in several cancer cell lines [18,36]. Negative controls were carried out by omitting the reverse transcriptase [39].

Accordingly, the Ca²⁺ response to NAADP was significantly (p < 0.05) reduced by silencing TPC1 with a selective small interfering RNA (siRNA) (Figure 5B), which significantly (p < 0.05) reduced both the percentage of mCRC cells responding to NAADP (1:20) (Figure 5C) and the magnitude of the Ca²⁺ peak (Figure 5D). The efficacy of gene silencing on TPC1 expression has been illustrated in Figure S3. The same blocking effect was achieved by NED-19 (100 μM, 30 min), a selective NAADP antagonist (Figure 5B–D). Collectively, these findings demonstrate that NAADP mobilizes the EL Ca²⁺ store by activating TPC1 in mCRC cells.

2.4. InsP₃-Induced ER Ca²⁺ Release Sustains NAADP-Induced Intracellular Ca²⁺ Signals in Metastatic Colorectal Cancer Cells

As described above, ER-embedded InsP₃Rs amplify lysosomal Ca²⁺ release via the CICR mechanism. Therefore, we challenged mCRC cells with NAADP-containing liposomes (1:20) upon depletion of the ER Ca²⁺ store with CPA (30 μM, 30 min) under 0Ca²⁺ conditions [39]. As shown in Figure 6A, this treatment significantly (p < 0.05) reduced the intracellular Ca²⁺ response to NAADP by diminishing the percentage of activated cells (Figure 6B) and dampening the magnitude of the Ca²⁺ transient (Figure 6C). The same effect was achieved by blocking InsP₃Rs with 2-APB (50 μM, 30 min), as depicted in Figure 6A and summarized in Figure 6B,C. Overall, these findings endorse the view that InsP₃-dependent ER Ca²⁺ release sustains the lysosomal Ca²⁺ burst induced by NAADP.

2.5. TPC1 Triggers FBS-Induced Intracellular Ca²⁺ Release and Stimulates Proliferation in Metastatic Colorectal Cancer Cells

We have previously shown that 20% FBS causes InsP₃-dependent ER Ca²⁺ release that in turn results in depletion of ER Ca²⁺ levels and activates SOCs in mCRC cells [39]. As InsP₃-induced Ca²⁺ release from ER stores may be induced by lysosomal Ca²⁺ mobilization (see Figure 2), we analyzed the Ca²⁺ response to 20% FBS after disruption of acidic stores with GPN (200 μM, 30 min) and nigericin (50 μM, 30 min). These treatments impaired 20% FBS-induced endogenous Ca²⁺ release under 0Ca²⁺ conditions (Figure 7A) by significantly reducing the percentage of responding cells (Figure 7B) and magnitude of the intracellular Ca²⁺ peak (Figure 7C).

Likewise, the Ca²⁺ response to 20% FBS was dramatically reduced in mCRC cells deficient of TPC1 (Figure 8A–C) or pretreated with NED-19 (100 μM, 30 min) to block NAADP-induced Ca²⁺ mobilization (Figure 8A–C). The increase in [Ca²⁺]_i induced by FBS has been shown to drive proliferation in several cancer cell lines [49,50,51]. However, the pharmacological and genetic blockade of SOCs did not affect the proliferation response to 20% FBS in mCRC cells [39]. Conversely, mCRC cell proliferation was significantly (p < 0.05) reduced in mCRC cells lacking TPC1 or pretreated with NED-19 (100 μM, 30 min) (Figure 8D).

In order to assess the Ca²⁺-dependent decoders whereby TPC1 mediates mCRC proliferation, we focused on extracellular-signal related kinases (ERK) and protein kinase B (Akt), which were previously been shown to support NAADP-induced proliferation [25]. The Ca²⁺-dependent phosphorylation of ERK and Akt was investigated as recently shown [3] under control conditions and upon pharmacological blockade of TPC1 with NED-19 (100 μM, 30 min) or genetic deletion of TPC1 with the selective siRNA. As illustrated in Figure 9 and Figure 10, respectively, both treatments significantly (p < 0.05) reduced the extent of ERK and Akt phosphorylation, which is consistent with previous reports by Favia and colleagues [25]. Therefore, it is possible to conclude that NAADP promotes mCRC cell growth by stimulating TPC1 to mobilize their lysosomal Ca²⁺ content, thereby recruiting the ERK and phosphoinositide 3-kinase (PI3K)/Akt phosphorylation cascades.

