Mechanisms of Radiation Injury and Cytoprotection in Osteoblasts
Artur Gevorgyan, MD, MSc, Balram Sukhu, PhD, Benjamin A. Alman, MD, Robert G. Bristow, MD, PhD, Cho Pang, PhD, Christopher Forrest, MD, MSc
INTRODUCTION: Inhibition of craniofacial bone growth is a common side effect of therapeutic radiation in children. The infant rabbit orbito-zygomatic complex (OZC) was established as an animal model of this condition (1). Importantly, administration of the radioprotector Amifostine (WR-2721, Ethyol^®) resulted in a significant mitigation of craniofacial bone growth inhibition (2). However, the mechanisms of radiation effects and cytoprotection have remained unknown. The purpose of this work was to investigate the mechanisms of Amifostine radioprotection in vitro.
METHODS: A primary periosteal osteoblast-like cell culture was established from the infant rabbit OZC as an ex vivo model (3). The dose-dependent effects of radiation (0; 10; 15 Gy) were measured in OZC periosteum-derived cell cultures via cell numbers, viability, alkaline phosphatase activity, collagen type I expression and mineralization. Subsequently, animals received either radiation; pre-treatment with Amifostine (200mg/kg, intravenously, 20 minutes before radiation) with radiation; or no treatment. Cell cultures were developed and tested for cell numbers, viability and alkaline phosphatase activity.
In the second study, MC3T3-E1 newborn mouse calvarial osteoblasts underwent γ-radiation (0-10 Gy) in the presence or absence of either WR-2721 (Amifostine), or WR-1065, its active metabolite (10^-7-10^-3 M). The effects of radiation with and without radioprotectors were assessed using endpoints of colony forming ability, cell viability, alkaline phosphatase activity, and expression of osteoblastic phenotype genes (alkaline phosphatase, collagen type I, osteocalcin, osteopontin).
RESULTS: In the first study, radiation resulted in a significant (p<0.05) and dose-dependent inhibition of cell numbers and viability, and no statistically significant changes in alkaline phosphatase activity (Fig. 1) (4). Collagen type I expression and mineralization were significantly reduced at 15 Gy dose. Pre-treatment with Amifostine significantly (p<0.05) enhanced the number of surviving cells.
In the second study, radiation resulted in a dose-dependent inhibition of clonogenic survival (Fig. 2) (5). Pre-treatment with WR-1065, but not WR-2721, resulted in a significant improvement of osteoblast survival. Specifically, maximum radioprotection was observed with 10^-4 M WR-1065 at a clinically relevant 2 Gy dose of radiation. Furthermore, radiation suppressed the expression of genes of osteoblastic phenotype.
CONCLUSIONS: Amifostine and its active metabolite WR-1065 afford significant radioprotection in both rabbit OZC periosteum-derived osteoblast-like cells and mouse calvarial osteoblasts.
Extrapolating these data to a clinical model, a more than 3200-fold improved survival can be achieved in radioprotected cells over the entire course of radiotherapy. This is important in devising pharmacological strategies for preventing impaired craniofacial bone growth in the survivors of pediatric head and neck cancer.