Abstract
Beta-asarone is one of the main bioactive constituents in traditional Chinese medicine Acorus calamu. Previous studies have shown that it has antifungal and anthelmintic activities. However, little is known about its anticancer effects. This study aimed to determine inhibitory effects on LoVo colon cancer cell proliferation and to clarify the underlying mechanisms in vitro and in vivo. Dose-response and time-course anti-proliferation effects were examined by MTT assay. Our results demonstrated that LoVo cell viability showed dose- and time-dependence on β-asarone. We further assessed anti-proliferation effects as β-asarone-induced apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide assay using a flow cytometer and observed characteristic nuclear fragmentation and chromatin condensation of apoptosis by microscopy. Moreover, we found the apoptosis to be induced through the mitochondrial/caspase pathway by decreasing mitochondrial membrane potential (MMP) and reducing the Bcl-2-to-Bax ratio, in addition to activating the caspase-9 and caspase-3 cascades. Additionally, the apoptosis could be inhibited by a pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). When nude mice bearing LoVo tumor xenografts were treated with β-asarone, tumor volumes were reduced and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays of excised tissue also demonstrated apoptotic changes. Taken together, these findings for the first time provide evidence that β-asarone can suppress the growth of colon cancer and the induced apoptosis is possibly mediated through mitochondria/caspase pathways.
Highlights
Epidemiological studies have demonstrated that the incidence of colorectal cancer (CRC) rank third in men and second in women worldwide (Parkin et al, 2005; Jemal et al, 2011)
The transmission electron microscope showed that LoVo cells lost normal structure after treatment and exhibited expansion of the endoplasmic reticulum, vacuolization in the cytoplasm, shrinkage and fragmentation of the nucleus and densification of the chromatin, all of which are the signs of early-stage apoptosis
For the first time we demonstrated that: (1) β-asarone decreases the viability of LoVo colon cancer cells in a time- and dose-dependent manner; (2) Apoptosis and necrosis in β-asarone-treated LoVo cells were revealed with flow cytometry by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling and with microscope observations; (3) Mechanistic evidence of apoptosis was drawn from the results that β-asarone depleted membrane potential (MMP), activated caspase-9 and caspase-3, and decreased the ratio of the survival protein Bcl-2 or Bcl-xL to the pro-apoptotic protein Bax; (4) The β-asarone inhibits the tumor growth and induces apoptosis in mice tumor xenografts model
Summary
Epidemiological studies have demonstrated that the incidence of colorectal cancer (CRC) rank third in men and second in women worldwide (Parkin et al, 2005; Jemal et al, 2011). The overall incidence and mortality have steadily declined in recent decades, CRC remains one of the leading causes of cancer related deaths. Chemotherapy is well known as one of the effective treatments for CRC besides surgery. Conventional chemotherapy of CRC with 5-fluorouracil (5-Fu) in combination with other anti-cancer agents improves overall and disease-free survival of patients after surgery (Machover, 1997). More and more apoptosis-targeted drugs or agents are exploited for therapeutic benefit (Cummings et al, 2004). Little is known about its inhibitory effect on cancer cells and the precise mechanism behind it remains unclear
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