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.

Keywords: NAADP; TPC1; cancer; colorectal carcinoma; lysosomal Ca2+ signalling; proliferation.

Figure 1

A functional lysosomal Ca²⁺ store is present in metastatic colorectal cancer (mCRC) cells. (A) the lysosomotropic compound, glycyl-l-phenylalanine-2-naphthylamide (GPN) (200 µM), caused a robust increase in [Ca²⁺]_i in the absence of external Ca²⁺ (0Ca²⁺) in mCRC cells loaded with the Ca²⁺-sensitive fluorophore, Fura 2/AM (4 µM, 30 min). (B) nigericin (50 µM), which dissipates the ΔpH that drives Ca²⁺ refilling of acidic organelles, induced a transient elevation in [Ca²⁺]_i in mCRC cells. (C) mean ± SE of the percentage of responding cells to GPN and nigericin, respectively. (D) mean ± SE of the amplitude of the peak Ca²⁺ response to GPN and nigericin, respectively.

Figure 2

The endoplasmic reticulum (ER) Ca²⁺ store supports the Ca²⁺ response to GPN and nigericin. (A) depletion of the ER Ca²⁺ store with cyclopiazonic acid (CPA) (30 μM, 30 min) impaired GPN-induced Ca²⁺ signals in mCRC cells. GPN was administered at 200 µM. (B) CPA (30 μM, 30 min) also impaired the transient Ca²⁺ response to nigericin (50 μM). (C) mean ± SE of the percentage of responding cells under the designated treatments. The asterisk indicates p < 0.05. (D) mean ± SE of the amplitude of the Ca²⁺ response to GPN and nigericin under the designated treatments. The asterisk indicates p < 0.05.

Figure 3

InsP₃Rs support the Ca²⁺ response to GPN. (A) 2-Aminoethoxydiphenyl borate (2-APB) (50 μM, 30 min), a selective InsP₃R blocker under 0Ca²⁺ conditions, impaired the Ca²⁺ response to GPN (200 µM) in mCRC cells. (B) mean ± SE of the percentage of mCRC cells responding to GPN in the absence and in the presence of 2-APB. The asterisk indicates p < 0.05. (C) mean ± SE of the amplitude of the Ca²⁺ response to GPN in the absence and in the presence of 2-APB. The asterisk indicates p < 0.05.

Figure 4

NAADP induces lysosomal Ca²⁺ release in mCRC cells. (A) liposomal delivery of NAADP (1:20 dilution) caused a transient elevation in [Ca²⁺]_i in the absence of external Ca²⁺ (0Ca²⁺), while restoration of extracellular Ca²⁺ did not cause any additional Ca²⁺ signal. Control liposomes did not induce and detectable increase in [Ca²⁺]_i. (B) disrupting the lysosomal Ca²⁺ store with GPN (200 µM, 30 min) severely affected the Ca²⁺ response to NAADP (1:20 dilution). (C) mean ± SE of the percentage of responding cells in the absence and in the presence of GPN. The asterisk indicates p < 0.05. (D) mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP in the absence and in the presence of GPN. The asterisk indicates p < 0.05.

Figure 5

Two-pore channel 1 (TPC1) mediates NAADP-induced lysosomal Ca²⁺ release in mCRC cells. (A) qRT-PCR analysis of TPCs revealed that TPC1 transcripts are more abundant as compared to those encoding for TPC2. Data are expressed as Fold change (mean ± SE) of qRT-PCR runs performed in triplicate. The asterisk indicates p < 0.05. (B) the Ca²⁺ response to liposomal delivery of NAADP (1:20) NAADP was inhibited by NED-19 (100 μM, 30 min), a selective TPC inhibitor, and by the genetic silencing of TPC1 by a specific siRNA. (C) mean ± SE of the percentage of responding cells under the designated treatments. The asterisk indicates p < 0.05. (D) mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP under the designated treatments. The asterisk indicates p < 0.05.

Figure 6

InsP₃Rs support the Ca²⁺ response to NAADP. (A) the intracellular Ca²⁺ response to liposomal delivery of NAADP (1:20) was dramatically reduced by pharmacological depletion of the ER Ca²⁺ pool with CPA (30 μM, 30 min) and by pharmacological blockade of InsP₃Rs with 2-APB (50 µM, 30 min). (B) mean ± SE of the percentage of responding cells under the designated treatments. The asterisk indicates p < 0.05. (C) mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP under the designated treatments. The asterisk indicates p < 0.05.

Figure 7

Fetal bovine serum (FBS)-induced intracellular Ca²⁺ release is impaired by disruption of acidic stores in mCRC cells. (A) intracellular Ca²⁺ signals induced by FBS 20% were dramatically reduced upon depletion of the lysosomal Ca²⁺ pool with either GPN (200 μM, 30 min) or nigericin (50 μM, 30 min). (B) mean ± SE of the percentage of responding cells under the designated treatments. The asterisk indicates p < 0.05. (C) mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP under the designated treatments. The asterisk indicates p < 0.05.

Figure 8

TPC1 mediates FBS-induced lysosomal Ca²⁺ release and proliferation in mCRC cells. (A) 20% FBS induced an intracellular Ca²⁺ transient that was significantly reduced by NED-19 (100 μM, 30 min) and by deleting TPC1 with the specific siTPC1. (B) mean ± SE of the percentage of responding cells under the designated treatments. The asterisk indicates p < 0.05. (C) mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP under the designated treatments. The asterisk indicates p < 0.05. (D) mean ± SE of the percentage of 20% FBS-induced cell proliferation under control conditions and upon pharmacological (NED-19) and genetic (siTPC1) blockade of NAADP-induced Ca²⁺ release. The asterisk indicates p < 0.05.

Figure 9

TPC1 stimulates extracellular-signal related kinases (ERK) phosphorylation in mCRC cells. p-ERK and ERK (Aa) bands in mCRC silenced for TPC1 or treated with NED-19 (100 μM, 30 min). Blots representative of four were shown. Lanes were loaded with 30 μg of proteins, probed with affinity purified antibodies and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-β-2-microglobulin (β2M) antibody, as housekeeping. Major bands of the expected molecular weights were shown (A). Bands were acquired, the densitometric analysis of the bands was performed by Total Lab V 1.11 computer program (Amersham Biosciences Europe, Italy) and the results were normalized to non-phosphorylated ERK (B). * p < 0.05 versus the respective control (Student’s t test). The asterisk indicates p < 0.05.

Figure 10

TPC1 stimulates Akt phosphorylation in mCRC cells. p-Akt and Akt (A) (Ba) bands in mCRC silenced for TPC1 or treated with NED-19 (100 μM, 30 min). Blots representative of four were shown. Lanes were loaded with 30 μg of proteins, probed with affinity purified antibodies and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-β2M antibody, as housekeeping. Major bands of the expected molecular weights were shown. Bands were acquired, the densitometric analysis of the bands was performed by Total Lab V 1.11 computer program (Amersham Biosciences Europe, Italy) and the results were normalized to non-phosphorylated Akt (B). * p < 0.05 versus the respective control (Student’s t test). The asterisk indicates p < 0.05.