• Molecules and Cells
• /
• v.24 no.1
• /
• pp.95-104
• /
• 2007

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition ($IC_{50}$) of MCF-7 cells at $26.4{\pm}0.7{\mu}M$ over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with $100{\mu}M$ acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun $NH_4$-terminal kinase 1/2 (SAPK/JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.

• Journal of Life Science
• /
• v.25 no.11
• /
• pp.1255-1264
• /
• 2015

Cnidium officinale, a traditional herb, has diverse beneficial pharmacological activities, such as anti-inflammatory, antioxidant, anticancer, and antiangiogenesis effects. However, the cellular and molecular mechanisms of apoptosis by C. officinale are poorly defined. The present study investigated the proapoptotic effects of water, ethanol, and methanol extract of C. officinale (WECO, EECO, and MECO, respectively) in human leukemia U937 cells. The antiproliferative activity of EECO was higher than that of WECO and MECO. The antiproliferative effect of EECO treatment in U937 cells was associated with the induction of apoptotic cell death, including increased populations of annexin-V positive cells, the formation of apoptotic bodies, DNA fragmentation, and increased numbers of cells with a loss of mitochondrial membrane potential (MMP, Δψm). EECO-induced apoptotic cell death was associated with upregulation of death receptor 4 (DR4) and down-regulation of cellular inhibitor of apoptosis protein-1 (cIAP-1), Bcl-2, and total Bid. The EECO treatment also induced the proteolytic activation of caspases (-3, -8, and -9), and degradation of caspase-3 substrate proteins, such as poly(ADP-ribose) polymerase (PARP), β-catenin, and phospholipase C-γ1 (PLCγ1). In addition, the EECO treatment effectively activated the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. However, compound C, a specific inhibitor of AMPK, significantly reduced EECO-induced apoptosis. These results indicate that AMPK is a key regulator of apoptosis in response to EECO in human leukemia U937 cells.

• Journal of Life Science
• /
• v.21 no.8
• /
• pp.1109-1119
• /
• 2011

In the present study, the pro-apoptotic effects of methanol extract of Prunus mume fruits (MEPM) in human leukemia U937 cells were investigated. It was found that exposure to MEPM resulted in growth inhibition in a concentration-dependent manner by inducing apoptosis. The induction of apoptotic cell death in U937 cells by MEPM was correlated with a down-regulation of inhibitor of apoptosis protein (IAP) family, such as X-linked inhibitor of apoptosis protein (XIAP) and survivin, anti-apoptotic Bcl-2, up-regulation of FasL and cleavage of Bid. MEPM treatment also induced the proteolytic activation of caspase-3, caspase-8 and caspase-9, and degradation of caspase-3 substrate proteins, such as poly (ADP-ribose) polymerase (PARP) and ${\beta}$-catenin. In addition, apoptotic cell death induced by MEPM was significantly inhibited by z-DEVD-fmk, a caspase-3 specific inhibitor, which demonstrates the important role of caspase-3 in the apoptotic process by MEPM in U937 cells. Taken together, these findings suggest that P. mume extracts may be a potential chemotherapeutic agent for the control of human leukemia cells and further studies will be needed to identify the active compounds.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.10
• /
• pp.4369-4376
• /
• 2015

Background: To investigate in-vitro antagonistic effect of low-dose liquiritigenin on gemcitabine-induced capillary leak syndrome (CLS) in pancreatic adenocarcinoma via inhibiting reactive oxygen species (ROS)-mediated signalling pathways. Materials and Methods: Human pancreatic adenocarcinoma Panc-1 cells and human umbilical vein endothelial cells (HUVECs) were pre-treated using low-dose liquiritigenin for 24 h, then added into gemcitabine and incubated for 48 h. Cell viability, apoptosis rate and ROS levels of Panc-1 cells and HUVECs were respectively detected through methylthiazolyldiphenyl-tetrazoliumbromide (MTT) and flow cytometry. For HUVECs, transendothelial electrical resistance (TEER) and transcellular and paracellular leak were measured using transwell assays, then poly (ADP-ribose) polymerase 1 (PARP-1) and metal matrix proteinase-9 (MMP9) activity were assayed via kits, mRNA expressions of p53 and Rac-1 were determined through quantitative polymerase chain reaction (qPCR); The expressions of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and PARP-1 were measured via western blotting. Results: Low-dose liquiritigenin exerted no effect on gemcitabine-induced changes of cell viability, apoptosis rate and ROS levels in Panc-1 cells, but for HUVECs, liquiritigenin ($3{\mu}M$) could remarkably elevate gemcitabine-induced decrease of cell viability, transepithelial electrical resistance (TEER), pro-MMP9 level and expression of ICAM-1 and VCAM-1 (p<0.01). Meanwhile, it could also significantly decrease gemcitabine-induced increase of transcellular and paracellular leak, ROS level, PARP-1 activity, Act-MMP9 level, mRNA expressions of p53 and Rac-1, expression of PARP-1 and apoptosis rate (p<0.01). Conclusions: Low-dose liquiritigenin exerts an antagonistic effect on gemcitabine-induced leak across HUVECs via inhibiting ROS-mediated signalling pathways, but without affecting gemcitabine-induced Panc-1 cell apoptosis. Therefore, low-dose liquiritigenin might be beneficial to prevent the occurrence of gemcitabine-induced CLS in pancreatic adenocarcinoma.

