J Pharmacol Exp Ther. 2006 Sep 21.
Reactive Oxygen Species Mediate Caspase Activation and Apoptosis Induced by Lipoic Acid in Human Lung Epithelial Cancer Cells through Bcl-2 Downregulation.
Moungjaroen J, Nimmannit U, Callery PS, Wang L, Azad N, Lipipun V, Chanvorachote P, Rojanasakul Y.
West Virginia University.
[ PubMed ] [ Free full text ( pdf) ]

Abstract: The antioxidant alpha-lipoic acid (LA) is a naturally-occurring compound that has been shown to possess promising anticancer activity due to its ability to preferentially induce apoptosis and inhibit proliferation of cancer cells relative to normal cells. However, the molecular mechanisms underlying the apoptotic effect of LA are not well understood. We report here that LA induced reactive oxygen species (ROS) generation and a concomitant increase in apoptosis of human lung epithelial cancer H460 cells. Inhibition of ROS generation by ROS scavengers or by overexpression of antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD) effectively inhibited LA-induced apoptosis, indicating the role of ROS, especially hydroperoxide and superoxide anion, in the apoptotic process. Apoptosis induced by LA was found to be mediated through the mitochondrial death pathway which requires caspase-9 activation. Inhibition of caspase activity by pan-caspase inhibitor (z-VAD-fmk) or caspase-9-specific inhibitor (z-LEHD-fmk) completely inhibited the apoptotic effect of LA. Likewise, the mitochondrial respiratory chain inhibitor rotenone potently inhibited the apoptotic and ROS inducing effects of LA, supporting the role of mitochondrial ROS in LA-induced cell death. LA induced downregulation of mitochondrial Bcl-2 protein through peroxide-dependent proteasomal degradation and overexpression of the Bcl-2 protein prevented the apoptotic effect of LA. Together, our findings indicate a novel pro-oxidant role of LA in apoptosis induction and its regulation by Bcl-2, which may be exploited for the treatment of cancer and related apoptosis disorders.

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Introduction

alpha-Lipoic acid (LA) is a naturally-occurring essential co-enzyme in mitochondrial multienzyme complexes catalyzing the oxidative decarboxylation of alpha-keto acids such as pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acid (Packer et al., 1995; Bilska and Wlodex, 2005). LA has been shown to combat oxidative stress by quenching a variety of intracellular reactive oxygen species (ROS) (Suzuki et al., 1991; Bilska and Wlodex, 2005). In addition to ROS scavenging, LA has also been shown to be involved in the recycling of other cellular antioxidants including vitamins C and E, and glutathione (Biewenga et al., 1997). LA has been demonstrated to be effective in preventing pathology in various experimental models in which ROS have been implicated, such as ischemia-reperfusion injury (Coombes et al., 2000), diabetes (Kocak and Karasu, 2002; Da Ros et al., 2005), diabetic neuropathy (Vincent et al., 2005), neurodegeneration (Pirlich et al., 2002), hypertension (de Champlain et al., 2004; Vasdev et al., 2005), radiation injury (Demir et al., 2005), and HIV activation (Patrick, 2000). On the other hand, LA has been reported to possess pro-oxidant activities (Dicter et al., 2002; Gorolska et al., 2003; Cakatay et al., 2005). For examples, LA dose dependently increases intramuscular ROS production and stimulates glucose uptake into adipocytes by increasing intracellular oxidant levels (Dicter et al., 2002). In cancer cells, ROS also play a crucial role in cell growth and apoptosis regulation. LA and its reduced form dihydrolipoic acid (DHLA) have been shown to inhibit proliferation and induce apoptosis of several cancer and transformed cell lines, while being less active toward normal non-transformed cells (Sen et al., 1999; Pack et al., 2001; Mark et al., 2003; Wenzel et al., 2005).

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LA has been shown to act as a pro-oxidant (Dicter et al., 2002; Gorolska et al., 2003; Cakatay et al., 2005) as well as anti-oxidant (Suzuki et al., 1991; Bilska and Wlodex, 2005), depending on cell type and cellular oxidative status. The antioxidant role of LA is commonly associated with cells under oxidative stress and this action of LA has been attributed to its ability to regenerate other cellular antioxidants such as vitamin C and E, and glutathione (4). The pro-oxidant role of LA is generally observed under non-oxidative stress conditions, which is also supported by this study. In human colon cancer HT29 cells, LA was shown to act a pro-oxidant by increasing mitochondrial O2•- generation (Wenzel et al., 2005). The colon cancer cells were also shown to possess a lower antioxidant status and are more susceptible to LA-induced apoptosis as compared to normal non-transformed cells, thus providing a basis for the selective effect of LA on cancer cells.

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The therapeutic potential of LA in cancer treatment has been demonstrated in several studies (Sen et al., 1999; Pack et al., 2001; Mark et al., 2003; Wenzel et al., 2005). Previously, chemotherapeutic agents such as doxorubicin, cisplatin, vincristine, and the alkaloid taxol have commonly been used as anti-tumor agents. However, at high concentrations these drugs are toxic to cells and cause adverse side-effects. In contrast, LA is an endogenous agent that has been widely used as a dietary supplement. It is known to increase cellular glutathione levels, regulate cellular redox balance and help protect against diabetic complications (Sen et al., 1997; Da Ros et al., 2005; Kocak and Karasu, 2002).