• Clinical and Experimental Reproductive Medicine
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• v.50 no.4
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• pp.213-222
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• 2023

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

• Herbal Formula Science
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• v.13 no.1
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• pp.71-83
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• 2005

Chelidonii Herba (Baekgulchae in Korean: CHE), a commonly used herb in Korea, Japan and China, is widely used in the treatment of stomach cancer, jaundice, gastric ulcer, edema and pain of stomach. In the present study, we demonstrated that CHE induces apoptosis in AGS cells, human stomach adenocarcinoma cell line. One of the most important recent advances in cancer research is the recognition that apoptosis plays a major role in both tumor formation and treatment response, In this study, CHE caused a decrease of viability in AGC cells. When AGS cells were treated with CHE, cells showed dose-dependent manner apoptotic cell death. Increased apoptotic cell death, exposured to CHE, resulted from induction of Bad translocation to mitochondria, cytochrome-c release from mitochondria to cytosol, activation of caspase-3, 8, 9, and PARP cleavage. These results suggest that CHE may be potential therapeutic approach in the clinical management of stomach adenocarcinoma.

• Molecular & Cellular Toxicology
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• v.3 no.1
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• pp.31-35
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• 2007

Hesperidin, known as a flavonoid constituent of citrus, has been known to reduce the proliferation of several cancer cells. We investigated whether hesperidin-induced cell death on SNU-668, human gastric cancer cells. The cytotoxicity of hesperidin on SNU-668 cells was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay at the concentration of 1, 10, 50, and 100 ${\mu}M$. Cell viability by hesperidin was 53.18$\pm$2.85% of control value at 100 ${\mu}M$. The cell death by hesperidin showed apoptotic features, which were confirmed using a combination of 4, 6-diamidino-2-phenylindole (DAPI) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. In the apoptosis-regulating genes, treatment of hesperidin decreased mRNA expression of B-cell CLL/lymphoma 2 (BCL2), whereas expression of BCL2-associated X protein (BAX) was increased. The mRNA expression and the activity of caspase3 (CASP3), a major apoptotic factor, was significantly increased by hesperidin treatment. These results suggest that hesperidin could induce apoptosis through CASP3 activation on SNU-668, human gastric cancer cells.

• Archives of Pharmacal Research
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• v.26 no.11
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• pp.937-942
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• 2003

Nitric oxide(NO) induces apoptosis in human osteoblasts. Treatment with exogenous NO donors, SNAP (S-Nitroso-N-acelylpenicillamine) and SNP (sodium nitroprusside), to MG-63 osteoblasts resulted in apoptotic morphological changes, as shown by a bright blue-fluorescent condensed nuclei and chromatin fragmentation by fluorescence microscope of Hoechst 33258-staining. The activities of caspase-9 and the subsequent caspase-3-like cysteine proteases were increased during NO-induced cell death. Pretreatment with Z-VAD-FMK (a pancaspase inhibitor) or Ac-DEVD-CHO (a specific caspase-3 inhibitor) abrogated the NO-induced cell death. The NO donor markedly activated JNK, a stress-activated protein kinase in the human osteoblasts. This study showed that the inhibition of the JNK pathway markedly reduced NO-induced cell death. But neither PD98059 (MEK inhibitor) nor SB203580 (p38 MAPK inhibitor) had any effect on NO-induced death. Taken together, these results suggest that JNK/SAPK may be related to NO-induced apoptosis in MG-63 human osteoblasts.

• Parasites, Hosts and Diseases
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• v.49 no.1
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• pp.17-23
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• 2011

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined, Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FAGS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 ${\mu}M$ and 11 ${\mu}M$ for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 ${\mu}M$ and 4.2 ${\mu}M$, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.

• Journal of fish pathology
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• v.16 no.1
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• pp.39-49
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• 2003

Cytosolic oxidation by 4-hydroxy-2-nonenal (4HNE) and tert-butyl hydroperoxide (t-BHP) results in cell death of bovine aortic endothelial cells (BAEC). In this study, we have investigated the roles of antioxidants such as 2,3,6-tribromo-4,5-dihydroxy benzyl methyl ether (TDB) and phloroglucinol in preventing cell death. After treatment with oxidants for 6h, cells became compact and showed nuclear condensation, which were characteristics of early apoptosis. After l2h treatment, morphologic features including severe cytoplasm condensation, membrane blebbing, and apoptotic bodies were prominent and these findings were interpreted as characteristics of late-apoptosis. When the apoptotic cells were treated with antioxidants for 12h, both early and late apoptotic cells did show no significant change. After oxidant treated cells were incubated with antioxidant for 24h, the characteristics of early-apoptosis were eliminated but cells in lateapoptosis could not return to normal cells. These results suggest that TDB and phloroglucinol prevent the cells from dying through apoptosis induced by 4HNE and t-BHP in early stage.

