Faris Pawan, Pellavio Giorgia, Ferulli Federica, Di Nezza Francesca, Shekha Mudhir, Lim Dmitry, Maestri Marcello, Guerra Germano, Ambrosone Luigi, Pedrazzoli Paolo, Laforenza Umberto, Montagna Daniela, Moccia Francesco
Laboratory of General Physiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, 27100 Pavia, Italy.
Research Centre, Salahaddin University-Erbil, 44001 Erbil, Kurdistan-Region of Iraq, Iraq.
Cancers (Basel). 2019 Apr 15;11(4):542. doi: 10.3390/cancers11040542.
Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca release. NAADP-induced EL Ca signals may be amplified by the endoplasmic reticulum (ER) through the Ca-induced Ca release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca refilling of acidic organelles, caused massive Ca release in the presence of a functional inositol-1,4,5-trisphosphate (InsP₃)-sensitive ER Ca store. Liposomal delivery of NAADP induced a transient Ca release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca 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 Ca 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.
烟酰胺腺嘌呤二核苷酸磷酸（NAADP）开启双孔通道1和2（TPC1和TPC2）以引发内溶酶体（EL）钙释放。NAADP诱导的EL钙信号可能通过内质网（ER）经钙诱导钙释放机制（CICR）得以放大。在此，我们旨在通过利用钙成像和分子生物学技术，首次评估EL钙信号在人转移性结直肠癌（mCRC）原代培养物中的作用。溶酶体促渗剂甘氨酰 - 苯丙氨酸β - 萘酰胺（GPN）以及尼日利亚菌素（可消除驱动酸性细胞器钙再填充的ΔpH）在存在功能性肌醇 - 1,4,5 - 三磷酸（InsP₃）敏感的内质网钙库的情况下，会引起大量钙释放。脂质体递送NAADP会诱导短暂的钙释放，而GPN和选择性TPC拮抗剂NED - 19可使其减少。药理学和基因操作表明，对NAADP的钙反应由TPC1触发，TPC1是mCRC细胞中表达最多的TPC亚型，且需要内质网嵌入的InsP₃受体。最后，NED - 19和TPC1的基因沉默降低了胎牛血清诱导的mCRC细胞中的钙信号、增殖以及细胞外信号调节激酶和Akt磷酸化。这些数据表明，NAADP门控的TPC1可被视为治疗mCRC替代疗法的新靶点。
