Diallyl disulfide induces apoptosis in human colon cancer cell line (COLO 205) through the induction of reactive oxygen species, endoplasmic reticulum stress, caspases casade and mitochondrial-dependent pathways

Abstract

In this study, we investigated the effects of DADS on human colon cancer cell line COLO 205 on cell cycle arrest and apoptosis in vitro. After 24 h treatment of COLO 205 cells with DADS, the dose- and time-dependent decreases of viable cells were observed and the IC₅₀ was 22.47 μM. The decreased percentages of viable cells are associated with the production of ROS. Treatment of COLO 205 cells with DADS resulted in G2/M phase arrest and apoptosis occurrence through the mitochondrial-pathway (Bcl-2, Bcl-xL down-regulation and Bak, Bax up-regulation). DADS increased cyclin B, cdc25c-ser-216-9 and Wee1 but did not affect CDK1 protein and gene expression within 24 h of treatment. DADS-induced apoptosis was examined and confirmed by DAPI staining and DNA fragmentation assay. DADS promoted caspase-3, -8 and -9 activity and induced apoptosis were accompanied by increasing the levels of Fas, phospho-Ask1 and -JNK, p53 and decreasing the mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-9 and -3. The COLO 205 cells were pre-treated with JNK inhibitor before leading to decrease the percentage of apoptosis which was induced by DADS. Inhibition of caspase-3 activation blocked DADS-induced apoptosis on COLO 205 cells.

Introduction

Apoptosis plays an essential role as a protective mechanism against tumor cells which are damaged or excess cells that have been improperly produced. Many studies have focused on selectively killing tumor cells through the induction of apoptosis (Ferreira et al., 2002). Apoptosis is a kind of cell death that upon receiving specific signals instructing the cells and causes specific morphological change such as plasma and nuclear membrane blebbings, chromatin condensation, proteases activation and DNA fragmentation that are considered as landmarks of the apoptotic process (Fadeel et al., 1999, Jacobson et al., 1997). Apoptosis can be divided into caspase-dependent and -independent pathways (Yu et al., 2002). Activation of caspase-dependent signaling pathways was via cleavage of the procaspase-9 or -8 results in the downstream activation of caspase-3, -6, -7, finally leading to apoptosis (Slee et al., 1999).

In Taiwan, about 15.03 persons per 100 thousand people die per year in colon cancer from the reports of the “People Health Bureau of Taiwan”. Surgery, radiotherapy and chemotherapy are used for clinical therapy in human colon cancer. Currently the strategies for treatment of human colon cancer are not yet satisfactory. It was reported that enhanced garlic consumption is closely related with reduced cancer incidence (Buiatti et al., 1989, Haenszel et al., 1972). Diallyl disulfide (DADS), an important oil-soluble organosulfur component of garlic (Allium sativum), has been reported to inhibit the growth of human cancer cells such as colon, lung, skin and breast (Hong et al., 2000, Kwon et al., 2002, Sundaram and Milner, 1996). DADS induced apoptosis in HL-60, HCT-15 and neuroblastoma cells through the production of ROS, induction of p53 and activation of caspase-3 (Filomeni et al., 2003, Hong et al., 2000, Kwon et al., 2002, Park et al., 2002). Recently, DADS and the specific inhibitors of MAPKs induced apoptosis in HepG2 hepatoma cells (Wen et al., 2004). DADS had been shown as an inhibiting potential in colon and renal carcinogenesis which is induced by N-diethylnitrosamine in F344 rats in vivo (Takahashi et al., 1992). Although DADS as an anti-tumor agent has been established, the exact mechanism of cytotoxic effect in the apoptotic pathways is not completely clear and the potential mechanism of apoptosis through which the signal pathway within the cell remains to be evaluated. In this present study, the aim was to dissect the mechanisms underlying DADS cytotoxicity and apoptosis in human colon cancer COLO 205 cells.