Study On The Apoptotic Effect Of α-bisabolol On Human Hepatoma HepG2 Cell Line

Terpenoid is a widespread fat-soluble secondary metabolites exist in plants and animals. Essential oil contains a lot of monoterpenes and sesquiterpenes, some of which have biological activities. Chamomile essential oil has anti-inflammatory antispasmodic, anti-fungal, anti-virus and anti-cancer function, and its main ingredient is sesame oil olympic blue andα-bisabolol. a-Bisabolol, a sesquiterpene alcohol with very low toxicity, has been widely used in fragrances and cosmetic preparations for hundreds of years. Recent years, this oily compound has been studied for its effect of increasing bacterial resistance to antibiotics and antimicrobials, antimutagenic activity, inhibitory effect on the genotoxic damage, depigmenting effect and apoptosis-inducing and anti-tumor activities.Liver cancer (LC) is one of the most common cancers with an annually increasing occurrence worldwide. LC has a two-to three-fold higher incidence rates in developing countries than in developed countries. Since most of current therapeutic treatments are ineffective and limited to treat the tumor, great effort has been making to find novel compounds to treat the tumors.Apoptosis, a basic biological phenomenon, is mostly mediated through extrinsic (death receptor) pathway and/or intrinsic (mitochondrial) pathway. The death receptor pathway is stimulated by the binding of cell surface death receptor such as Fas and tumor necrosis factor (TNF) receptor, which leads to the formation of a death-inducing signaling complex and the activation of caspase-8. Activated caspase-8 can not only directly activates downstream effector caspases, but also cleave Bid to tBid, which is important in Fas receptor pathway and can mediates cross-talk between these two pathways. The mitochondrial pathway is dependent on the release of cytochrome c from the mitochondria by mitochondrial outer membrane permeabilization (MOMP). MOMP is controlled by the BCL-2 family, which is composed of both pro-apoptotic molecules (Bax, Bcl-Xs, Bak, Bid, Bad, Bim and Bik) and antiapoptotic molecules (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Al). Once released to cytosol, cytochrome c combines with Apaf-1 and procaspase-9 to form the apoptosome. Activated caspase-9 can cleave and activate downstream effector caspases, such as caspase-3 and-7, leading to apoptotic cell death.In this study, the apoptotic effect of a-bisabolol against human liver carcinoma cell line HepG2 was investigated. MTT assay showed a-bisabolol could effectively induce cytotoxicity in several human cancer cell lines (PC-3, Hela, ECA-109 and HepG2), especially in HepG2 cells. The results of nuclei morphology examination, DNA fragmentation detection indicated HepG2 cells exhibited typical apoptotic features after treated with a-bisabolol. The results of flow cytometry analysis showed up to nearly 40%. The analysis of the cleavage of poly(ADP ribose) polymerase and caspases indicated a-bisabolol might induce dose-and time-dependent apoptosis in HepG2 cells. Western blot data also showed a cascade activation of caspase-8,-9,-3 and an evidently promoted expression of Fas, implying caspase-8 might function as an upstream regulator, and the Fas-related pathway might be involved in this process. Preparation of mitochondrial/cytosol fraction followed with immunoblot analysis showed the release of chromosome c from mitochondria, down-regulated expression of Bcl-2 and translocation of Bax, Bak and Bid, suggesting the mitochondrial-related pathway might be involved in a-bisabolol-induced apoptosis either. Detection of accumulation of nuclear wild-type p53 and up-regulated expression of NFkB after cells were treated with a-bisabolol indicated these two key regulator with transcriptional decision-making function in various signaling pathways might also play a role in a-bisabolol-induced apoptosis in HepG2 cells. These results provide further insight into a-bisabolol-induced apoptosis and deepen our previous cognition of the toxicity and anticancer activity of a-bisabolol, and can increase the possibility of developing a-bisabolol to be a promising future chemotherapeutic agent.Bcl-2 family proteins are key regulatory factors, and also are hot targets of a new generation of anti-cancer drugs in developing. Studies on the structure-function of these proteins have revealed the critical role of the surface cavity of anti-apoptotic Bcl-2 and Bcl-xL in the interaction with pro-apoptotic proteins and in the inhibition of cell death signaling pathway. Several classes of small molecule inhibitors of Bcl-2 have been reported:(1) synthesized BH3 peptides and mimic small molecules which could penetrate cell membrane; (2) natural products identified by random screening methods; (3) organic compounds designed by structrue-based and computer-aided technique. These various small molecules binding with Bcl-2 provides a powerful tool to explore the function of Bcl-2, and very likely lead to the develpoment of new anti-cancer drugs. To further explore the anti-cancer mechanism of a-bisabolol and to search for the potential target of which in cell, we selected five anti-apoptotic members of Bcl-2 family(Al, Bcl-2, Bcl-w, Bcl-xL and Mcl-1), using computer modeling and Autodock to dock a-bisabolol with these five proteins. We performed a preliminary study on the potential binding site of a-bisabolol on these five Bcl-2 family proteins, and try to find a new way to study the molecular mechanisms of the anti-cancer effect of a-bisabolol and to explore a new generation of anticancer drugs.