Cancer Letters

Cancer Letters

Volume 182, Issue 1, 8 August 2002, Pages 83-92
Cancer Letters

Se-methylselenocysteine induces apoptosis through caspase activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells

https://doi.org/10.1016/S0304-3835(02)00075-7Get rights and content

Abstract

Se-methylselenocysteine (Se-MSC) is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis, but its mechanism of action is still not well understood. The present study was designed to assess the mechanism of Se-MSC on the induction of apoptosis in SKOV-3 ovarian cancer cells. Se-MSC displayed strong inhibitory effects on cell proliferation and viability of SKOV-3 cells in dose and time dependent manners and induced apoptosis. Investigation of the mechanism of Se-MSC-induced apoptosis revealed that treatment with Se-MSC produced morphological features of apoptosis and DNA fragmentation. This was associated with caspase-3 activation and cleavage of poly(ADP-ribose) polymerase and phospholipase C-γ1 proteins. However, SKOV-3 cells treated with Se-MSC did not demonstrate cytochrome c accumulation in the cytosol during apoptosis induction. Pretreatment of cells with the caspase inhibitors (z-VAD-fmk and DEVD-CHO) prevented Se-MSC-induced apoptosis. These results suggested that Se-MSC induces apoptosis through cytochrome c-independent caspase-3 activation in SKOV-3 cells. In late stage of apoptosis, p18 kDa fragment of Bax was generated with the down-regulation of the expressions of survivin, X-linked inhibitor of apoptosis protein, and human inhibitor of apoptosis protein 1 following Se-MSC treatment, suggesting that the modulation of Bax and IAP (inhibitors of apoptosis) family proteins play some role in Se-MSC-mediated apoptosis. Pre-treatments of z-VAD-fmk and the calpain inhibitor, calpeptin inhibited Bax cleavage. These results suggested that Bax cleavage is mediated by calpain, and calpain activation may be a caspase-dependent one. Taken together, the chemopreventive effects of Se-MSC may be related in part to the caspase-3 activation, the down-regulation of IAP family proteins, and Bax cleavage mediated by caspase-dependent calpain activation.

Introduction

Ovarian cancer is the leading cause of death among gynecological malignancy. When the disease is still confined to the ovary, surgery alone can be curative. Due to a lack of powerful diagnostic tests and absence of any overt symptoms, the majority of patients with ovarian cancer are diagnosed at an advanced stage, a time when the therapeutic measures now available are often ineffective.

Chemoprevention is coined for cancer prevention and cancer control by use of synthetic or naturally occurring chemical agents [1]. Chemoprevention is promising because chemotherapy and surgery have not been fully effective against the high incidence and low survival rate of ovarian cancer [2]. Recently, the list of agents with chemopreventive activity is growing rapidly. One important class of chemopreventive compounds comprises organic or inorganic form of selenium [3], [4]. Selenium is an essential trace mineral element in mammals, and many epidemiological studies have suggested a possible causal relationship between selenium consumption and a reduced risk for some cancers [5], [6], [7], [8]. Among selenium compounds, Se-methylselenocysteine (Se-MSC) is one of the most effective compounds for chemoprevention based on low toxicity, easy conversion to methylselenol, and substantial anticarcinogenic properties through mechanisms distinct from selenite [4], [9], [10], [11]. Many therapeutic and chemopreventive agents eliminate tumor cells by inducing apoptotic cell death. Se-MSC was also shown to induce apoptosis in cancer cell lines [11], [12], [13], [14], [15]. However, these reports showed conflicting results of its anti-cancer effects.

Apoptosis is essential for development, maintenance of tissue homeostasis, and elimination of unwanted or damaged cells from multicellular organisms [16], [17]. Several genes have been identified as either inducers or repressors of apoptosis. Among these, caspases, a growing family of cysteine proteases that cleave specific substrates at aspartic acid residues, have been identified as major components of apoptosis [18]. Caspase activation is regulated by various proteins, including the inhibitory proteins of the IAP (inhibitors of apoptosis) family and the Bcl-2 family. Although caspases play a major role in apoptosis, there are evidences that other proteases including calpain may also be involved in this process [19], [20].

Apoptosis has been shown to be an important determinant of the response of cancer cells to chemotherapeutic agents. In the present study, we focus on the potential mechanism of Se-MSC-induced apoptosis. We have confirmed that Se-MSC-mediated apoptosis is associated with cytochrome c-independent caspase-3 activation, PARP (poly(ADP-ribose) polymerase) degradation, and the down-regulation of IAP family proteins in SKOV-3 cells. In addition, the cleavage of Bax protein was detected after caspase activation and PARP cleavage. Bax cleavage was found to be mediated by caspase-dependent activation of calpain. Therefore, Se-MSC induces apoptosis of SKOV-3 cells through caspase activation and the down-regulation of IAP family proteins, and subsequently enhances apoptosis through Bax cleavage mediated by caspase-dependent calpain activation.

Section snippets

Cell culture

SKOV-3 cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2 in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum, 2 mM glutamine, and 100 U/ml penicillin and 100 μg/ml streptomycin. Typically, 105/ml SKOV-3 cells were seeded in T-25 flasks as 4 ml cultures, and were maintained in the tissue culture incubator for 18–20 h before the addition of Se-MSC. At the indicated times, cells were harvested and washed 2× with phosphate-buffered saline (PBS). Cell

Se-MSC-induced caspase-3 activation and PARP degradation

Se-MSC-induced cytotoxicity, measured as the percentage of viable cells, is shown in Fig. 1A. When cells were treated with Se-MSC, in agreement with previous reports [12], [13], a significant decrease of viable cells was observed in both Se-MSC concentration and culture time-dependent. Nucleic acid staining with PI revealed typical apoptotic nuclei in Se-MSC treated cells, but control cells did not show any feature of apoptosis (Fig. 1B). In order to quantify the degree of apoptosis, we

Discussion

Various forms of selenocompounds with diverse chemical structures have been studied for their chemopreventive properties in epidemiological and experimental studies [4], [5], [9], [10]. Although a number of selenocompounds are effective chemopreventive agents, Se-MSC continues to attract great attention because of its chemical nature, low toxicity, and significant chemopreventive effects [4], [9], [10]. Previous studies demonstrated that Se-MSC induces apoptosis mediated by reactive oxygen

Acknowledgments

This work supported in part by Ministry of Science and Technology (MOST) and the Korean Science and Engineering Foundation (KOSEF) through the Center for Traditional Microorganism Resources (TMR) at Keimyung University.

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