Green tea polyphenols-induced apoptosis in human osteosarcoma SAOS-2 cells involves a caspase-dependent mechanism with downregulation of nuclear factor-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.
References (45)
- et al.
Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells vs. normal cells
Arch. Biochem. Biophys.
(2000) - et al.
Malignant reversion of a human osteosarcoma cell line, Saos-2, by inhibition of NFkappaB
Biochem. Biophys. Res. Commun.
(2002) - et al.
Green tea polyphenol (−)-epigallocatechin 3-gallate inhibits MMP-2 secretion and MT1-MMP-driven migration in glioblastoma cells
Biochim. Biophys. Acta
(2002) - et al.
Involvement of NF-kappaB signalling in skin physiology and disease
Cell. Signal.
(2003) - et al.
Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts
Cancer Lett.
(1998) - et al.
A review of the health effects of green tea catechins in in vivo animal models
J. Nutr.
(2004) NF-kappa B: arresting a major culprit in cancer
Drug Discov. Today
(2002)- et al.
Involvement of caspase-3 in epigallocatechin-3-gallate-mediated apoptosis of human chondrosarcoma cells
Biochem. Biophys. Res. Commun.
(2000) - et al.
Growth factor-dependent induction of p21(CIP1) by the green tea polyphenol, epigallocatechin gallate
Cancer Lett.
(2000) - et al.
Nuclear factor-kappa B is upregulated in colorectal cancer
Surgery
(2001)
Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol
Mutat. Res.
Regulation of the transcriptional activity of the nuclear factor-kappaB p65 subunit
Biochem. Pharmacol.
Oral consumption of green tea polyphenols inhibits insulin-like growth factor-I-induced signaling in an autochthonous mouse model of prostate cancer
Cancer Res.
Role of NF-kappaB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death
Oncogene
Growth inhibitory and antimetastatic effect of green tea polyphenols on metastasis-specific mouse mammary carcinoma 4T1 cells in vitro and in vivo systems
Clin. Cancer Res.
The role of apoptosis in cancer development and treatment response
Nat. Rev., Cancer
Medicinal benefits of green tea: Part I. Review of noncancer health benefits
J. Altern. Complement. Med.
Medicinal benefits of green tea: Part II. Review of anticancer properties
J. Altern. Complement. Med.
Nuclear transcription factor-kappaB as a target for cancer drug development
Leukemia
The Re1/NF-kappa B/I kappa B signal transduction pathway and cancer
Cancer Treat Res.
Essential role of caspases in epigallocatechin-3-gallate-mediated inhibition of nuclear factor kappa B and induction of apoptosis
Oncogene
Overview of sarcomas in the adolescent and young adult population
J. Pediatr. Hematol. Oncol.
Cited by (50)
Pharmacological evaluation of phytochemicals from South Indian Black Turmeric (Curcuma caesia Roxb.) to target cancer apoptosis
2017, Journal of EthnopharmacologyCitation Excerpt :Ahmed et al. (2015) has proved that petroleum ether fraction of the oleo-gum resin extract of Boswellia serrata has IC50 value (1.58 μg/mL at 48 h.) in HepG2 cell line compared to the more efficacious HRE. Induction of apoptosis is always associated with the activation of caspases (Nhan et al., 2006) that have been shown to be involved via several pathways (Hafeez et al., 2006) including activation of downstream effector molecules such as PARP (Zimmermann et al., 2001). It is known that the anti-apoptotic proteins Bcl-2 and Bcl-xL prohibit Cytochrome C release whereas, the pro-apoptotic Bcl-2 proteins Bad, Bid, Bax, and Bim enhance its release.
The role of polyphenols on bone metabolism in osteoporosis
2015, Food Research InternationalTea and bone health: Steps forward in translational nutrition<sup>1-5</sup>
2013, American Journal of Clinical NutritionGreen Tea and other Fruit Polyphenols Attenuate Deterioration of Bone Microarchitecture
2013, Polyphenols in Human Health and DiseaseMinnelide reduces tumor burden in preclinical models of osteosarcoma
2013, Cancer LettersCitation Excerpt :A previous study has shown that NF-κB levels were affected by triptolide in multiple myeloma cells [35]. Further, inhibitors of NF-κB pathway used on different OS cell lines have also shown significant cytotoxicity [36–38] in other studies. In osteosarcoma cells, NF-κB regulated pathways have often been reported as a potential drug target [37,39–41].
Catechin-rich oil palm leaf extract enhances bone calcium content of estrogen-deficient rats
2013, NutritionCitation Excerpt :The GT (−)-catechin repressed bone resorption. EGCG is among the most active catechins and significantly inhibited the survival of differentiated osteoclasts and increased osteoclast apoptosis [25,26]. EGCG induced osteoclast cell death by single-strand DNA damage, without affecting the osteoblasts [26], and decreased the differentiation and formation of osteoclasts [26,27].