Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer

Carcinogenesis, Mar 2005

Aspirin-induced apoptosis is one of the important mechanisms for its antitumour effect against gastric cancer. We aimed at investigating the involvement of bcl-2 family members in the apoptotic pathway in gastric cancer. Gastric cancer cell line AGS and MKN-45 were observed as to cell growth inhibition and induction of apoptosis in response to treatment with aspirin. Cell proliferation was measured by MTT assay. Apoptosis was determined by 4′-6-diamidino-2-phenylindole staining. Protein expression was determined by western blotting. We showed that aspirin activated caspase-8, caspase-9 and capase-3, cleaved and translocated Bid, induced a conformational change in and translocation of Bax and cytochrome c release. In addition, suppression of caspase-8 with the specific inhibitor z-IETD-fmk, as well as the pan-caspase inhibitor z-VAD-fmk, prevented Bid cleavage and subsequent apoptosis. The caspase inhibitors failed to abolish the effects on Bax activation. In conclusion, our results identify a role of caspase-8/Bid and activation of Bax as a novel mechanism for aspirin-induced apoptosis in gastric cancer.

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Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer

Carcinogenesis Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer Qing Gu 1 2 Ji De Wang 1 Harry H.X.Xia 1 Marie C.M.Lin 0 Hua He 1 Bing Zou 1 Shui Ping Tu 1 Yi Yang 1 Xin Guang Liu 2 Shiu Kum Lam 1 Wai Man Wong 1 Annie O.O.Chan 1 Man Fung Yuen 1 Hsiang Fu Kung 0 Benjamin Chun-Yu Wong 1 0 Institute of Molecular Biology, University of Hong Kong , Hong Kong 1 Department of Medicine, University of Hong Kong, Queen Mary Hospital , Hong Kong 2 Department of Gastroenterology, First Hospital, Peking University , Beijing , People's Republic of China 4To whom correspondence should be addressed. Tel: þ86 852 2855 4541; Fax: þ86 852 2872 5828; Email: Aspirin-induced apoptosis is one of the important mechanisms for its antitumour effect against gastric cancer. We aimed at investigating the involvement of bcl-2 family members in the apoptotic pathway in gastric cancer. Gastric cancer cell line AGS and MKN-45 were observed as to cell growth inhibition and induction of apoptosis in response to treatment with aspirin. Cell proliferation was measured by MTT assay. Apoptosis was determined by 40-6-diamidino-2-phenylindole staining. Protein expression was determined by western blotting. We showed that aspirin activated caspase-8, caspase-9 and capase-3, cleaved and translocated Bid, induced a conformational change in and translocation of Bax and cytochrome c release. In addition, suppression of caspase-8 with the specific inhibitor z-IETD-fmk, as well as the pan-caspase inhibitor z-VAD-fmk, prevented Bid cleavage and subsequent apoptosis. The caspase inhibitors failed to abolish the effects on Bax activation. In conclusion, our results identify a role of caspase-8/Bid and activation of Bax as a novel mechanism for aspirin-induced apoptosis in gastric cancer. Introduction Apoptosis is mediated through two major pathways, the death receptor pathway (extrinsic) and the mitochondrial pathway (intrinsic). In the extrinsic pathway, stimulation of death receptors such as Fas and TNFR1 leads to clustering and formation of a death-inducing signaling complex which includes the adapter protein FADD (Fas-associated death domain) and initiator caspases such as caspase-8. Caspase-8 drives its activation through self-cleavage and then activates downstream effector caspases such as caspase-9 and caspase-3 (1). In the intrinsic pathway death receptors transmit the death signals to mitochondria, resulting in the release of several mitochondrial intermembrane space proteins, such as cytochrome c, which associate with Apaf-1 and procaspase-9 to form the apoptosome (2). Abbreviations: DAPI, 40-6-diamidino-2-phenylindole; GFP, green fluorescent protein; PARP, poly(ADP-ribose) polymerase; PBS, phosphatebuffered saline; tBid, truncated Bid; TNF, tumor necrosis factor. The mitochondrial pathway is controlled and regulated by the Bcl-2 family proteins. The anti-apoptotic subfamily comprises Bcl-2 and Bcl-xL. The multidomain pro-apoptotic subfamily consists of Bax and Bak and the BH3 domainonly proteins include Bad and Bim (3). Pro- and antiapoptotic Bcl-2 family members converge on mitochondria in response to the death signal and compete to regulate the release of cytochrome c (2). The pro-apoptotic proteins Bax and Bak are required for induction of apoptosis by the mitochondrial pathway. Bax is localized in the cytosol of normal cells and translocated to mitochondria after apoptotic stimulation (3,4). Bax-dependent Smac and Omi release plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer (5). Most of the BH3 domain-only proteins are localized outside the mitochondria in living cells and translocate to mitochondria by several different mechanisms after apoptotic stimulation, leading to activation of proapoptotic proteins such as Bax and Bak (3). However, It is not clear how the BH3 domain-only proteins (e.g. Bid) transmit the apoptotic signal to the pro-apoptotic family such as Bax and Bak. Aspirin has potent analgesic, anti-pyretic and antiinflammatory actions and reduces the risk of gastric and colon cancer (6,7). Some studies have reported that aspirin induces apoptosis with release of cytochrome c from mitochondria (8--11). However, others suggested that aspirin induces cell cycle arrest and causes necrosis at high concentrations in vitro but does not induce apoptosis (12). Therefore, whether or not aspirin induces apoptosis and the molecular mechanisms involved are not clear. In the present study we have confirmed that aspirin induces apoptosis in gastric cancer cells and demonstrated that caspase-8/Bid and a conformational change in and translocation of Bax are involved in aspirin-induced apoptosis. Materials and methods Chemicals Aspirin and Anti-Bax 6A7 monoclonal antibody were purchased from Sigma (St Louis, MO). Anti-Bax (N-20) polyclonal antibody, anti-caspase3, anti-PARP, anti-cytochrome c were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anticaspa (...truncated)


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Qing Gu, Ji De Wang, Harry H.X. Xia, Marie C.M. Lin, Hua He, Bing Zou, Shui Ping Tu, Yi Yang, Xin Guang Liu, Shiu Kum Lam, Wai Man Wong, Annie O.O. Chan, Man Fung Yuen, Hsiang Fu Kung, Benjamin Chun-Yu Wong. Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer, Carcinogenesis, 2005, pp. 541-546, 26/3, DOI: 10.1093/carcin/bgh345