• International Journal of Oral Biology
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• v.38 no.3
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• pp.101-110
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• 2013

We investigated the synergistic apoptotic effects of co-treatments with Chios gum mastic (CGM) and eugenol on G361 human melanoma cells. An MTT assay was conducted to investigate whether this co-treatment efficiently reduces the viability of G361 cells compared with each single treatment. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining, and analyses of DNA hypoploidy. Western blot analysis and immunofluorescent staining were also performed to evaluate expression and translocation of apoptosis-related proteins following CGM and eugenol co-treatment. Proteasome activity and mitochondrial membrane potential (MMP) changes were also assayed.The results indicated that the co-treatment of CGM and eugenol induces multiple pathways and processes associated with an apoptotic response in G361 cells. These include nuclear condensation, DNA fragmentation, a reduction in MMP and proteasome activity, an increase of Bax and decrease of Bcl-2, a decreased DNA content, cytochrome c release into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, and the activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD). In contrast, separate treatments of $40{\mu}g/ml$ CGM or $300{\mu}M$ eugenol for 24 hours did not induce apoptosis. Our present data thus suggest that a combination therapy of CGM and eugenol is a potential treatment strategy for human melanoma.

• International Journal of Oral Biology
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• v.45 no.4
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• pp.169-178
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• 2020

L-ascorbic acid (L-AA; vitamin C) induces apoptosis in cancer cells. This study aimed to elucidate the molecular mechanisms of L-AA-induced apoptosis in human laryngeal epidermoid carcinoma Hep-2 cells. L-AA suppressed the viability of Hep-2 cells and induced apoptosis, as shown by the cleavage and condensation of nuclear chromatin and increased number of Annexin V-positive cells. L-AA decreased Bcl-2 protein expression but upregulated Bax protein levels. In addition, cytochrome c release from the mitochondria into the cytosol and activation of caspase-9, -8, and -3 were enhanced by L-AA treatment. Furthermore, apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were translocated into the nucleus during apoptosis of L-AA-treated Hep-2 cells. L-AA effectively inhibited the constitutive nuclear factor-κB (NF-κB) activation and attenuated the nuclear expression of the p65 subunit of NF-κB. Interestingly, L-AA treatment of Hep-2 cells markedly activated Akt and mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase [JNK]) and and LY294002 (Akt inhibitor), SB203580 (p38 inhibitor) or SP600125 (a JNK inhibitor) decreased the levels of Annexin V-positive cells. These results suggested that L-AA induces the apoptosis of Hep-2 cells via the nuclear translocation of AIF and EndoG by modulating the Bcl-2 family and MAPK/Akt signaling pathways.

• The Korea Journal of Herbology
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• v.28 no.4
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• pp.33-40
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• 2013

Objectives : To study the apoptosis effects of fermented Undaria pinnatifida extracts(FUP) against HCT-15 colon cancer cells. Method : By measuring cell proliferation, DNA fragmentation, cell cycle, morphology, and western blot from FUP, the study investigated the effects of the extractions had upon the HCT-15 colon cancer cells, and concluded that the inhibiting effects upon cells were induced by apoptosis. Result : FUP effectively inhibited the growth of HCT-15 colon cancer cells. After analyzing the DNA fragmentation, the study observed a DNA ladder, while examining the cells, and found an increase of sub-G1 hypodiploid cells. On the changes regarding the nucleus of the cells, a condensation of cells and chromatin, as well as an apoptotic body was clearly observed. By observing through western blot from FUP, the study found a decreased level of Bcl-2 from HCT-15 colon cancer cells, but the increased level of Bax and cleaved caspase-3, which as a result induced apoptosis, inhibiting the growth of HCT-15 colon cancer cells. FUP increased the natural death of HCT-15 colon cancer cells by the induction of apoptosis. FUP seemed to have no suppressing effect upon HL-60/MX2 cells. However, compared to the fucoidan, the study was able to clearly observe morphological changes of HCT-15 cells apoptosis, in a 1/2 concentration. Conclusion : FUP had antiproliferative effects on different kinds of cancer cells, while proving especially efficacious against colon cancer cells.

