Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells

Ta-Wei Tai,Yuan-Kun Tu,I.-Ming Jou,Fong-Chin Su,Tsung-Ting Tsai,Ching-Yu Chen,Chiou-Feng Lin

doi:10.1038/srep44245

Abstract

Inhibiting osteoclasts and osteoclast precursors to reduce bone resorption is an important strategy to treat osteoclast-related diseases, such as osteoporosis, inflammatory bone loss, and malignant bone metastasis. However, the mechanism by which apoptosis is induced in the osteoclasts and their precursors are not completely understood. Here, we used nitrogen-containing bisphosphonate zoledronic acid (ZA) to induce cell apoptosis in human and murine osteoclast precursors and mature osteoclast-like cells. Caspase-3-mediated cell apoptosis occurred following the ZA (100 μM) treatment. Reactive oxygen species (ROS) were also generated in a time-dependent manner. Following knock-down of the p47phox expression, which is required for ROS activation, or co-treatment with the ROS inhibitor, N-acetyl-L-cysteine, ZA-induced apoptosis was significantly suppressed in both osteoclast precursors and mature osteoclast-like cells. The ROS-activated mitogen-activated protein kinases pathways did not trigger cell apoptosis. However, a ROS-regulated Mcl-1 decrease simultaneously with glycogen synthase kinase (GSK)-3β promoted cell apoptosis. These findings show that ZA induces apoptosis in osteoclast precursors and mature osteoclast-like cells by triggering ROS- and GSK-3β-mediated Mcl-1 down-regulation.

Highlights

Tartrate-resistant acid phosphatase (TRAP), and transferase dUTP nick end labeling (TUNEL) staining followed by fluorescent and light microscopic observation were used to detect the formation of osteoclast-like cells and cell apoptosis
The results showed that zoledronic acid (ZA) treatment induced apoptosis in mouse macrophage cell line RAW264.7 and human monocytic cell line THP-1, in a time-dependent manner (Fig. 1A, top and middle)
Further terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining revealed ZA-induced DNA fragmentation, a marker of cell apoptosis, in the RAW264.7-derived osteoclast-like cells (Fig. 1B, bottom). These results suggest that ZA induces apoptosis in monocytes, macrophages, and differentiated osteoclast-like cells

Summary

Introduction

TRAP, and TUNEL staining followed by fluorescent and light microscopic observation were used to detect the formation of osteoclast-like cells and cell apoptosis. ZA inhibits the farnesyl diphosphate-mediated mevalonate pathway, thereby inhibiting osteoclast proliferation and inducing apoptotic cell death in osteoclasts[4,9]. Previous studies have shown that the use of ZA may significantly enhance apoptosis by elevating reactive oxygen species (ROS) levels in prostate carcinoma, multiple myeloma, and salivary adenoid cystic carcinoma cell models[10,11]. Beyond a certain threshold, chronic exposure of osteoclasts to elevated oxidative stress results in cytotoxic effects due to the increased oxidative damage of DNA, proteins, and lipids, which can lead to apoptosis via the caspase-dependent pathway[23]. We hypothesized that ROS could promote apoptosis of osteoclast precursors and osteoclasts via intracellular signal pathways. The purpose of this study was, to investigate the ROS-mediated intracellular signal pathways in ZA-treated osteoclast precursors

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Conclusion