Cancer Letters

Cancer Letters

Volume 271, Issue 1, 18 November 2008, Pages 117-128
Cancer Letters

The novel isoflavone 7-hydroxy-3′,4′-benzoisoflavone induces cell apoptosis in human osteosarcoma cells

https://doi.org/10.1016/j.canlet.2008.05.037Get rights and content

Abstract

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system and is characterized by an extremely aggressive clinical course that lacks an effective treatment. This study is the first to investigate the anti-cancer effects of a new isoflavone-derived 7-hydroxy-3′,4′-benzoisoflavone (HBI) in human osteosarcoma cells. HBI-induced cell apoptosis in human osteosarcoma cell lines. The accumulation of reactive oxygen species (ROS) is a critical mediator in HBI induced cell death. HBI also induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation, p38, JNK and p53 phosphorylation. Transfection with ASK1, p38 and JNK small interfering RNA (siRNA) antagonized HBI-induced cell apoptosis. HBI also triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl2 ratio. Bax knockdown using a Bax siRNA strategy reduced Bax expression and subsequent cell death. In addition, ASK1, p38 and JNK siRNA reduced HBI-induced p53 phosphorylation and Bax expression. These results suggest that the ROS-ASK1-p38/JNK-p53 and Bax pathway plays a critical role in HBI’s anti-cancer effects.

Introduction

Osteosarcoma is one of the most common malignant bone cancers, especially in children. These tumors are characterized by a highly malignant and metastatic potential [1]. Although chemotherapeutic strategies have improved, the survival rate of patients who relapse on therapy has not improved during the last decade. Aberrant regulation of cell growth and deregulation of apoptosis occur commonly in cancer cells and may play an essential role in tumor development. Accordingly, current anti-cancer agents act by inhibiting cell growth or inducing cell apoptosis [1].

Reactive oxygen species (ROS) are derived from the metabolism of molecular oxygen. Aerobic respiration coupled to the generation of ATP leads to the formation of the superoxide anion radical (O2). Superoxide anion radicals can then form other ROS, such as hydrogen peroxide (H2O2) and highly reactive hydroxyl radicals [2], [3]. Oxidative stress occurs when this critical balance is disrupted because of excess ROS, antioxidant depletion, or both. Accumulating evidence indicates that chemotherapeutic agents may be selectively toxic to tumor cells because they increase oxidant stress and enhance already stressed cells beyond their limit [4], [5]. It has been reported that ROS is involved in activation of the apoptosis signal-regulating kinase 1 (ASK1)/mitogen activated protein kinase (MAPK) signaling pathway [6], [7], [8]. The upstream activator of MAPK signaling is ASK1, a member of the MAPK kinase kinase (MAPKKK) family [8], [9]. ASK1 is activated in response to various stresses, including tumor necrosis factor, serum withdrawal, endoplasmic reticulum stress, Fas ligation and H2O2 [10], [11], [12], [13]. ASK1 activity is regulated at various points, including dimerization, phosphorylation, and protein–protein interactions [13], [14]. Phosphorylation of the ASK1 Ser967 residue is required for formation of the ASK-14-3-3 complex and keep ASK1 inactive [15], [16], [17]. ROS has been reported to trigger the dissociation of 14-3-3 from ASK1 [18], [19]. ASK1 can induce cell death by activating several proapoptotic signaling proteins, including c-jun-NH2-kinase (JNK) and p38 MAPK [20], [21].

Phyto-estrogens have been suggested as having a preventive effect for various cancers. Natural isoflavone phyto-estrogen has been reported to exhibit multiple functions resulting in anti-tumor effects [22]. The main phyto-estrogens derived from the diet are genistein, daidzein, and glycitein which are isoflavones found in soybeans [23]. Although the effects of isoflavone-induced tumor apoptosis have been studied in some cancers [24], the role of isoflavone in the process of cell apoptosis in osteosarcoma remains large unknown. Here, we synthesized the new isoflavone derived 7-hydroxy-3′,4′-benzoisoflavone (HBI) (Fig. 1A) and investigated the anti-cancer activity in human osteosarcoma cells. Our data provide evidences that HBI induced apoptosis of osteosarcoma cells (U2OS) via a mechanism involving the generation of ROS and the activation of ASK1-p38/JNK-p53-Bax pathway.

Section snippets

Materials

7-hydroxy-3′,4′-benzoisoflavone [HBI; chemical name: 7-hydroxy-3-(naphthalen-2-yl)-4H-chromen-4-one; C19H12O3] was synthesized at the Graduate Institute of Pharmaceutical Chemistry, School of Pharmacy, China Medical University (Taichung, Taiwan). Protein A/G beads, anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for cytochrome c, Bax, Bak, Bcl-2, Bcl-XL, ASK1, 14-3-3, p38, JNK, α-tubulin and the small interfering RNAs (siRNAs) against

HBI induced cell apoptosis in human osteosarcoma cells

To investigate the potential cell death of HBI in human osteosarcoma cells, we first examined the effect of HBI on cell survival in human osteosarcoma cells (U2OS). Treatment of U2OS with HBI-induced cell death in a concentration-dependent manner by using MTT assay (Fig. 1B). The IC50 value of HBI was 17.3 μM for U2OS cells. Next we investigated the anti-cancer effect of HBI in other osteosarcoma cell lines. Table 1 also shows that HBI-induced cell death in other osteosarcoma cell lines (MG-63,

Discussion

Osteosarcoma is the most common primary malignant tumor of bone, especially in children. The principles of treatment of osteosarcoma have undergone dramatic changes in the past 20 years. Until recently, a 5-year survival rate of 20% with surgical treatment alone was considered acceptable. This outcome suggested that 80% of patients had pulmonary metastasis at time of presentation. [1]. The development of novel therapeutic agents targeting the malignant behavior of osteosarcoma cells, is

Conflict of Interest

The authors have no conflict of interest.

Role of the Funding Source

This work was supported by Grants from the National Taiwan University Hospital, Yun-Lin Branch (NTUHYL97.S0012; NTUHYL97.S009 and NTUHYL 97.N004).

Acknowledgments

This work was supported by Grants from the National Taiwan University Hospital, Yun-Lin Branch (NTUHYL97.S0012; NTUHYL97.S009 and NTUHYL 97.N004).

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