Apoptosis is a process of active cell death characterized by cell surface blebbing, changes in membrane permeability, nuclear DNA condensation, and internucleosomal fragmentation.1) Several apoptotic pathways exist in cells responsive to apoptotic stimuli, and although each pathway is initiated by different mechanisms, they share a common final phase of apoptosis, which involves the activation of executioner caspases and the dismantling of substrates critical for cell survival.2) Classical apoptosis can be initiated via two distinct biochemical mechanisms, both of which result in caspase activation: (i) the extrinsic pathway that involves ligation of cell surface death receptors by their respective ligands, and (ii) the intrinsic mitochondrial pathway, which is largely controlled by the Bcl-2 family of pro-and anti-apoptotic proteins. It has been well established that the expressions of many gene products, such as Bid, Bax and Bcl-2, are critical for programmed cell death. Bcl-2 and Bcl-xl have anti-apoptotic functions, whereas Bid, Bax, Bim and Bcl-xs promote apoptosis. The balance between these two groups of genes determines the fate of cells in many systems, 3) and in particular, determines the depolarization of the mitochondrial membrane and the release of cytochrome c from mitochondria into the cytosol.4) This released cytochrome c then binds to apoptotic protease activating factor-1 (Apaf-1), which causes its oligomerization. 5) Procaspase-9 then binds to Apaf-1 oligomers to form a high-molecular-mass complex called apoptosome.6) In addition, this interaction activates caspase-9, which activates caspase-3 and thus triggers the irreversible apoptotic program. 7)Donepezil (R,S-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methylpiperidine hydrochloride) is prescribed for the treatment of cognitive dysfunction in Alzheimer's disease (AD).8) It prevents the hydrolysis of acetylcholine (ACh) and elevates ACh concentrations in the synaptic cleft, which increases cholinergic transmission.9) Previous studies haveshown that donepezil has neuroprotective effects in addition to its AChE-inhibiting action, and that these effects are due to the stimulation of nicotinic acetylcholine receptors, the activation of the phosphoinositide-3 kinase/Akt pathway, and the up-regulation of the anti-apoptotic protein Bcl-2. 10,11) However, no report has been issued on the molecular mechanism underlying the anti-proliferative effects of donepezil.In the present study, we demonstrate that donepezilinduced apoptosis involves the mitochondrial pathway via loss of mitochondrial membrane potential, a hallmark of permeability transition, and that this causes the release of cytochrome c to cytosol, and the sequential activations of caspases-8, -9, and -3 via the modulation of Bcl-2 in human promyelocytic leukemia HL-60 cells. Furthermore, this is the first report to show that donepezil induces apoptosis through a mitochondria-mediated caspase-dependent pathway. Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea: and d Eisai Korea ...

Despite of the widespread use of the Papanicolaou smear and HPV (human papollomavirus) vaccine, cervical cancer remains the most common cancer in women under 45 years of age in developing countries. Furthermore, almost half of all women with invasive cervical cancer are diagnosed at a late stage. Accordingly, there is an urgent need to identify an agent that can be used alone or in combination with a conventional anti-cancer drug like cisplatin, to improve the prognosis of this disease. 1)Apoptosis, a morphologically and biochemically defined form of cell death, plays a key role in the regulation of the growth of normal and neoplastic tissues.2) Recent studies have amply documented that two major pathways are involved in the regulation of apoptosis. The extrinsic apoptotic pathway involves cell surface death receptors such as Fas/CD95 and tumor necrosis factor receptor 1, 2 (TNFR1, 2), which upon activation up-regulate downstream signaling cascade including the activation of caspase-8.2) The intrinsic pathway is dependent on various cell stress stimuli leading to altered ratio of Bcl-2 family members affecting the release of cytochrome c and apoptotic protease activating factor-1 (Apaf-1) that leads to caspase-9 activation.3) The active forms of caspase-8 and -9 may activate downstream effector caspases-3, -6, and -7, resulting in the cleavage of several intracellular polypeptides (e.g., PARP-1) as well as activation of DNase that leads to DNA fragmentation.Endoplasmic reticulum (ER) is a central organelle engaged in lipid synthesis and protein folding and maturation. A variety of toxic insults, including failure of protein synthesis, folding, and degradation and Ca 2ϩ overload, can disturb the ER function and result in ER stress. 4,5) Under normal conditions, the ER stress sensors (IRE1, PERK, and ATF6) reside with binding to glucose-regulated protein 78 (GRP78). When ER stress occurred, the sensors dissociate with GRP78 and transfer downstream signals to the cytoplasm. As a consequence, these compensatory responses contribute to the reduction of ER overload and functions to protect cells against ER stress. However, if the ER functions are severely affected, cells are removed by apoptosis. ER stress-mediated apoptosis is triggered by the activation of ER membrane resident caspase-12 and induction of growth arrest DNA damageinducible gene 153 (GADD153).6,7) Calpains (a family of Ca 2ϩ -dependent intracellular cysteine proteases) are also implicated in ER stress-induced apoptosis. There are ubiquitously expressed calpain I (m-calpain) and calpain II (m-calpain) proteases. A recent study has shown that ubiquitous calpains promote caspase-12 and c-Jun N-terminal kinase (JNK) activation during ER stress-induced apoptosis. 8)Numerous naturally occurring substances are recognized to be anti-oxidants, cancer preventive agents, or even antitumor agent such as paclitaxel.9) Triterpenes are part of a family of naturally occurring compounds and present a wide spectrum of pharmacological activities, with one of the most elu...

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In our previous study, we reported the isolation of pennogenin-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranoside (spiroconazol A), a steroidal saponin, from the flowers of Dioscorea bulbifera L. var. sativa. In the present study, we aimed to investigate the effects of spiroconazol A on autophagy and its underlying mechanisms in A549 and NCI-H358 human non-small cell lung cancer (NSCLC) cells. Spiroconazol A inhibited the proliferation of NSCLC cells in a concentration- and time-dependent manner. To determine the type of programmed cell death induced by spiroconazol A, we performed a characterization of apoptosis in spiroconazol A-treated A549 cells. Our results showed that spiroconazol A significantly suppressed A549 cell viability but did not influence cell apoptosis because phosphatidylserine and caspase activation were not detected. Furthermore, spiroconazol A treatment upregulated the expression of LC3-II and autophagy-related Beclin-1 protein, suggesting that spiroconazol A induces autophagy in A549 cells. Moreover, spiroconazol A activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but did not affect the phosphorylation of Janus kinase or ERK1/2. Notably, SB203580, a p38 MAPK inhibitor, had a significant inhibitory effect on spiroconazol A-induced autophagic cell death in A549 cells. Our results indicated that spiroconazol A-induced autophagy is dependent on p38 MAPK signaling and has potential as a therapeutic target in NSCLC.

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