Pennogenin-3-O-α-L-Rhamnopyranosyl-(1→2)-[α-L-Rhamnopyranosyl-(1→3)]-ß-D-Glucopyranoside (Spiroconazol A) Isolated from Dioscorea bulbifera L. var. sativa Induces Autophagic Cell Death by p38 MAPK Activation in NSCLC Cells.

In our previous study, we reported the isolation of pennogenin-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-ß-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.

CWJ-081, a novel 3-arylisoquinoline derivative, induces apoptosis in human leukemia HL-60 cells partially involves reactive oxygen species through c-Jun NH2-terminal kinase pathway.

In the present study, we investigated the effect of a novel 3-arylisoquinoline derivative 3-(6-ethyl-benzo[1,3]dioxol-5-yl)-7,8-dimethoxy-2-methyl-2H-isoquinolin-1-one (CWJ-081) on the induction of apoptosis and the putative molecular mechanism of its action in human leukemia cells. Treatment with CWJ-081 exhibited a characteristic feature of apoptosis including externalization of phosphatidylserine and formation of DNA fragmentation in human leukemia cell lines (HL-60, U-937, K-562). In addition, stimulation of HL-60 cells with CWJ-081 induced a series of intracellular events: (1) the activations of caspase-8, -9, and -3; (2) the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1); (3) the loss of mitochondrial membrane potential (ΔΨ(m)); (4) the release of cytochrome c; and (5) the modulation of Bcl-2 family proteins. We further demonstrated that CWJ-081 induces reactive oxygen species (ROS) production and c-Jun NH(2)-terminal kinase (JNK) activation. Pretreatment with the antioxidant N-acetyl-L-cysteine (NAC) markedly inhibited the CWJ-081-induced JNK activation and apoptosis. Moreover, CWJ-081-induced apoptosis was suppressed in the presence of SP600125, a specific JNK inhibitor. Taken together, these data suggest that CWJ-081 induces apoptosis via the mitochondrial apoptotic pathway in HL-60 cells, and ROS-mediated JNK activation plays a key role in the CWJ-081-induced apoptosis.

3α,23-isopropylidenedioxyolean-12-en-27-oic acid, a triterpene isolated from Aceriphyllum rossii, induces apoptosis in human cervical cancer HeLa cells through mitochondrial dysfunction and endoplasmic reticulum stress.

In the present study, we investigated the effect of 3alpha,23-isopropylidenedioxyolean-12-en-27-oic acid (IPA), an active compound isolated from Aceriphyllum rossii, on the apoptotic activity and the molecular mechanism of the action in human cervical cancer HeLa cells. Treatment with IPA significantly increased externalization of phosphatidylserine residues and apoptotic DNA fragmentation as shown by Annexin V staining and 4',6-diamidino-2-phenylindole-dihydrochloride (DAPI) staining, respectively. In addition, IPA induced the activations of caspase-8, -9, -3, and cleavage of poly(ADP ribose) polymerase (PARP-1) in HeLa cells. Pretreatment with a specific caspase-8, -9, or -3 inhibitor neutralized the pro-apoptotic activity of IPA in HeLa cells. Furthermore, IPA was found to induce the loss of mitochondrial membrane potential, the release of cytochrome c to the cytosol, and the increased ratio of mitochondrial Bax/Bcl-2. Moreover, we demonstrated that IPA triggered endoplasmic reticulum (ER) stress, as shown by changes in cytosol-calcium level, activation of mu-calpain and caspase-12, and up-regulation of glucose-regulated protein 78 (GRP78) and growth arrest DNA damage-inducible gene 153 (GADD153). IPA-induced apoptosis was substantially reduced in the presence of an intracellular calcium chelator BAPTA/AM. Taken together, these results suggest that both mitochondrial dysfunction and ER stress contribute to IPA-induced apoptosis of human cervical cancer HeLa cells.

Donepezil, a potent acetylcholinesterase inhibitor, induces caspase-dependent apoptosis in human promyelocytic leukemia HL-60 cells.

Although donepezil, a potent acetylcholinesterase (AChE) inhibitor, has been used to treat Alzheimer's disease (AD) due to its neuroprotective effects, its mode of action to inhibit the growth of cancer cells is poorly understood. In the present study, we investigated the pro-apoptotic activities of donepezil in HL-60 human promyelocytic leukemia cells and the underlying molecular mechanism involved. It was found that donepezil induced the apoptosis of HL-60 and U937 cells in a dose- and time-dependent manner, as evidenced by the formation of DNA fragmentation and the accumulation of positive cells for Annexin V. In addition, the activations of caspase-8, -9, and -3 were significantly increased 36 h after donepezil treatment. Furthermore, the broad caspase inhibitor (z-VAD-fmk) blocked donepezil-induced apoptosis. In addition, donepezil was found to cause the loss of mitochondrial membrane potential (DeltaPsi(m)), to increase the release of cytochrome c to the cytosol, and to alter the expressions of Bcl-2 family proteins. Taken together, these results demonstrate for the first time that donepezil displayed an induction of apoptosis in HL-60 cells via a mitochondria-mediated caspase-dependent pathway.