3. Discussion

Herein, we demonstrated for the first time that a functional lysosomal Ca²⁺ store is present in mCRC cells and functionally coupled to ER-dependent Ca²⁺ release through InsP₃Rs. We further showed that the novel second messenger, NAADP, stimulates TPC1 to mobilize the EL Ca²⁺ store, thereby engaging InsP₃Rs to generate a transient increase in [Ca²⁺]_i. We finally illustrated that NAADP-dependent TPC1-mediated Ca²⁺ signals underlie FBS-induced endogenous Ca²⁺ release and proliferation in mCRC cells. These data endorse the view that TPCs may provide alternative targets to design novel anticancer therapies and that the therapeutic outcome of their inhibition should be investigated in vivo.

Recent studies uncovered the role played by TPC1 and TPC2 during tumorigenesis [33,34]. These investigations depicted a general pattern according to which TPC1 is more abundant than TPC2 in the majority of cancer cell lines examined [18,36]. Although the involvement of TPCs in cancer growth, vascularization and metastasis was unequivocally demonstrated [35,36,37], several issues remained to be addressed in cancer cells. First, are acidic EL vesicles able to trigger a global increase in [Ca²⁺]_i? Second, is the lysosomal Ca²⁺ store functionally coupled to the main ER Ca²⁺ store, as observed in normal cell types? Third, is NAADP per se able to mobilize lysosomal Ca²⁺ through TPCs? Herein, we first demonstrated that a robust endogenous Ca²⁺ release could be evoked by challenging mCRC cells with two structurally and mechanistically unrelated agents which target the lysosomal Ca²⁺ content. Accordingly, both the lysosomotropic compound GPN and the ionophore nigericin induced a transient increase in [Ca²⁺]_i under 0Ca²⁺ conditions that was associated to the loss of Lysotracker Red fluorescence. These findings strongly suggested that an acidic Ca²⁺ store was present in mCRC cells and could be depleted to elevate intracellular Ca²⁺ levels. Acidic vesicles may establish quasi-synaptic MCSs with the ER, which confers them with the unique role of triggering physiologically relevant Ca²⁺ signals by inducing ER-dependent Ca²⁺ release via CICR [22,52,53]. In agreement with previous findings in human fibroblasts [41], the Ca²⁺ response to GPN was dramatically halted by depletion of the ER Ca²⁺ store with CPA and pharmacological blockade of InsP₃Rs with 2-APB. Likewise, CPA dramatically affected nigericin-induced intracellular Ca²⁺ mobilization. These observations confirm that ER-embedded InsP₃Rs are required to globalize the increase in [Ca²⁺]_i evoked by lysosomal Ca²⁺ release also in mCRC cells. Notably, a recent mathematical model confirmed that local lysosomal Ca²⁺ leak may be amplified into a regenerative Ca²⁺ wave by juxtaposed ER-resident InsP₃Rs [45]. These data collectively demonstrate that a Ca²⁺-dependent functional cross-talk between acidic and ER Ca²⁺ stores also exist in cancer cells, such as mCRC cells. Future work will have to assess the occurrence of MCSs in mCRC cells at structural level.

