Flavonoids purified from Rhus verniciflua Stokes actively inhibit cell growth and induce apoptosis in human osteosarcoma cells

https://doi.org/10.1016/j.bbagen.2005.08.010Get rights and content

Abstract

Many studies have suggested that dietary flavonoids are anticancer agents that induce the apoptosis of cancer cells. However, the effects of flavonoids on the induction of apoptosis in osteosarcoma cells are unclear. Previously, a flavonoid fraction, consisting mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein, herein named RCMF (the RVS chloroform-methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS). This study evaluated the effects of RCMF on the proliferation and apoptosis using human osteosarcoma (HOS) cells. The mechanism of growth inhibition of the HOS cells by the flavonoid fraction, RCMF, was also assessed. The results demonstrated that RCMF exhibited sensitive growth inhibition and induced apoptosis in HOS cells. PARP cleavage was closely associated with the RCMF-induced apoptosis of the HOS cells. Furthermore, the activation of caspase 8 and Bax, the inhibition of Bcl-2 expression, and the release of cytochrome c are believed to be involved in the RCMF-mediated apoptosis. Collectively, these findings suggest that RCMF is an agent which may be capable of inducing sensitive growth inhibition and apoptosis in HOS cells.

Introduction

Cell death can occur through several different mechanisms of which its most widely described forms are apoptosis and necrosis. A significant physiological consequence of cell death by apoptosis is that the apoptotic cells are immediately phagocytosed by macrophages. Therefore, the release of intracellular molecules that cause a secondary disturbance to the surrounding tissue is limited to a low level compared with necrosis, which causes further tissue destruction and inflammation [1], [2].

Recently, there has been a global trend toward the use of natural bioactive substances found in fruits, vegetables, and herbs, as antioxidants and functional foods [3], [4], [5], [6]. Some of these substances are believed to have potential as cancer chemopreventive or therapeutic agents [7], [8], [9]. Most of these substances exert their chemotherapeutic activity by blocking the cell cycle progression and triggering apoptotic cell death. Therefore, the induction of apoptosis in tumor cells has become an indicator of the tumor-treating ability of naturally-derived bioactive substances [10], [11]. Recent attention has also focused on the development of target organ-specific apoptosis inducers as novel cancer-preventive and therapeutic approaches [9].

Flavonoids are commonly found in most plants and are integral parts of the human diet [12]. Flavonoids exert a remarkable spectrum of biological activities affecting the basic cell functions, such as growth [13], differentiation [14], and apoptosis [15]. Flavonoids are known to have anticarcinogenic, anti-inflammatory, antibacterial, immune-stimulating, and antiviral activities [16], [17], [18], and the beneficial effects of flavonoids have been attributed to the inhibition of the enzymes involved in signal transduction and to their antioxidant properties. Indeed, many studies have shown that flavonoids inhibit PI3-kinase, protein kinase C, protein tyrosine kinase, and some transcriptional factors, and that such inhibition leads to cell growth arrest and tumor cell death [12], [19], [20]. However, the effects of flavonoids on inducing apoptosis in osteosarcoma cells have not been widely reported. In addition, the mechanism(s) by which flavonoids induce cell death in osteosarcoma cells are not clearly defined.

Previously, a flavonoid fraction, consisting mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein, herein named RCMF (RVS chloroform-methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS) which is traditionally used as a food additive and an herbal medicine [21]. In Korea, much attention has been paid to RVS because it is a well-known antioxidant and antitumor agent [22]. In addition, our previous findings indicated that RVS extract is protective against oxidative damage by scavenging radicals or by altering the oxidized forms of chemicals, and flavonoids are the active compounds responsible for the antioxidant property of RVS [23]. Preliminary experiments also found that RCMF had antitumor activity in lymphoma and hepatoma cell lines [21], [24]. This study evaluated the effects of RCMF on the proliferation and apoptosis using human osteosarcoma (HOS) cells. In addition, the mechanism of the growth inhibition of the HOS cells by the flavonoid fraction, RCMF, was assessed.

