Alteration of cancer pain-related signals by radiation: Proteomic analysis in an animal model with cancer bone invasion

Abstract

PURPOSE: Although radiotherapy is highly effective in relieving bone pain due to cancer invasion, its mechanism remains unclear. The aim of this study was to explore this mechanism in an animal model system. METHODS AND MATERIALS: A hind paw model of cancer pain was developed by transplanting a murine hepatocarcinoma, HCa-1, into the periosteal membrane of the foot dorsum of C3H/HeJ mice. Bone invasion from HCa-1 was histopathologically confirmed from sequential tumor sampling. For three experimental groups, a control (N), tumor without radiation (T), and tumor with radiation (TR), the development and level of pain were objectively examined in mice with a growing tumor by assessing pain-associated behavior. The differential expression of pain-related signals in the spinal cord was analyzed by proteomic analysis using high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry, and those of proteins by Western blotting. The pain-mediating neurotransmitters in the spinal cord were also examined by immunohistochemical staining for calcitonin gene-related peptide (CGRP) and substance P. RESULTS: In the histopathologic examinations, bone invasion from HCa-1 was seen from Day 7 and was evident at Day 14 after transplantation, and measurable pain-associated behaviors were developed from Day 7. After 25 Gy of radiation to the tumors, the objective level of pain in the TR group decreased, with higher thresholds to mechanical and thermal stimulation than in the T group. From the 2-DE of spinal cord, 107 spots were identified; 12 proteins were changed more than fivefold because of tumor formation but then reversed after radiation in the tumor-bearing mice. The proteins involved included secretagogin, syntenin, P2X purinoreceptor 6 (P2X6), and Ca(2+)/Calmodulin-dependent protein kinase 1 (CaM kinase 1), the functions of which have been known to be involved in the Ca(2+)-signaling cascade, ATP-mediated fast synaptic transmission, or control of vesicular trafficking. Validations using Western blotting were successful for the CaM kinase and P2X6. In immunohistochemical staining of the spinal cord, a significant decrease after irradiation was shown in the expression of CGRP, but not in substance P. CONCLUSIONS: We developed a novel model for bone pain due to cancer invasion, which was confirmed by histopathologic examination and measurable pain-associated behaviors. Radiotherapy decreased the objective level of pain. The underlying mechanism seems to be related to the Ca(2+)-signaling cascade or control of vesicular trafficking.