• Herbal Formula Science
• /
• v.25 no.1
• /
• pp.51-68
• /
• 2017

Objectives : Oxidative stress due to excessive accumulation of reactive oxygen species (ROS) is one of the risk factors for the development of several chronic diseases, including neurodegenerative diseases. Methods : In the present study, we investigated the protective effects of cheongnoemyeongsin-hwan (CNMSH) against oxidative stress‑induced cellular damage and elucidated the underlying mechanisms in neuronal-derived SH-SY5Y cells. Results : Our results revealed that treatment with CNMSH prior to hydrogen peroxide (H2O2) exposure significantly increased the SH-SY5Y cell viability, indicating that the exposure of the SH-SY5Y cells to CNMSH conferred a protective effect against oxidative stress. CNMSH also effectively attenuated H2O2‑induced comet tail formation, and decreased the phosphorylation levels of the histone ${\gamma}H2AX$, as well as the number of apoptotic bodies and Annexin V‑positive cells. In addition, CNMSH exhibited scavenging activity against intracellular ROS generation and restored the mitochondria membrane potential (MMP) loss that were induced by H2O2, suggesting that CNMSH prevents H2O2‑induced DNA damage and cell apoptosis. Moreover, H2O2 enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase, a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with CNMSH. Furthermore, CNMSH increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, CNMSH is able to protect SH-SY5Y cells from H2O2-induced apoptosis throughout blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating Nrf2/HO-1 signaling pathway. Conclusions : Therefore, we believed that CNMSH may potentially serve as an agent for the treatment and prevention of neurodegenerative diseases caused by oxidative stress.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.45 no.6
• /
• pp.911-917
• /
• 2016

Lespedeza cuneata G. Don is an edible perennial herb used in traditional Korean medicine. We investigated the anti-proliferative properties and mechanism of L. cuneata extract. The ethanolic extract of L. cuneata dose-and time-dependently inhibited human colorectal cancer cell proliferation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to test the effect of the extract on proliferation of HT-29 colorectal cancer cells. The extract inhibited HT-29 cell proliferation with an $IC_{50}$ value of $554.26{\pm}8.81{\mu}g/mL$. L. cuneata extract suppressed production of pro-inflammatory cytokines interleukin-6 and tumor necrosis $factor-{\alpha}$. Apoptosis was evaluated by analysis of DNA fragmentation, poly(ADP-ribose) polymerase cleavage, caspase-3 activity, and protein expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2). Our results demonstrated that the extract induced DNA fragmentation and characteristic morphological changes associated with apoptosis in HT-29 colorectal cancer cells. The extract also time- and dose-dependently up-regulated expression of the Bax and down-regulated expression of the Bcl-2. Furthermore, the extract dose- and time-dependently enhanced caspase-3 activity. Our findings provide evidence that L. cuneata extract may mediate its anti-proliferative effect via modulation of apoptosis.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.17 no.2
• /
• pp.394-402
• /
• 2003