Subsequently, we used a liposomal preparation to load mCRC cells with the membrane-impermeant NAADP and examine the following increase in [Ca²⁺]_i. NAADP induced a transient Ca²⁺ signal that was associated to endogenous Ca²⁺ release, but not to extracellular Ca²⁺ entry. To the best of our knowledge, this was the first demonstration that NAADP per se was able to increase the [Ca²⁺]_i in a cancer cell line other than cervical cancer HeLa cells [14]. The Ca²⁺ response to NAADP was impaired by disrupting acidic stores with GPN, by genetic silencing of TPC1, and by NED-19, a selective antagonist that prevents NAADP from binding to TPCs [54]. Moreover, our results confirmed previous results obtained in cancer cells by showing that TPC1 was ≈50-fold more abundant than TPC2 transcript. These data, therefore, hint at TPC1 as the molecular target recruited by NAADP to mobilize the acidic Ca²⁺ in mCRC cells and provide the first evidence that NAADP directly triggers intracellular Ca²⁺ signals in cancer cells. As expected, the Ca²⁺ response to NAADP was impaired by depleting the ER Ca²⁺ pool with CPA or by inhibiting InsP₃Rs with 2-APB. This observation is consistent with the functional coupling between the acidic and ER Ca²⁺ stores described above and demonstrates that InsP₃Rs sustain NAADP-evoked Ca²⁺ signals also in mCRC cells. Likewise, NAADP-induced Ca²⁺ signals are severely dampened by blocking InsP₃Rs and/or RyRs in a variety of normal cells, including primary human cytotoxic T lymphocytes [55], rat pulmonary arterial myocytes [56], HeLa cells [14], and mouse pancreatic acinar cells [44]. Our findings, therefore, extend to cancer cells the requirement of ER-dependent Ca²⁺ release for NAADP to trigger a global increase in [Ca²⁺]_i.

Intracellular Ca²⁺ signals support many cancer hallmarks, including aberrant proliferation, enhanced migration and metastasis, resistance to apoptosis and angiogenesis [5,57,58,59]. Although TPC1 is generally more abundant in cancer cell lines, as mentioned above, a clear role in tumorigenesis has been uncovered for TPC2 [18,35,36,37]. Recent investigations demonstrated that NAADP may deliver Ca²⁺ signals which stimulate cancer cells to grow by recruiting a number of Ca²⁺-dependent decoders, including ERK, Akt and c-Jun N-terminal kinase (JNK) [25]. It has long been known that FBS stimulates cancer cell proliferation and migration by inducing InsP₃-dependent Ca²⁺ release followed by store-operated Ca²⁺ entry (SOCE) [49,50,51]. However, genetic and pharmacological impairment of Stim1, Stim2, Orai1 and Orai3, which mediate SOCE in mCRC cells, do not affect their proliferation rate [39]. This finding strongly suggests that SOCE is not the Ca²⁺-permeable pathway that underlies the proliferation effect of FBS in mCRC cells. In the present investigation, we provided the evidence that NAADP-induced TPC1 activation triggers 20% FBS-induced intracellular Ca²⁺ release in mCRC cells. This conclusion is supported by the evidence that the intracellular Ca²⁺ response to 20% FBS is impaired (1) by disrupting acidic Ca²⁺ stores with GPN/nigericin and (2) by genetic (with a specific siRNA) and pharmacological (with NED-19) blockade of TPC1-mediated Ca²⁺ release. We, therefore, propose that NAADP triggers the Ca²⁺ response to FBS, which is then amplified by InsP₃-dependent ER Ca²⁺ release and sustained over time by SOCE activation. Unlike SOCE, however, TPC1 supports mCRC growth as the proliferation rate of mCRC cells is dramatically reduced upon genetic silencing of TPC1 or in the presence of NED-19 to prevent lysosomal Ca²⁺ release. We further showed that pharmacological and genetic blockade of TPC1 remarkably reduced ERK and Akt phosphorylation, which have long been known to support Ca²⁺-dependent proliferation in both normal and neoplastic cells [3,60,61]. The 5-year survival of CRC patients showing disease recurrence or being metastatic at diagnosis falls from 80–90% to 10–20% despite development of novel chemotherapeutics or the introduction of targeted therapies against VEGF and EGF signaling [62]. Therefore, TPC1 may be regarded as a novel promising target to develop alternative treatments for individual suffering from mCRC.

