Green tea polyphenols-induced apoptosis in human osteosarcoma SAOS-2 cells involves a caspase-dependent mechanism with downregulation of nuclear factor-κB

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Abstract

Development of chemotherapy resistance and evasion from apoptosis in osteosarcoma, a primary malignant bone tumor, is often correlated with constitutive nuclear factor-κB (NF-κB) activation. Here, we investigated the ability of a polyphenolic fraction of green tea (GTP) that has been shown to have antitumor effects on various malignant cell lines to inhibit growth and induce apoptosis in human osteosarcoma SAOS-2 cells. Treatment of SAOS-2 cells with GTP (20–60 μg/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-κB/p65 and lowering of NF-κB/p65 and p50 levels in the cytoplasm and nucleus. GTP treatment of cells reduced IκB-α phosphorylation but had no effect on its protein expression. Furthermore, GTP treatment resulted in the inhibition of IKK-α and IKK-β, the upstream kinases that phosphorylate IκB-α. The increase in apoptosis in SAOS-2 cells was accompanied with decrease in the protein expression of Bcl-2 and concomitant increase in the levels of Bax. GTP treatment of SAOS-2 cells also resulted in significant activation of caspases as was evident by increased levels of cleaved caspase-3 and caspase-8 in these cells. Treatment of SAOS-2 cells with a specific caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) rescued SAOS-2 cells from GTP-induced apoptosis. Taken together, these results indicate that GTP is a candidate therapeutic for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-κB.

Introduction

Osteosarcoma, a primary malignant bone tumor, is most common in children and adolescents, accounting for 0.3% of pediatric-cancer-related deaths in the United States (Herzog, 2005). Osteosarcoma is thought to be derived from osteoblasts which secrete bone matrix. Despite aggressive multimodal therapy, this devastating tumor often acquires drug resistance and metastasizes (Marina et al., 2004). Therefore, our goal is to identify agents that could promote apoptosis of osteosarcoma cells which do not exert any toxic effects on normal cells.

Green tea polyphenols (GTP) has received much attention over the last few years as cancer chemopreventive and chemotherapeutic agent (Adhami et al., 2004, Shimizu et al., 2005). These dietary polyphenols have shown to possess antitumor effects in several malignant cell lines including breast, skin, liver, pancreas, lung, prostate and bladder (Adhami et al., 2004, Shimizu et al., 2005, Cooper et al., 2005a). Furthermore, it has been shown that GTP possesses anti-inflammatory, anti-oxidant, anti-clastogenic and anti-mutagenic activities in a variety of malignant cells and preclinical models of cancer (Cooper et al., 2005b, Crespy and Williamson, 2004). Green tea polyphenols have shown differential effects in inhibiting cell growth, causing cell cycle arrest and inducing apoptosis in cancer cells without affecting normal cells (Ahmad et al., 2000, Chen et al., 1998). GTP-mediated effects include inhibition of various kinases viz. MAPK, protein kinase B/AKT, (Siddiqui et al., 2004); loss of mitochondrial transmembrane potential (Nakazato et al., 2005); modulation of Bax and Bcl-2 family members (Baliga et al., 2005), induction of p21/WAF1 and p27/SDI1, inhibition of cyclin D1-associated pRB (Liberto and Cobrinik, 2000), activation of caspases (Qanungo et al., 2005) and inhibition of various growth factors including IGF-1, VEGF and FGF; and inhibition of serine proteases and matrix metalloproteinases (Annabi et al., 2002), critical for cancer progression.

