Involvement of reactive oxygen species/c-Jun NH2-terminal kinase pathway in kotomolide A induces apoptosis in human breast cancer cells

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Abstract

The anticancer effects of kotomolide A (KTA), a new butanolide constituent isolated from the leaves of Cinnamomum kotoense (Lauraceae), on the two human breast cancer cell lines MCF-7 and MDA-MB-231, were first investigated in our study. KTA exhibited selectively antiproliferative effects in cancer cell lines without showing any toxicity in normal mammary epithelial cells. Treatment of cancer cells with KTA to trigger G2/M phase arrest was associated with increased p21/WAF1 levels and reduced amounts of cyclin A, cyclin B1, cdc2 and cdc25C. KTA induced cancer cell death treatment by triggering mitochondrial and death receptor 5 (DR5) apoptotic pathways, but did not act on the Fas receptor. Exposure of MCF-7 and MDA-MB-231 cells to KTA resulted in cellular glutathione reduction and ROS generation, accompanied by JNK activation and apoptosis. Both antioxidants, NAC and catalase, significantly decreased apoptosis by inhibiting the phosphorylation of JNK and subsequently triggering DR5 cell death pathways. The reduction of JNK expression by siRNA decreased KTA-mediated Bim cleavage, DR5 upregulation and apoptosis. Furthermore, daily KTA i.p. injections in nude mice with MDA-MB-231 s.c. tumors resulted in a 50% decrease of mean tumor volume, compared with vehicle-treated controls. Taken together, the data show that cell death of breast cancer cells in response to KTA is dependent upon ROS generation and JNK activation, triggering intrinsic and extrinsic apoptotic pathways. The ROS/JNK pathway could be a useful target for novel approaches in breast cancer chemotherapy.

Introduction

Breast cancer is one of the most common human malignancies and the second leading cause of cancer-related deaths in women (Jemal et al., 2007). This pathology is currently controlled by surgery and radiotherapy, and is frequently supported by adjuvant chemo- or hormonotherapies (Bange et al., 2001). However, breast cancer is highly resistant to radiation and conventional chemotherapeutic agents, and this resistance is associated with a poor prognosis for this metastatic disease, especially in cases of hormone-independent cancer (Bange et al., 2001, Roy et al., 2005). About 30%–40% of women with this form of cancer will develop metastases and eventually die from their disease (Jemal et al., 2007). Novel therapies are therefore needed to deal with the increasing incidence of human breast cancer.

All aerobic organisms are subject to physiological oxidant stress as a consequence of aerobic metabolism. Aerobic respiration coupled to the generation of ATP leads to the formation of the superoxide anion radical (O2radical dot). Superoxide anion radicals can then form other reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and the highly reactive hydroxyl radical (radical dotOH) (Feig et al., 1994, Fujino et al., 2006). The redox status of all aerobic cells is balanced by enzyme and nonenzyme systems (Feig et al., 1994, Fujino et al., 2006, Schumacker, 2006, Kondo et al., 2006). Oxidative stress occurs when this critical balance is disrupted because of excess ROS production and/or antioxidant depletion (Schumacker, 2006, Kondo et al., 2006). Evidence is accumulating which indicates that many chemotherapeutic agents may be selectively toxic to tumor cells because they increase oxidant stress and enhance these already stressed cells beyond their limit (Ham et al., 2006, Moungjaroen et al., 2006). Cytotoxic ROS signaling appears to be triggered by the activation of the mitochondrial-dependent cell death pathway through activation of the mitogen-activated protein kinase (MAPK) pathways and the proapoptotic Bcl-2 proteins Bax or Bak, with subsequent mitochondrial membrane permeabilization and cell death (Feig et al., 1994, Kim et al., 2005, Zhang and Chen, 2004, Kuo et al., 2007).

Cinnamomum kotoense Kanehira and Sasaki (Lauraceae) is a small evergreen tree, native to Lanyu Island of Taiwan, which has more recently been cultivated as an ornamental plant. Kotomolide A (KTA) (Fig. 1A), is a new butanolide constituent isolated from the leaves of C. kotoense, and its properties as an anticancer agent have previously been described (Chen et al., 2006). This study is the first to determine the cell growth inhibition activity of KTA and examine its effect on cell cycle distribution and apoptosis in two human breast cancer cell lines, MCF-7 and MDA-MB-231. Our data has found that KTA increases the level of ROS in the human breast cancer cells, and this increase is responsible for its apoptotic effects. KTA-induced ROS activated JNK, and subsequently triggered mitochondrial and DR5 apoptotic pathways. Also, KTA's anticancer activity was further examined in a xenograft model. On the other hand, KTA did not exhibit any toxicity in the normal mammary epithelial cell line H184B5F5/M10, or in the nude mice's body weight and organs. These data suggest that KTA may be of value in the development of novel therapeutic approaches for the treatment of breast cancer.

Section snippets

Test compound

Kotomolide A (KTA) (purity > 90%) was isolated from the leaves of C. kotoense as described previously (Chen et al., 2006). Briefly, the air-dried leaves were extracted with MeOH at room temperature, and the MeOH extract was obtained upon concentration under reduced pressure. The MeOH extract, suspended in H2O, was partitioned with CHCl3 to give fractions soluble in CHCl3 and H2O. The CHCl3 soluble fraction was chromatographed over silica gel using n-hexane-EtOAc-acetone as eluent to produce five

KTA inhibits cell proliferation and clonogenic survival in both MCF-7 and MDA-MB-231 cell lines

To investigate the potential cell proliferative inhibition activity of KTA in breast cancer, we first examined the effect of KTA on cell proliferation and clonogenic survival in both MCF-7 and MDA-MB-231 cell lines. As shown in Fig. 1B, exposure of MCF-7 and MDA-MB-231 to KTA for 48 h inhibited the growth of each cell line in a dose-dependent manner. The IC50 values of KTA were 4.98 µM for MCF-7 and 4.67 µM for MDA-MB-231. In contrast, KTA failed to affect the proliferation of H184B5F5/M10 at

Discussion

Breast cancer is the most common malignancy and the second leading cause of cancer mortality in women (Jemal et al., 2007). Unfortunately, there are limited treatment options available for this disease, because chemotherapy and radiation therapy are largely ineffective, and metastasis frequently occurs even after potentially curative surgery (Bange et al., 2001). This study is the first to show that KTA inhibits the growth of two human breast cancer cell lines, MCF-7 and MDA-MB-231, both in

Acknowledgment

This study was supported by a research grant from the National Science Council of Taiwan (NSC 95-2320-B-037-043).

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