• Toxicological Research
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• v.29 no.4
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• pp.257-261
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• 2013

${\alpha}$-Curcumene is one of the physiologically active components of Curcuma zedoaria, which is believed to perform anti-tumor activities, the mechanisms of which are poorly understood. In the present study, we investigated the mechanism of the apoptotic effect of ${\alpha}$-curcumene on the growth of human overian cancer, SiHa cells. Upon treatment with ${\alpha}$-curcumene, cell viability of SiHa cells was inhibited > 73% for 48 h incubation. ${\alpha}$-Curcumene treatment showed a characteristic nucleosomal DNA fragmentation pattern and the percentage of sub-diploid cells was increased in a concentration-dependent manner, hallmark features of apoptosis. Mitochondrial cytochrome c activation and an in vitro caspase-3 activity assay demonstrated that the activation of caspases accompanies the apoptotic effect of ${\alpha}$-curcumene, which mediates cell death. These results suggest that the apoptotic effect of ${\alpha}$-curcumene on SiHa cells may converge caspase-3 activation through the release of mitochondrial cytochrome c.

• Journal of Nutrition and Health
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• v.48 no.5
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• pp.451-456
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• 2015

Purpose: Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimer's disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride ($CoCl_2$)-mediated hypoxic death in human neuroblastoma SH-SY5Y cells. Methods: Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2',7'-dichlorofluorescin diacetate (DCF-DA) and 4',6-diamidino-2-phenylindole (DAPI) staining. Results: Exposure to $CoCl_2$, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the $CoCl_2$-induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel. Conclusion: Based on these data, fermented C. sunki peel extract might have a protective effect against $CoCl_2$-induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis.

• Journal of Ginseng Research
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• v.39 no.1
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• pp.22-28
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• 2015

Background: Sun ginseng (SG), a specific formulation of quality-controlled red ginseng, contains approximately equal amounts of three major ginsenosides (RK1, Rg3, and Rg5), which reportedly has antitumor-promoting activities in animal models. Methods: MTT assay was used to assess whether SG can potentiate the anticancer activity of epirubicin or paclitaxel in human cervical adenocarcinoma HeLa cells, human colon cancer SW111C cells, and SW480 cells; apoptosis status was analyzed by annexin V-FITC and PI and analyzed by flow cytometry; and apoptosis pathway was studied by analysis of caspase-3, -8, and -9 activation, mitochondrial accumulation of Bax and Bak, and cytochrome c release. Results: SG remarkably enhances cancer cell death induced by epirubicin or paclitaxel in human cervical adenocarcinoma HeLa cells, human colon cancer SW111C cells, and SW480 cells. Results of the mechanism study highlighted the cooperation between SG and epirubicin or paclitaxel in activating caspase-3 and -9 but not caspase-8. Moreover, SG significantly increased the mitochondrial accumulation of both Bax and Bak triggered by epirubicin or paclitaxel as well as the subsequent release of cytochrome c in the targeted cells. Conclusion: SG significantly potentiated the anticancer activities of epirubicin and paclitaxel in a synergistic manner. These effects were associated with the increased mitochondrial accumulation of both Bax and Bak that led to an enhanced cytochrome c release, caspase-9/-3 activation, and apoptosis. Treating cancer cells by combining epirubicin and paclitaxel with SG may prove to be a novel strategy for enhancing the efficacy of the two drug types.

• Journal of Life Science
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• v.19 no.2
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• pp.163-168
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• 2009

To understand cytotoxic activity of Rubia cordifolia L. (Rubiaceae), which has been used as a traditional oriental medicine, the mechanism underlying cytotoxic effect of its extract on human acute Jurkat T cells was investigated. The methanol extract of roots (3 kg) of R. codifolia was evaporated, dissolved in water, and then extracted by dichloromethane. The substances in the chloroform extract showing the most cytotoxic activity were further purified by a series of preparative HPLC. The extracted active substance (65 mg) was designated as CCH1. When Jurkat T cells were treated with CCH1 at concentration ranging from 0.5 to 2.0 ${\mu}g$/ml, apoptotic phenomena of cells companying several subsequent biochemical reactions such as mitochondria cytochrome c release, activation of casapase-8, -9, and caspase- 3, degradation of PARP and DNA fragmentation occurred via mitochondria-dependent pathway. However, abrogation of apoptosis was observed in an ectopic expression of Bcl-xL, which is a suppressor for mitochondrial cytochrome c release. These results demonstrate that the cytotoxicity of CCH1 against Jurkat T cells is attributable to apoptosis mediated by mitochodria-dependent death-signaling regulated by Bcl-xL. In addition, the CCH1 is more potent to leukemia Jurkat T cell than to human peripheral blood monocyte cells (PBMC).

