Inhibition of cyclooxygenase-2 and induction of apoptosis in estrogen-nonresponsive breast cancer cells by Antrodia camphorata

Abstract

The objective of this study was to investigate the fermented culture broth of Antrodia camphorata (A. camphorata) to induce apoptosis and inhibit cyclooxygenase-2 (COX-2) in estrogen-nonresponsive (MDA-MB-231) human breast cancer cells. Treatment of the highly invasive MDA-MB-231 cells with A. camphorata (40–240 μg/ml) resulted in dose and time-dependent sequences of events marked by apoptosis, as evidenced by loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. Apoptosis in the MDA-MB-231 cells was accompanied by release of cytochrome c, activation of caspase-3, -8, and -9, and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). Although the A. camphorata-induced apoptosis was associated with a reduction in Bcl-2 protein levels, negligible Bax increase was observed. Furthermore, A. camphorata treatment inhibited COX-2 protein expression and prostaglandin E2 (PGE2) production in MDA-MB-231 cells. Analysis of the study data suggests that A. camphorata exerts growth inhibition on (highly invasive) estrogen-nonresponsive human breast cancer cells through apoptosis induction associated with COX-2 inhibition, and that it may possess anticancer properties potentially valuable for application in drug products.

Introduction

A new basidiomycete, Antrodia camphorata, in the polyporaceae (Aphyllophorales), which causes brown heart rot in Cinnamonum kanehirai hay (Lauraceae) in Taiwan, has been identified as a new genus of the Antrodia species (Zang and Su, 1990, Wu et al., 1997). A. camphorata is rare and expensive as it grows only on the inner heart-wood wall of the C. kanehirai and cannot be cultivated. It has been utilized in traditional Chinese medicine for the treatment of food and drug intoxication, diarrhea, abdominal pain, hypertension, skin itches and liver cancer (Tsai and Liaw, 1985), however, very few biological activity tests are reported.

Recently, the relationship between apoptosis and cancer has been emphasized, with increasing evidence suggesting that the related processes of neoplastic transformation, progression and metastasis involve alteration of the normal apoptotic pathways (Bold et al., 1997). Morphological hallmarks of this process include loss of cell volume, hyperactivity of the plasma membrane, and condensation of peripheral heterochromatin, followed by cleavage of the nucleus and cytoplasm into multiple membrane-enclosed bodies containing chromatin fragments (Wyllie et al., 1980). Apoptosis is controlled by two major pathways including a mitochondrial (Green and Reed, 1998) and a membrane death receptor (DR) pathway (Ashkenazi and Dixit, 1999). Cell-free systems have been used to demonstrate that mitochondrial release of cytochrome c into the cytosol is rate limiting in terms of the activation of caspases and endonucleases (Martinou et al., 2000). Cytosolic cytochrome c activates procaspase-9 by binding to Apaf1 in the presence of dATP, leading to activation of caspase-9 and, subsequently, of downstream effector caspases (including caspase-3), with triggering of apoptosis (Li et al., 1997). Membrane DRs are activated by their respective ligands and engage adaptor molecules and caspases, including proximal caspase-8 (Ashkenazi and Dixit, 1999, Serhidan et al., 1997). Caspase-8 is a proximal caspase that plays a critical role in the DR-mediated apoptotic pathway, used by Fas, tumor necrosis factor α, and tumor necrosis factor-related apoptosis-inducing ligand, that is independent of cytochrome c release (Ashkenazi and Dixit, 1999). Apoptosis provides a number of clues with respect to effective anticancer therapy, and many chemotherapeutic agents reportedly exert their antitumor effects by inducing apoptosis in cancer cells.

Recently, the link between cyclooxygenase (COX)-2 and cancer has been the subject of investigation (Wang and Dubois, 2004). COX, referred to as prostaglandin endoperoxide synthase, is the rate-limiting enzyme for the metabolic conversion of arachidonic acid to prostaglandins (PGs) and related eicosanoids. Isoforms of the COX gene include constitutively expressed COX-1 and inducible COX-2 (Williams et al., 1999). Unlike the former, the COX-2 gene has been characterized as an immediate-early gene associated with neoplastic transformation (Eberhart et al., 1994), cell growth (Fujita et al., 2002), angiogenesis (Chang et al., 2004), invasiveness and metastasis (Pai et al., 2003). Evidence from in vitro and in vivo studies suggests an important role for prostaglandins and their synthesizing enzyme COX-2 in mammary carcinogenesis (DuBois and Smalley, 1996, Liu et al., 2001). Some human breast cancers, notably those that are estrogen-independent with high metastatic potential, express constitutive and inducible COX-2, and produce high prostaglandin E2 (PGE2) levels (Liu and Rose, 1996). Currently, the inhibition of COX-2 expression and the blockade of the prostaglandin (PG) cascade with chemotherapy agents is being proposed for cancer treatment trials.

Antrodia camphorata (A. camphorata) is well known in Taiwan as a traditional Chinese medicine, and it has been shown to exhibit antioxidant and anticancer effects. In our previous study, A. camphorata was used for the inhibition of AAPH-induced oxidative hemolysis and lipid/protein peroxidation of normal human erythrocytes (Hseu et al., 2002). A. Camphorata in submerged culture protects low-density lipoproteins against oxidative modification and may provide effective protection from atherosclerosis (Yang et al., 2006a). Our previous results also indicated that A. camphorata inhibits LPS induction of cytokine, inducible nitric oxide synthase (iNOS) and COX-2 expression by blocking NF-κB activation in RAW 264.7 macrophages (Hseu et al., 2005). Interestingly, A. camphorata exhibits significant apoptotic cell death against estrogen-responsive MCF-7 cells and premyelocytic leukemia (HL-60) cells (Yang et al., 2006b, Hseu et al., 2004). The effects were observed in MCF-7 cells and leukemia HL-60 cells, but not in healthy breast cells (HBL100), erythrocytes, and human umbilical vein endothelial cells (Yang et al., 2006b, Hseu et al., 2004).

Breast cancer is the most common malignancy in American and northwestern European women. Approximately one-third of all women with breast cancer develops metastases and ultimately expires due to the effects of the disease. Despite the fact that many tumors initially respond to chemotherapy, breast cancer cells can subsequently survive and gain resistance to the treatment. In this study, the effects of the fermented broth of A. camphorata harvested from submerged cultures on estrogen-nonresponsive (MDA-MB-231) human breast cancer cells was investigated, based on the interesting biological activities reported and their potential clinical application. We also evaluated the mechanisms by which A. camphorata inhibition of COX-2 affects tumor growth in estrogen-independent, highly invasive, metastatic MDA-MB-231 cells, because inhibitors of COX-2 are being extensively studied as anticancer agents.