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)