Apigenin induces apoptosis in Hep G2 cells: Possible role of TNF-α and IFN-γ

Abstract

Flavonoids are one of the biologically active plant food constituents, possessing potential chemopreventive properties against a wide variety of chronic diseases. Apigenin, a common dietary flavonoid abundantly present in fruits and vegetables is believed to possess preventive and therapeutic potential against various cancers. In the present study, we have evaluated regulation of apoptotic cell death by apigenin (25 and 50 μM) in human hepatoblastoma derived cell line Hep G2. Apigenin-induced programme cell death in terms of TNF-α, IFN-γ release and induction of caspases activity. TNF-α and IFN-γ levels in apigenin-pretreated groups were significantly and dose dependently elevated as compared to the control values (28–39% and 66–85%), (208–336% and 579–1088%), respectively. Treatment of apigenin significantly induced caspase-3, -7, -10 and caspase-9 activity (160–209% and 203–270%) in a dose-dependent manner. The effects on caspases, TNF-α, and IFN-γ processes mediate the plausible mechanism of apoptosis induction of apigenin.

Introduction

Cancer is a disease, where the treatment can be as debilitating as the disease. Therefore, prevention could be considered as important as treatment in cancer. Diet can play a vital role in cancer prevention. Studies have shown that a diet high in fruits and vegetables is associated with a reduced risk of cancer (Khan et al., 2005, Park et al., 2005, Doss et al., 2005).

Hepatocellular carcinoma (HCC) is one of the most common malignancies leading to over one million deaths annually worldwide (Seow et al., 2001, Kern et al., 2002). The majority of the patients diagnosed with HCC have low recovery rates, and conventional and modified therapies now available are rarely beneficial (Kern et al., 2002). Therefore, it is essential to search novel agents that are efficient and have minimum side effects.

Apoptosis is an inducible form of controlled cell death that is essential for normal human embryogenesis as well as homeostatic state and protective processes (Raff, 1998). It has an essential role in controlling cell number in many developmental and physiological conditions. Apoptosis is impaired in many human tumors, suggesting that disruption of apoptotic function contributes substantially to the transformation of a normal cell into a tumor cell. It is an important event in chemotherapy-induced tumor-cell killing. Components of the apoptosis signaling cascade include caspases along with several other triggers and regulators (Nicholson et al., 1995, Talanian et al., 1997). Agents that are capable of inducing selective apoptosis of cancer cells are being given great interest in developing novel cancer preventive approaches. Studies have suggested that high fruits and vegetable consumption is related with a decreased risk of numerous types of cancer, including breast, colon, lung larynx, pancreas, oral and prostate cancer (Lin et al., 2005, van Dijk et al., 2005, Michels et al., 2005). Apigenin is a bioflavone, considered to have a bioactive effect on the human health as an antioxidant, radical scavenger. It also acts as a carbohydrate metabolism promoter, immune system modulator. Also plays a role in anti-platelet aggregation, as Ca²⁺ channel blocker and as an anti-inflammatory agent (Lee et al., 2005, Verbeek et al., 2005, Chen et al., 2005). Previously have shown to inhibit the proliferation of several human cancer cell lines, including U937, HL-60 and Hela cells (Gupta et al., 2002, Kobayashi et al., 2002, Kim et al., 1998). Studies in several human carcinoma cells including breast, colon cancer cells and leukemic cells have shown that apigenin induces growth inhibition, cell cycle arrest and apoptosis (Wang and Kurzer, 1997, McVean et al., 2000, Wang et al., 2000). Apigenin treatment resulted in G1 cell cycle arrest in synchronized human diploid fibroblasts and G2/M arrest in rat neuronal cells (Lepley and Pelling, 1997, Sato et al., 1994). Tumor necrosis factor alpha (TNF-α) is a multifunctional cytokine involved in the expression of many genes integral to the inflammatory response. It induces apoptosis of a variety of tumor cell types. The anti-tumor effect of TNF-α is often augmented by interferon (IFN)γ (Sugarman et al., 1985). It has demonstrated previously that TNF-α and IFN-γ synergistically induce apoptosis of tumor cells (Sugarman et al., 1985, Koshiji et al., 1998, Sasagawa et al., 2000). IFN-γ exerts its biological activities by binding to cell surface receptors. IFN-γ is a potent activator of caspases, leading to subsequent cell death (Boehm et al., 1997). Apoptosis the maintenance the homeostasis cell (Kerr et al., 1972). The transduction and execution of apoptotic signals requires the coordinated action of a cascade of caspases (aspartate-specific cysteine proteases) (Cotter et al., 1992). The caspases are present in cells as inactive procaspases, with the active tetramer being formed by removal of the prodomain and cleavage between the large and small subunits (Martin and Green, 1995). Caspase activity is responsible, either directly or indirectly, for cleavage of cellular proteins, which are characteristically proteolysed during apoptosis.

Although a recent study has shown that apigenin inhibits growth of human hepatoma Hep G2 cells (Yee et al., 2003, Chianga et al., 2005) information on its anti-apoptotic properties and cellular mechanism remain limited. It has been seen that lactatedehydrogenase (LDH) release is a feature of late apoptotic cells (Grub et al., 2000). Apoptosis is an active process of cell death characterized by cellular shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation (McDermott et al., 1998, Fladmark et al., 1999, Ding et al., 2000). Impairment of apoptosis has been implicated in many human diseases including malignancies (Talanian et al., 1997).