4. Materials and Methods

4.1. Expansion of Tumor Cells

After signing an informed consent, patients (>18 years) affected by mCRC who had undergone surgery intervention to remove primary tumor and/or liver metastases, were enrolled. All procedures were performed according to the guidelines prescribed for the treatment of CRC neoplasia, and no patient was subjected to unnecessary invasive procedures. The present study was approved by the Foundation IRCCS Policlinico San Matteo (Ethical code 20110000996, 17/01/2011). Tumor specimens were processed as previously described with the GentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) after being treated, with Tumor dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturers’ instructions [38,39]. Tumor cells were filtered to remove clusters, checked for viability with trypan blue die exclusion and resuspended at a concentration of 0.5–1 × 10⁶ cells/mL of CellGro SCGM (Cell Genix, Freiburg, Germany), supplemented with 20% FBS, 2 mM L-glutamine, (complete medium) (Life Technologies Inc, Carlsbad, CA, USA) and cultured in 25 cm² tissue flasks (Corning, Stone Staffordshire, England) at 37 °C and 5% CO₂. Viable tumor cells attached to the flask within 12–24 h. Cultures at 75% to 100% confluence were selected for subculture by trypsinization with 0.25% trypsin and 0.02% EDTA (Life Technologies Inc) in a calcium/magnesium-free balanced solution. The culture medium was changed twice a week and cellular homogeneity evaluated microscopically every 24–48 h. Cells were cryopreserved in 90% FBS and 10% dimethyl sulfoxide and stored in liquid nitrogen for further experiments. To confirm the neoplastic origin of cultured cells obtained after 3–5 passages underwent to morphological and immunocytochemical analysis [38,63]. To confirm the neoplastic origin of cultured cells, at least 3 cytospins were performed using 1 × 10⁵ cultured cells/cytospin obtained after 4–6 passages, for morphologic and immunocytochemical analysis. Cells were fixed in alcohol 95° and 1 slide was stained with hematoxylin-eosin to identify malignant cells on the basis of cytomorphology. To distinguish the tumor from hyperplastic mesothelial cells, the other slides were tested with monoclonal antibodies against cytokeratin CAM 5.2 (Dako, Glostruo, Denmark) using indirect immunoenzymatic staining according to the manufacturers’ instructions. Figure S4 reports two representative images with regard to immunohistochemistry performed to evaluate the neoplastic origin of mCRC cells.

For proliferation assays, tumor cells were thawed and plated at the concentration of 10–20 × 10⁵/mL and evaluated after 3–4 days when reached the optimal confluency, as described in [39]. Results are expressed as mean ± SE under each condition and the cells were obtained from all four patients. Differences were assessed by the Student t-test for unpaired values as related to controls. All statistical tests were carried out with GraphPad Prism 4 (San Diego, CA, USA).

4.2. Solutions for Intracellular Ca²⁺ Recordings

Physiological salt solution (PSS) had the following composition (in mM): 150 NaCl, 6 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 Glucose, 10 Hepes. In Ca²⁺-free solution (0Ca²⁺), Ca²⁺ was substituted with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of PSS as measured with an osmometer (Wescor 5500, Logan, UT, USA) was 338 mmol/kg.

4.3. Preparation of NAADP-Containing Liposomes

NAADP-containing liposomes were prepared from lecithin by a thin film hydration method, as recently shown in [48]. A thin film was formed by dissolving the lecithin in chloroform/methanol solution (2:1, v/v) in a round bottom flask and following removal of the solvent under vacuum condition at room temperature, which ensured complete removal of the solvents. The film was then hydrated with PBS buffer (10 mM, pH 7.4) to make a 20 mL of lipid coarse dispersion. Liposomes were prepared by adding cholesterol in a 89:20 lecithin:cholesterol molar ratio, codissolved in chloroform and then dried. The dried film from a flask was suspended in 4 mL of rehydration solution. The resulting liposomal dispersion was sonicated [64] for 3 min (Ultrasound Homogenizer-Biologics) and extruded 21 times with 100 nm filter. Finally, the mixture was dialyzed in PBS bulk for 24 h with 3 bulk-changes. Properties of liposomes were modulated by varying the rehydration solution composition. Liposomes were prepared from PBS solution containing only 70 g of NAADP. Moreover, liposomes free to NAADP were prepared directly from the PBS solution, this type of liposome referred as FL was used as reference. NAADP was diluted at 1:20, as shown elsewhere [48].

