Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Anti-Lung Cancer Activity of the Curcumin Analog JZ534 In Vitro.

This study investigated the anticancer effect of the curcumin analog JZ534 on lung cancer cell lines H460, A549, H1975, and HCC827. The antiproliferation effect of JZ534 was measured through the methylthiazoletetrazolium assay, and cell colony formation was observed. Cell cycle and apoptosis were determined by flow cytometry, and the preliminary mechanism was studied by Western blot. Results showed that JZ534 significantly inhibited the vitality and colony formation of lung cancer cells. JZ534 induced the G2/M cell cycle arrest of the cancer cells and suppressed the expression of cycle-related proteins, including cyclin B1 and Cdc2. Meanwhile, JZ534 induced cell apoptosis and upregulated the expression of apoptosis-related proteins, including cleaved caspase-3, Bax, and p53. At the same dose, JZ534 showed better antitumor activity than curcumin. These results suggest that JZ534 exhibits excellent anti-lung cancer activity by inhibiting the growth and inducing the apoptosis of lung cancer cells. Therefore, JZ534 is a promising lead compound for cancer treatment.