The members of Rel/nuclear factor (NF)-κB family form hetero- and homodimers and control the expression of a number of genes that regulate cell survival, proliferation, immune response and apoptosis (Gilmore, 2003). In unstimulated cells, NF-κB is sequestered in the cytoplasm as heterodimers, composed of p50/c-Rel and p50/p65 subunits, bound by IκB-α, thus preventing their translocation into nucleus. In response to various stimuli, IκB-α subunit is phosphorylated by an upstream kinase, IKK-α, at serine residues 32 and 36, triggering ubiquitination and degradation of IκB-α by the 26S proteosome. This signal facilitates the release and translocation of the NF-κB heterodimer into the nucleus, where it binds with specific DNA motifs in the promoter regions of target genes and activates their transcription (Vermeulen et al., 2002, Pahl, 1999). The diverse signals (several hundred described so far) that can trigger the NF-κB activation highlight its pivotal role in several biological processes including neoplastic progression and possess high activity in transformed and malignant cells (Liu et al., 2001). NF-κB has been shown to be constitutively activated in most types of human cancer including breast, colon, skin, lung, esophagus, pancreas, prostate and gliomas and plays a critical role in the regulation of cell survival, proliferation and apoptosis (Sovak et al., 1997, Lind et al., 2001, Bell et al., 2003, Mukhopadhyay et al., 1995, Wang et al., 1999, Nair et al., 2003, Tselepis et al., 2002, Suh et al., 2002). In recent years, NF-κB has emerged as a major therapeutic target in cancer because of its ability to cause chemotherapy resistance and evasion from apoptosis (Yamamoto and Gaynor, 2001, Garg and Aggarwal, 2002). Studies have shown that the inhibition of constitutively active NF-κB leads to reversion of malignancy in human osteosarcoma cells (Andela et al., 2002). Therefore, sustained inhibition of NF-κB may be a rational strategy for effective management of this disease. Non-toxic agents that have the ability to inhibit NF-κB activity may be ideal candidates as therapeutics for osteosarcoma. Since human osteosarcoma SAOS-2 cells possess high constitutive levels of activated NF-κB and are resistant to chemotherapy and apoptosis, we investigated whether GTP has the potential to induce apoptosis along with its mechanism of action. Our results demonstrate that GTP is a candidate therapeutic agent for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-κB.

Section snippets

Cell lines and reagents

Human osteosarcoma SAOS-2 cells were a kind gift of Dr. Brian Johnstone, Department of Orthopedics, Case Western Reserve University. Green tea extract was obtained from Mitsui Norin Co. (Polyphenon-E®, Tokyo, Japan) and is subsequently referred to as GTP. Polyphenon-E contains epicatachin-3-gallate (EGC) 6.4%, epicatechin (EC) 10.7%, epigallocatechin-3-gallate (EGCG) 63%, gallocatechin-3-gallate (GCG) 2.0%, epicatechin-3-gallate (ECG) 6.1%, catechin-3-gallate (CG) 0.1%, gallocatechin-3-gallate

GTP treatment reduces the viability of human osteosarcoma SAOS-2 cells

To ascertain the effect of GTP on the viability of SAOS-2 cells, we performed MTT assay. As shown in Fig. 1, treatment of cells with 10–80 μg/ml of GTP resulted in dose- and time-dependent inhibition of cell growth compared to control group. The cell growth inhibitory effect was more pronounced at 48 h post-GTP treatment as compared to 16 and 24 h. Compared to controls, significant inhibition in cell growth was observed at the GTP doses of 60 and 80 μg/ml for 16 and 24 h (P < 0.001). In

Discussion

In this study, we have demonstrated the anti-proliferative effects of GTP against human osteosarcoma SAOS-2 cells. Recent studies indicate that green tea polyphenols exert inhibitory effects on the activity of several enzymatic and metabolic pathways of relevance to the development and progression of cancer (Cooper et al., 2005b, Crespy and Williamson, 2004, Ahmad et al., 2000, Chen et al., 1998, Siddiqui et al., 2004, Nakazato et al., 2005, Baliga et al., 2005, Liberto and Cobrinik, 2000,

Acknowledgments

This work was supported in part by USPHS/NIH/NCCAM grant R21-AT02258 and USPHS/NIH/NIAMS grant RO1-AR48782.

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