4.4. [Ca²⁺]_i Measurements

mCRC cells were loaded with 4 µM fura-2 acetoxymethyl ester (Fura-2/AM; 1 mM stock in dimethyl sulfoxide) in PSS for 30 min at 37 °C and 5% CO₂, as shown in [39]. After washing in PSS, the coverslip was fixed to the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany), usually equipped with a Zeiss ×40 Achroplan objective (water-immersion, 2.0 mm working distance, 0.9 numerical aperture). The cells were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. A first neutral density filter (1 or 0.3 optical density) reduced the overall intensity of the excitation light and a second neutral density filter (optical density = 0.3) was coupled to the 380 nm filter to approach the intensity of the 340 nm light. A round diaphragm was used to increase the contrast. The excitation filters were mounted on a filter wheel (Lambda 10, Sutter Instrument, Novato, CA, USA). Custom software, working in the LINUX environment, was used to drive the camera (Extended-ISIS Camera, Photonic Science, Millham, UK) and the filter wheel, and to measure and plot on-line the fluorescence from 10 up to 40 rectangular “regions of interest” (ROI). Each ROI was identified by a number. Since cell borders were not clearly identifiable, a ROI may not include the whole cell or may include part of an adjacent cell. Adjacent ROIs never superimposed. [Ca²⁺]_i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “ratio”). An increase in [Ca²⁺]_i causes an increase in the ratio [39]. Ratio measurements were performed and plotted on-line every 3 s. The experiments were performed at room temperature (22 °C).

4.5. RNA Isolation and Real Time RT-PCR (qRT-PCR)

Total RNA was extracted from mCRC cells using the QIAzol Lysis Reagent (QIAGEN, Milan, Italy). Single cDNA was synthesized from RNA (1 μg) using random hexamers and M-MLV Reverse Transcriptase (Promega, Milan, Italy). Reverse transcription was always performed in the presence or absence (negative control) of the reverse transcriptase enzyme. qRT-PCR was performed in triplicate using 1 μg cDNA and specific primers (intron-spanning primers) for TPCN1 and TPCN2, and for RyR1, RyR2 and RyR3 (Table S1). Briefly, GoTaq qPCR Mastermix (Promega, Milan, Italy) was used according to the manufacturer instruction and qRT-PCR performed using Rotor Gene 6000 (Corbett, Concorde, NSW, Australia). The conditions were as follows: initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 95 °C for 30 s; annealing at 58 °C for 30 s, and elongation at 72 °C for 40 s. The qRT-PCR reactions were normalized using β-actin (BAC) as housekeeping genes. The triplicate threshold cycles (Ct) values for each sample were averaged resulting in mean Ct values for both the gene of interest and the housekeeping genes. The gene Ct values were then normalized to the housekeeping gene by taking the difference: ΔCt = Ct_[gene] − Ct_{[housekeeping]}, with high ΔCt values reflecting low mRNA expression levels. Melting curves were generated to detect the melting temperatures of specific products immediately after the PCR run. However, PCR products were also separated with agarose gel electrophoresis, stained with ethidium bromide. The molecular weight of the PCR products was compared to the DNA molecular weight marker VIII (Roche Molecular Biochemicals, Monza, Italy).

4.6. Membrane Preparation and Immunoblotting

Cells were diluted in PBS and centrifuged 1000× g for 10 min. The cell pellets were resuspended with RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) containing 0.1 mg/mL PMSF, Protease Inhibitor Cocktails (P8340, Sigma-Aldrich Inc., Milan, Italy) and Phosphatase Inhibitor (1 mM Sodium Orthovanadate). The homogenates were solubilized in Laemmli buffer [65] and 30 µg proteins were separated on precast gel electrophoresis (4–20% Mini-PROTEAN TGX Stain-Free Gels, Bio-Rad, Milan, Italy) and transferred to the Hybond-P PVDF Membrane (GE Healthcare, Milan, Italy) by electroelution. After 1 h blocking with Tris buffered saline (TBS) containing 3% BSA and 0.1% Tween (blocking solution) the membranes were incubated overnight at 4 °C with the following antibodies: affinity purified rabbit anti-TPC1 (SAB2104213; Sigma-Aldrich In.), phospho-Akt (p-Akt; Cell Signaling Technology, 4060, Pero (Mi), Italy) or phospho-Erk (p-Erk; Cell Signaling Technology, 4377), diluted 1:1000 in 3% BSA in T-TBS. The membranes were washed and incubated for 1 h with peroxidase-conjugated goat anti-rabbit IgG (Chemicon, AP132P, Merck Millipore, Milan, Italy) or peroxidase-conjugated rabbit anti-mouse IgG (Dakocytomation, P0260, Agilent, Cernusco sul Naviglio (Mi), Italy), diluted 1:100,000 in blocking solution. The bands were detected with ECL™ Select western blotting detection system (GE Healthcare Europe GmbH, Italy). Prestained molecular weight markers (ab116028, Abcam-Prodotti Gianni, Milan, Italy) were used to estimate the molecular weight of the bands. Blots were stripped as shown in and re-probed with RabMAb anti β-2-microglobulin antibody ([EP2978Y] ab75853; ABCAM) as housekeeping. The antibody was diluted 1:10,000 in blocking solution.