This study was designed to investigate the protective effect of Bojungmyunyuk-dan(BJMY-Dan) on the cisplatin-induced cytotoxicity of primary rat astrocytes. BJMY-Dan is an oriental herbal prescription for its ability to recover protective effects against anti-cancer chemotherapies. After astrocytes were treated cisplatin, MTT assay was performed for cell viability test. To explore the mechanism of cytotoxicity, I used the several measures of apoptosis to determine whether this processes was involved in cisplatin-induced cell damage in astrocytes. Also, astrocytes were treated with BJMY-Dan and then, followed by the addition of cisplatin. Cisplatin decreased the viability of astrocytes in a dose and time-dependent manner. BJMY-Dan increased the viability of astrocytes treated cisplatin. Astrocytes treated cisplatin were revealed as apoptosis characterized by nuclear staining and flow cytometry. BJMY-Dan protected astrocytes from cisplatin-induced nuclear fragmentation and chromatin condensation. Also, caspase-3 and caspase-9 proteases were activated in astrocytes by cisplatin. BJMY-Dan inhibited the activation of caspase proteases in cisplatin-treated astrocytes. Cleavage of [poly(ADP-ribose) polymerase](PARP) was occurred at 12hr after treatment of cisplatin in astrocytes. BJMY-Dan recovered the cleavage of PARP in cisplatin-treated astrocytes. Also, BJMY-Dan inhibited the activation of pro-apoptotic factor, Bak by cisplatin. Lastly, astrocytes stained with JC-1 and Rhodamine 123 were photographed by fluorescence microscope to visualize changes of mitochondrial membrane permeability transition(MPT) during treatment with cisplatin for 24hr. BJMY-Dan recovered the change of MPT by cisplatin in astrocytes. According to above results, BJMY-Dan may protect astrocytes from cytotoxicity induced by chemotherapeutic agents, including cisplatin.

• Journal of Life Science
• /
• v.25 no.1
• /
• pp.93-100
• /
• 2015

Pachymic acid (PA) is a lanostane-type triterpenoid derived from the Poria cocos mushroom. Several beneficial biological features of PA provide medicine with a wide variety of valuable effects, such as anticancer and anti-inflammatory activity; it also has antioxidant effects against oxidative stress. Nonetheless, the biological properties and mechanisms that produce this anti-cancer action of PA remain largely undetermined. In this study, we investigated the pro-apoptotic effects of PA in T24 human bladder cancer cells. It was found that PA could inhibit the cell growth of T24 cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies and chromatin condensation and accumulation of cells in the sub-G1 phase. The induction of apoptotic cell death by PA was connected with an up-regulation of pro-apoptotic Bax and Bad protein expression and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL proteins, and inhibition of apoptosis family proteins. In addition, apoptosis-inducing concentrations of PA induced the activation of caspase-9, an initiator caspase of the mitochondrial-mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. PA also induced apoptosis via a death receptor-mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. Taken together, the present results suggest that PA may be a potential chemotherapeutic agent for the control of human bladder cancer cells.

• Journal of Life Science
• /
• v.18 no.6
• /
• pp.804-813
• /
• 2008

Cordyceps militaris, the Chinese medicinal fungal genus Cordyceps, is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, anti-virus and anti-infection activities. However, the molecular mechanisms of C. militaris on biochemical actions in cancer have not been clearly elucidated yet. In the present study, we investigated the anti-proliferative activity of the water extract of C. militaris (WECM) in human hepatocellular carcinoma HepG2 cells. It was found that WECM could inhibit the cell growth in a dose-dependent manner, which was associated with morphological changes and apoptotic cell death such as formation of apoptotic bodies and increased populations of apoptotic sub-G1 phase. Apoptotic cell death of HepG2 cells by WECM was connected with a up-regulation of pro-apoptotic Bax expression, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21 (WAF1/CIP1). In addition, WECM treatment induced the proteolytic activation of caspase-3 and a concomitant degradation and/or inhibition of poly (ADP-ribose) polymerase (PARP), ${\beta}-catenin$ and phospholipase $(PLC)-{\gamma}1$ protein. Furthermore, caspase-3 inhibitor, z-DEVD-fmk, significantly inhibited WECM-induced apoptosis demonstrating the important role of caspase-3 in the observed cytotoxic effect. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of C. militaris.

• Journal of Life Science
• /
• v.23 no.8
• /
• pp.1057-1063
• /
• 2013

Citri Pericarpium is one of the most commonly used traditional herbal medicines in Korea, China, and Japan. Its extracts have many properties including the treatment of indigestion and inflammatory respiratory syndromes such as bronchitis and asthma. However, the underlying molecular mechanisms of anti-cancer activity and molecular targets are not fully understood. In this work, we investigated the anti-proliferative activity of Citri Pericapium (EMCP) methanol extract on reactive oxygen species (ROS) production and the association of these effects with apoptotic cell death using U937 human leukemia cells in vitro. EMCP treatment decreased cell proliferation in a dose-dependent manner following an increase of the sub-G1 phase, the down-regulation of Bax proteins, the activation of caspases, the degradation of poly (ADP-ribose) polymerase proteins (PARP), and the induction of ROS generation. However, the quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the EMCP-induced apoptosis effects. In addition, heme oxygenase-1 expression also recovered by inhibiting the nuclear translocation of phosphorylated NF-E2-related factor 2. Taken together, our data indicate that ROS are involved as key mediators in the early molecular events in the EMCP-induced apoptotic pathway.