Section snippets

Chemicals and laboratory wares

Unless otherwise specified, all chemicals used in this study were obtained from the Sigma Chemical Co. (St. Louis, MO) and all the laboratory wares were purchased from Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ). A purified flavonoid sample, RCMF, was prepared from a crude acetone extract of RVS wood, according to previously described procedures [21]. The RCMF was freshly dissolved in absolute ethanol and the final concentration of ethanol was not in excess of 0.1% (v/v) at any time

Effects of RCMF on proliferation of the HOS cells

The effect of RCMF treatment on cell proliferation was determined by tritium incorporation using HOS cells. As shown in Fig. 1, the addition of RCMF to the cultured HOS cells markedly inhibited the incorporation of tritium by the DNA in the cells, in a dose-dependent manner. For example, the level of tritium uptake by the cells treated with 20 μg/ml RCMF for 48 h was 10,745 ± 1000 cpm, which was a decrease of 64.6% from the 30,375 ± 3805 cpm of the untreated control cells. Treatment with 100 μg/ml

Discussion

Current studies involved in developing effective cancer-preventive approaches have focused mainly on the utilization of natural bioactive agents that can induce selective apoptosis in cancer cells [9]. Flavonoids exert various pharmacological properties and are believed to be beneficial compounds for cancer chemoprevention [14], [15], [26]. Previously, we prepared the purified flavonoid fraction, RCMF, and identified the active compounds contained in the RCMF [21]. In the present study, we

Acknowledgement

This work was supported by Korea Research Foundation Grant (KRF-2004-F00022).

References (41)

  • C.L. Miranda et al.

    Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines

    Food Chem. Toxicol.

    (1999)
  • Y.O. Son et al.

    Selective antiproliferative and apoptotic effects of flavonoids purified from Rhus verniciflua Stokes on normal versus transformed hepatic cell lines

    Toxicol. Lett.

    (2005)
  • J.C. Lee et al.

    Identification of Rhus verniciflua Stokes compounds that exhibit free radical scavenging and anti-apoptotic properties

    Biochim. Biophys. Acta

    (2002)
  • J.C. Lee et al.

    Extract from Rhus verniciflua Stokes is capable of inhibiting the growth of human lymphoma cells

    Food Chem. Toxicol.

    (2004)
  • R.T. Allen et al.

    Morphological and biochemical characterization and analysis of apoptosis

    J. Pharmacol. Toxicol. Methods

    (1997)
  • M. Germain et al.

    Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7

    J. Biol. Chem.

    (1999)
  • X. Li et al.

    Cleavage of Poly (ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis

    Exp. Cell Res.

    (2000)
  • X. Luo et al.

    Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors

    Cell

    (1998)
  • L. Annunziato et al.

    Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions

    Toxicol. Lett.

    (2003)
  • A.G. Yakovlev et al.

    A role of the Ca2+/Mg2+-dependent endonuclease in apoptosis and its inhibition by Poly (ADP-ribose) polymerase

    J. Biol. Chem.

    (2000)
  • Cited by (112)

    • Fisetin alleviates early brain injury following experimental subarachnoid hemorrhage in rats possibly by suppressing TLR 4/NF-κB signaling pathway

      2015, Brain Research
      Citation Excerpt :

      Fisetin (3,3′,4′,7-tetrahydroxyxflavone) is a bioactive flavonol molecule with a defined chemical structure (Fig. 1), which can be found in fruits and vegetables such as strawberry, apple, persimmon, grape, onion and cucumber at concentrations in the range of 2–160 μg/g (Arai et al., 2000). Accumulating evidence indicates that fisetin has multiple biological activities, such as antioxidant, anticarcinogenic, anti-inflammatory, antibacterial, immune-stimulating, antiviral (Duarte et al., 1993; Jang et al., 2005; Rice-Evans et al., 1996), neuroprotective and neurotrophic (Maher et al., 2006; Zbarsky et al., 2005). However, there is no data available about its cytotoxic and genotoxic (Lapchak, 2013).

    View all citing articles on Scopus
    1

    Contributed equally to this work.

    View full text