• Journal of Life Science
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• v.27 no.8
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• pp.951-957
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• 2017

To understand the cytotoxic activity of Machilus thunbergii, which has been used as a traditional oriental medicine, the mechanism underlying the cytotoxic effect of its extract on human acute Jurkat T cells was investigated. The methanol extract of roots (3 kg) of M. thunbergii was evaporated, dissolved in, and then extracted by water. The water-extracted active substance was designated MTWE. When Jurkat T cells were treated with MTWE at concentrations of 0, 25, 50, and $100{\mu}g/ml$, the apoptotic phenomenon of cells accompanying several subsequent biochemical reactions, such as mitochondrial cytochrome c release, activation of caspase-3, and ICAD degradation, was detected in the Jurkat T cells. Moverover. the expression of Bcl-xL, which is a suppressor for mitochondrial cytochrome c release pathway, was reduced in the Jurkat T cells. As DUSP6, a growth suppressor of cancer cells, ranged from 0, 25, 50, $100{\mu}g/ml$ of MTWE, the expression level was elevated in the Jurkat T cells. The apoptotic morphological change of the nuclei was observed by DAPI staining. Although the potential involvement of the other factors and DUSP6 is currently being investigated in more detail, these findings support the notion that MTWE is able to achieve the apoptosis of Jurkat T cells, and it seems that MTWE is useful as a method of evaluating a chemotherapeutic agent or tonic materials for human acute leukemia.

• Environmental Analysis Health and Toxicology
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• v.18 no.2
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• pp.89-94
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• 2003

Tributyltin (TBT) used world-wide in antifouling paints for ships is a widespread environmental pollutant and cause reproductive organs atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of toxicity in reproductive organs by TBT. In this study, we investigated that the mechanisms underlying DNA fragmentation induced by TBT in the rat leyding cell line, R2C. Effects of TBT on intracellular Ca$\^$2+/ level and reactive oxygen species (ROS) were investigated in R2C cells by fluorescence detector. TBT significantly induced intracellular Ca$\^$2+/ level in a time-dependent manner. The rise in intracellular Ca$\^$2+/ level was followed by a time-dependent generation of reactive oxygen species (ROS) at the cytosol level. Simultaneously, TBT induced the release of cytochrome c from the mitochondrial membrane into the cytosol. Furthermore, ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca$\^$2+/ chelator, indicating the important role of Ca$\^$2+/ in R2C during these early intracellular events. In addition, Z-DEVD FMK, a caspase-3 inhibitor, decreased apoptosis by TBT. Taken together, the present results indicated that the apoptotic pathway by TBT might start with an increase in intracellular Ca$\^$2+/ level, continues with release of ROS and cytochrome c from mitochondria, activation of caspases,and finally results in DNA fragmentation.

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

• Molecules and Cells
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• v.46 no.9
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• pp.545-557
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• 2023

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg²⁺-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Fe²⁺, and Fe³⁺ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg²⁺-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.2
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• pp.115-121
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• 2009

Functional defects in mitochondria are involved in the induction of cell death in cancer cells. We assessed the toxic effect of camptothecin against the human cervical and uterine tumor cell line SiHa with respect to the mitochondria-mediated cell death process, and examined the combined effect of camptothecin and anticancer drugs. Camptothecin caused apoptosis in SiHa cells by inducing mitochondrial membrane permeability changes that lead to the loss of mitochondrial membrane potential, decreased Bcl-2 levels, cytochrome c release, caspase-3 activation, formation of reactive oxygen species and depletion of GSH. Combination of camptothecin with other anticancer drugs (carboplatin, paclitaxel, doxorubicin and mitomycin c) or signaling inhibitors (farnesyltransferase inhibitor and ERK inhibitor) did not enhance the camptothecin-induced cell death and caspase-3 activation. These results suggest that camptothecin may cause cell death in SiHa cells by inducing changes in mitochondrial membrane permeability, which leads to cytochrome c release and activation of caspase-3. This effect is also associated with increased formation of reactive oxygen species and depletion of GSH. Combination with other anticancer drugs (or signaling inhibitors) does not appear to increase the anti-tumor effect of camptothecin against SiHa cells, but rather may reduce it. Combination of camptothecin with other anticancer drugs does not seem to provide a benefit in the treatment of cervical and uterine cancer compared with camptothecin monotherapy.

• Proceedings of the PSK Conference
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• 2002.04a
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• pp.124.1-124.1
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• 2002