• Toxicological Research
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• v.25 no.3
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• pp.113-118
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• 2009

Cell death induced by menadione (vitamin K-3,2-methyl-1,4-naphthoquinone) has been investigated in human promyelocytic leukemia HL-60 cells. Menadione was found to induce both apoptosis and necrosis in HL-60 cells. Low concentration ($1{\sim}$50 ${\mu}$M) of menadione induced apoptotic cell death, which was demonstrated by typical DNA ladder patterns on agarose gel electrophoresis and flow cytometry analysis. In contrast, a high concentration of menadione (100 ${\mu}$M) induced necrotic cell death, which was demonstrated by DNA smear pattern in agarose gel electrophoresis. Necrotic cell death was accompanied with a great reduction of cell viability. Menadione activated caspase-3, as evidenced by both increased protease activity and proteolytic cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP) into 85 kDa cleavage product. Caspase-3 activity was maximum at 50 ${\mu}$M of menadione, and very low at 100 ${\mu}$M of menadione. Taken together, our results showed that menadione induced mixed types of cell death, apoptosis at low concentrations and necrosis at high concentrations in HL-60 cells.

• Archives of Pharmacal Research
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• v.27 no.8
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• pp.834-839
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• 2004

In human neuroblastoma SK-N-BE(2) cells undergoing apoptotic death induced by ginsenos-ide Rh2, a dammarane glycoside that was isolated from Panax ginseng C. A. Meyer, caspase-1 and caspase-3 were activated. The expression of Bax was increased in the cells treated with ginsenoside Rh2, whereas Bcl-2 expression was not altered. Treatment with caspase-1 inhibi-tor, Ac-YVAD-CMK, or caspase-3 inhibitor, Z-DEVD-FMK, partially inhibited ginsenoside Rh2-induced cell death but almost suppressed the cleavage of the 116 kDa PARP into a 85 kDa fragment. When the levels of p53 were examined in this process, p53 accumulated rapidly in the cells treated early with ginsenoside Rh2. These results suggest that activation of caspase-1 and -3 and the up-regulation of Bax are required in order for apoptotic death of SK-N-BE(2) cells to be induced by ginsenoside Rh2, and p53 plays an important role in the pathways to promote apoptosis.

• Molecules and Cells
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• v.42 no.6
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• pp.448-459
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• 2019

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is a promising target for gastric cancer (GC) treatment; however the efficacy of PI3K/mTOR dual inhibitors in GC has not yet been maximized. Additionally, the effect of autophagy regulation by PI3K/mTOR dual inhibitors has not been clearly elucidated in GC treatment. We aimed to show that our newly developed PI3K/mTOR dual inhibitor, CMG002, when combined with an autophagy inhibitor, chloroquine (CQ), potently induces effective cancer cell death in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) cells, where both the PI3K/AKT/mTOR and autophagy pathways play important roles in disease pathogenesis. EBV- and mock-infected AGS and NUGC3 GC cell lines were treated with CMG002 +/- CQ. PI3K/AKT/mTOR signaling pathway mediators, cellular apoptosis and autophagy markers were confirmed by Western blot assay. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. CMG002 effectively blocked the PI3K/AKT/mTOR pathway by markedly decreasing phosphorylation of AKT and its downstream mediator S6. CMG002 induced G0/G1 cell cycle arrest and enhanced apoptotic cell death in AGS and NUGC3 cells, particularly EBV-infected cells compared with mock-infected cells, as confirmed by flow cytometric analyses and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The combination of CMG002 plus CQ synergistically increased apoptotic cell death in EBV-infected GC cell lines when compared with CMG002 alone (P < 0.05). Our results suggest that the new PI3K/mTOR dual inhibitor, CMG002, when used in combination with the autophagy inhibitor, CQ, provides enhanced therapeutic efficacy against EBVaGC.

• Biomolecules & Therapeutics
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• v.24 no.2
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• pp.147-155
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• 2016

Chaetominine is a quinazoline alkaloid originating from the endophytic fungus Aspergillus fumigatus CY018. In this study, we showed evidence that chaetominine has cytotoxic and apoptotic effects on human leukemia K562 cells and investigated the pathway involved in chaetominine-induced apoptosis in detail. Chaetominine inhibited K562 cell growth, with an $IC_{50}$ value of 35 nM, but showed little inhibitory effect on the growth of human peripheral blood mononuclear cells. The high apoptosis rates, morphological apoptotic features, and DNA fragmentation caused by chaetominine indicated that the cytotoxicity was partially caused by its pro-apoptotic effect. Under chaetominine treatment, the Bax/Bcl-2 ratio was upregulated (from 0.3 to 8), which was followed by a decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, and stimulation of Apaf-1. Furthermore, activation of caspase-9 and caspase-3, which are the main executers of the apoptotic process, was observed. These results demonstrated that chaetominine induced cell apoptosis via the mitochondrial pathway. Chaetominine inhibited K562 cell growth and induced apoptotic cell death through the intrinsic pathway, which suggests that chaetominine might be a promising therapeutic for leukemia.