Elsevier

Life Sciences

Volume 75, Issue 19, 24 September 2004, Pages 2303-2316
Life Sciences

Proliferative inhibition, cell-cycle dysregulation, and induction of apoptosis by ursolic acid in human non-small cell lung cancer A549 cells

https://doi.org/10.1016/j.lfs.2004.04.027Get rights and content

Abstract

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many types of medicinal plants and is present in human diet. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. Here, we report that UA inhibits the cell proliferation of human lung cancer cell line A549 and provide a molecular understanding of this effect. The results showed that UA blocked cell cycle progression in the G1 phase that was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner cdk2, 4, and 6 with concomitant induction of p21/WAF1. This accumulation of p21/WAF1 might be through a p53-dependent manner. Further, UA treatment also resulted in the triggering of apoptosis as determined by DNA fragmentation assay. This effect was found to correlate with the up-regulation of Fas/APO-1, Fas ligand, and Bax, and down-regulation of NF-κB, Bcl-2, and Bcl-XL. Taken together, our study indicated that UA might be a potential chemopreventive agent for lung cancer.

Introduction

Non-small cell lung cancers commonly develop resistance to radiation and chemotherapy, and they often present at stages beyond surgical remedy. Since current treatment modalities are inadequate, novel therapies are necessary to reduce the effects of the increasing incidence in pulmonary neoplasm (Kim et al., 2003). Ursolic acid (UA), a pentacyclic triterpene acid which is widely distributed in medical herbs and edible plants, like waxlike coating of apples and other fruits (Cha et al., 1998, Sohn et al., 1995). UA has been shown to possess anti-bacterial, antiviral, hepatoprotective, immunomodulatory activity (Liu, 1995, Novotny et al., 2000). Studies have shown that UA exhibits growth inhibition properties against many human cancer cell lines, including breast, leukemia, prostate, liver, colon and skin, and mouse melanoma B16 cancer cells (Andersson et al., 2003, Baek et al., 1997, Choi et al., 2000, Es-Saady et al., 1996a, Es-Saady et al., 1996b, Huang et al., 1994, Kim et al., 2000, Tokuda et al., 1986). In addition, UA has been reported that display a remarkable spectrum of biochemical activities to influence processes that are dysregulated during cancer development. These include inhibition of tumorigenesis, tumor promotion, invasion, metastasis and angiogenesis, and induction of tumor cell differentiation (Cha et al., 1996, Cha et al., 1998, Es-Saady et al., 1996a, Es-Saady et al., 1996b, Harmand et al., 2003, Huang et al., 1994, Lee et al., 1994, Novotny et al., 2000, Sohn et al., 1995, Tokuda et al., 1986). It also interrupts with DNA synthesis enzymes, including DNA polymerase and DNA topoisomerase (Novotny et al., 2000, Mizushina et al., 2000). However, the precise antitumorigenic mechanisms of UA in lung cancer cells remain largely unknown.

In this study, not only did we assay the effects of UA on cell proliferation, cell cycle distribution, and apoptosis in human lung cancer cell line A549, but we also established the cell cycle–related (p53, p21/WAF1, cyclin D1/D2, cyclin E, cdk2, 4, and 6) and apoptosis-associated (Fas/APO-1, Fas ligand, NF-κB, Bax, Bcl-2, and Bcl-XL) molecular pathways.

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Materials

Fetal bovine serum (FBS), penicillin G, streptomycin, and amphotericin B were obtained from GIBCO BRL (Gaithersburg, MD). Ursolic acid, dimethyl sulfoxide (DMSO), ribonuclease (RNase), and propidium iodide (PI) and RPMI-1640 were supplied by Sigma Chemical Co. (St. Louis, MO). XTT and p53 pan ELISA kit were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Nucleosome, WAF1, Fas Ligand and Fas/APO-1 ELISA kits, and the antibodies (human reactive anti-cyclin D1, cyclin D2, cyclin E, cdk2,

Effect of UA on A549 cell proliferation

We first tested the antiproliferative effect of UA in a lung cancer cell line, A549. As shown in Fig. 1, the proliferative inhibitory effect of UA was observed to be dose-dependent. The maximum proliferative inhibition of UA was 84.42% that occurred at 72 h using by 40 μM UA.

UA-induced cell cycle arrest and apoptosis in A549 cells

To determine whether interfering with cell cycle progression and inducing apoptosis mediate the growth inhibition of A549 cells by UA, we evaluated the effect of UA on cell cycle distribution and fragmentation of DNA.

Discussion

Many natural compounds, especially plant products and dietary constituents, have been found to possess chemopreventive activities both in vitro and in vivo (Kelloff et al., 2000). Ursolic acid (UA) is a pentacyclic triterpenoid compound, which occurs naturally in a large variety of vegetarian foods, medicinal herbs and plants (Lee et al., 1988). UA has been reported to arrest Hep G2 cell cycle at G0/G1 phase by inhibiting DNA replication and increasing p21/WAF1 expression (Kim et al., 2000).

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