Norcantharidin induces apoptosis of breast cancer cells: Involvement of activities of mitogen activated protein kinases and signal transducers and activators of transcription

Abstract

Involvement of activities of mitogen-activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs) remains unsolved in norcantharidin-associated breast cancer cell apoptosis. This study investigated the anti-cancer effect of norcantharidin and its underlying mechanism in two human breast cancer cell lines, estrogen receptor (ER)− HS-578T and ER+ MCF-7 cells. Norcantharidin induced potent cytotoxicity and arrested cell growth through increasing phosphorylation of Chk1, Chk2 and total p21^Waf1/Cip1 and reducing cyclin B and cdc25c expression. It also induced apoptosis through extrinsic death receptor and intrinsic mitochondrial pathways by cytochrome c release, caspase activation, oligonucleosome appearance, PARP cleavage, and aberration of Bcl-2 family protein expression and phosphorylation. Although norcantharidin did not affect STAT1, STAT3, and STAT5 protein expression, it suppressed STAT3 and STAT5 phosphorylation in HS-578T cells, whereas it up-regulated STAT1 phosphorylation and down-regulated STAT5 phosphorylation in MCF-7 cells. Moreover, norcantharidin activated MAPK family member proteins, extracellular signal-regulated kinase (ERK), p38^MAPK and c-Jun N-terminal kinase (JNK), were all phosphorylated by treatment. Pretreatment with selective kinase inhibitors significantly attenuated the norcantharidin-induced cytotoxicity in breast cancer cells. These findings suggest the potential involvement of MAPK and STAT pathways in norcantharidin-induced apoptogenesis. Norcantharidin may be an effective anti-cancer drug against breast cancer.

Introduction

Breast cancer (BCa) is the second most common cancer worldwide after lung cancer and the fifth most common cause of cancer-related death. Notably, morbidity of BCa in females is roughly 100 times higher than males, survival rates for both sexes are equal (Jemal et al., 2009). Unfortunately, severe morbidity of BCa has not been improved by surgery, radiotherapy, biological therapy, chemo- or hormonotherapies, as BCa is highly resistant to chemotherapy and no effective cure exists for patients with advanced disease stages (Andre et al., 2004, Orlando et al., 2007). Several biomarkers have been identified that predict response to chemotherapy to variable extent. Since estrogen is well known for its ability to directly modulate expression of growth factor receptor pathways and cell cycle regulatory genes (Dickson and Russo, 2000), status of estrogen receptor (ER) in a tumor significantly correlates with the likelihood of achieving pathologic complete response (Tewari et al., 2008). Patients who bear operable and ER-negative (ER−) tumors are more likely to achieve higher chemosensitivity than those ER+ cases (Guarneri et al., 2006, Ring et al., 2004).

Norcantharidin is a demethylated analog of cantharidin purified from the dried body of the Chinese blister beetle Mylabris, (Mylabris phalerata Pallas), which is a traditional Chinese medicine long been used for treating malignant tumors (Wang, 1989). In comparison with cantharidin, the major bioactive compound in Mylabris, norcantharidin has the advantage of easy synthesis and reduced intrinsic toxicity while retaining its anti-cancer activity. The anti-cancer mechanisms of norcantharidin have been illuminated by the evidence showing its anti-proliferation, pro-apoptotic and anti-migratory effects on many cancer cells. Norcantharidin inhibits the growth of numerous cancer cell lines via apoptosis, including oral cancer (Kok et al., 2003), hepatoma (Chen et al., 2002), leukemia (Liao et al., 2007), colorectal adenocarcinoma (Peng et al., 2002), melanoma (An et al., 2004b), glioblastoma (Hong et al., 2000) and gallbladder carcinoma (Fan et al., 2007). Besides, animal studies demonstrated that norcantharidin reduced pulmonary metastatic capacity of colorectal adenocarcinoma cells (Chen et al., 2005, Chen et al., 2009b) and prolonged survival of HepG2 tumor-bearing nude mice (Yang et al., 1997).

The well-studied mechanism for the anti-cancer activity of norcantharidin is its ability to induce tumor cell apoptosis, the process of which encompasses interruption of cell cycle progression of tumors at mitotic phase, suppression of cell adhesion and motility, expressional modulation of Bcl-2 superfamily member proteins and of caspase activity, and induction of eventual apoptotic cell death. In fact, the apoptotic induction by norcantharidin has been evidenced in many types of tumor cells and, more recently, in human breast cancer cells (Huang et al., 2009). In addition, the activities of mitogen-activated protein kinase (MAPK) family members, such as extracellular signal-regulated kinase (ERK), p38^MAPK, and c-Jun N-terminal kinase (JNK), have been mechanistically implicated in the apoptogenesis of norcantharidin-treated tumor cells (An et al., 2004a, Chen et al., 2002, Chen et al., 2003, Chen et al., 2005, Hong et al., 2000, Peng et al., 2002). Although Huang et al. (2009) has recently demonstrated that norcantharidin interferes with Akt phosphorylation in a highly metastatic human BCa cell line, however, not enough is known about the role that MAPKs play in the norcantharidin-elicited apoptosis of breast tumor cells. Moreover, aberrant activation of signal transducers and activators of transcription (STATs), mainly STAT1, STAT3, and STAT5 proteins, is involved in the pathogenesis of cancer (Bromberg, 2001). Overall, STAT1 functionally promotes apoptosis and tumor suppression, while STAT3 and STAT5 modulate cell growth and inhibit apoptosis (Battle and Frank, 2002, Bromberg, 2001). To date, the role of STATs in the apoptotic induction of BCa cells by norcantharidin remains unclear.

Due to lack of knowledge for the above-mentioned viewpoints on the anti-cancer activity of norcantharidin in BCa cells, this study thus aimed at validating the norcantharidin-driven cytotoxicity in two BCa cell lines with opposite ER status (i.e., ER− HS-578T and ER+ MCF-7 cells), delineating the responsive profiles of STATs and MAPKs activities to drug treatment therein, as well as elucidating the pharmacological mechanism underlying the drug action.