4.7. Protein Content

Protein contents of all the samples were determined by the Bradford’s [66] method using bovine serum albumin (BSA) as standard.

4.8. Gene Silencing

siRNA targeting TPC1 was purchased by Sigma-Aldrich Inc. MISSION esiRNA (human TPCA1, EHU016301). Scrambled siRNA was used as negative control. Briefly, once the monolayer cells had reached 50% confluency, the medium was removed and the cells were added with Opti-MEM I reduced serum medium without antibiotics (Life Technologies, Milan, Italy). siRNAs (100 nM final concentration) were diluted in Opti-MEM I reduced serum medium and mixed with Lipofectamine™ RNAiMAX transfection reagent (Life Technologies, Milan, Italy) pre-diluted in Opti-MEM), according to the manufacturer’s instructions. After 20 min incubation at room temperature, the mixes were added to the cells and incubated at 37 °C for 5 h. Transfection mixes were then completely removed and fresh culture media was added. The effectiveness of silencing was determined by immunoblotting (see Figure S3) and silenced cells were used 48 h after transfection.

4.9. Lysotracker Red Staining

Lysosomal compartment was visualized in vital cells by incubating the cultures in the presence of LysoTraker Red, as described in [67]. Briefly, cells were incubated with Lysotracker Red cells were incubated with (50 nM) for 20 min at 37 °C. To visualize lysosomes, a BX51 Olympus microscope (Segrate (Mi), Italy) equipped with a 100 W mercury lamp was used with the following configuration: 540 nm (excitation filter), 580 nm (dichroic filter) and 590 nm (barrier filter).

4.10. Statistics

All the data have been collected from mCRC cells deriving from at least three coverslips from three independent experiments. The amplitude of intracellular Ca²⁺ release in response to each agonist (NAADP and FBS) or drug (GPN and nigericin) was measured as the difference between the ratio at the peak of intracellular Ca²⁺ mobilization and the mean ratio of 1 min baseline before the peak. Pooled data are given as mean ± SE and statistical significance (p < 0.05) was evaluated by the Student’s t-test for unpaired observations. Data relative to Ca²⁺ signals are presented as mean ± SE, while the number of cells analyzed is indicated in the corresponding bar histograms.

4.11. Chemicals

Fura-2/AM and Lysotracker Red were obtained from Molecular Probes (Molecular Probes Europe BV, Leiden, The Netherlands). All the chemicals were of analytical grade and obtained from Sigma Chemical Co. (St. Louis, MO, USA).

5. Conclusions

In conclusion, this study demonstrated for the first time that a functional lysosomal Ca²⁺ store is present and functionally coupled to ER-dependent Ca²⁺ release through InsP₃Rs in mCRC cells. NAADP stimulates TPC1 to mobilize lysosomal Ca²⁺ and mediate 20% FBS-induced mCRC proliferation. TPC1 stimulates proliferation by recruiting the Ca²⁺-dependent ERK and PI3K/Akt phoshorylation cascades. These data extend to cancer cells previous findings on normal cells and lend further support to the notion that NAADP-induced TPC activation could be targeted to eradicate secondary metastasis in cancer patients.