• Animal Bioscience
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• v.37 no.2
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• pp.284-294
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• 2024

Objective: Pork is an important source of animal protein in many countries. Subtle physiochemical changes occur during pork postmortem aging. The changes of apoptosis and autophagy in pork at 6 h to 72 h after slaughter were studied to provide evidence for pork quality. Methods: In this article, morphological changes of postmortem pork was observed through Hematoxylin-eosin staining, apoptotic nuclei were observed by TdT-mediated dUTP nick end labeling assay, protein related to apoptosis and autophagy expressions were tested by western blot and LC3 level were expressed according to immunofluorescence assay. Results: In this study, we found the occurrence of apoptosis in postmortem pork, and the process was characterized by nucleus condensation and fragmentation, formation of apoptotic bodies, increase in apoptosis-related Bax/Bcl-2 levels, and activation of caspases. Autophagy reached its peak between 24 and 48 h after slaughter, accompanied by the formation of autophagosomes on the cell membrane and expression of autophagy-related proteins beclin-1, P62, LC3-I, LC3-II, and ATG5. Conclusion: Obvious apoptosis was observed at 12 h and autophagy reached its peak at 48 h. The present work provides the evidence for the occurrence of apoptosis and autophagy during postmortem aging of pork. In conclusion, the apoptosis and autophagy of muscle cells discovered in this study have important implications for pork in the meat industry.

• The Journal of Internal Korean Medicine
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• v.21 no.2
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• pp.259-266
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• 2000

Objectives : The effects of aqueous extracts of Cortex ulmi pumilae (a traditional medicine for cancer treatment in oriental medicine) on the induction of apoptotic cell death were investigated in human liver origm hepatoma cell lines, HepG2. Methods : The death of HepG2 cells was markedly induced by the addition of extracts of Cortex ulmi pumilae in a dose-dependent manner. The apoptotic characteristic ladder pattern of DNA strand break was not observed in cell death of HepG2. In addition, it was not shown nucleus chromatin condensation and fragmentation under hoechst staining. However, by the using annexin V staining assay, externalizations of phosphatidylserine in HepG2 cell which were treated with Cortex ulmi pumilae extracts were detected in the early time (at 9 hr after extract treatment). Furthermore, LDH release was not detected in this early stage. Therefore, Cortex ulmi pumilae extracts-induced cell death of HepG2 cells is mediated by apoptotic death signal processes. Result : The activity of caspase 3-like proteases remained in a basal level in HepG2 cells which treated with the extract of Cordyceps sinensis. However, it was markedly increased in HepG2 cells which treated with two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) which were differently extracted (respectively, 2.3 and 3.3 fold). On a while, the phosphotransferase activities of JNK1 was markedly induced in HepG2 cells which were treated with two extracts of Cortex ulmi pumilae. On the contrary, the activation of transcriptional activator, activating protein1(AP-1) and NF-kB were severely decreased by these two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K). In addition, antioxidants (GSH and NAC) and intracellular $Ca2^+$ level regulator (Bapta/AM and Thapsigargin) did not affect Cortex ulmi pumilae extracts-induced apoptotic death of HepG2 cells. Conclusions : In conclusion, our results suggest that two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) induces the apoptotic death of human liver origin hepatoma HepG2 cells via activation of caspase 3-like proteases as well as JNK1, and inhibition of transcriptional activators, AP-1 and $NK-{\kappa}B$.

• Korean Journal of Environmental Biology
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• v.31 no.3
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• pp.204-212
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• 2013

This study analyzed the occurrence of tunic softness, survival rate, metabolic rate and histopathologic changes arising from the effect of hypoxic environment in order to find the causes of occurrences of tunic softness, which manifests as the key phenomenon of mass mortality of Halocynthia roretzi. Regarding the survival of H. roretzi with reduction in dissolved oxygen, all the entities died on the 4th day of exposure to the dissolved oxygen concentration of $2mg\;L^{-1}$ while 50% mortality was observed on the 5th day of exposure to the dissolved oxygen concentration of $3mg\;L^{-1}$. Therefore the 5 days-$LC_{50}$ was found to be $3.55mg\;L^{-1}$ (1.86~$4.96mg\;L^{-1}$). However, occurrence of tunic softness was not observed during the period of exposure to low oxygen concentration. The oxygen consumption rate significantly decreases at the dissolved oxygen concentration of less than $5mg\;L^{-1}$ in comparison to the control group. Therefore, it is presumed that H. roretzi controls the respiration rate for prescribed period of time when exposed to hypoxic environment. Regarding the histopathologic changes in the gill, digestive gland and cyst of H. roretzi due to hypoxic environment, necrosis of epithelial layer, in filtration of blood cells, and condensation of nucleus that compose each of the organs were observed. Regarding morphological changes, the decrease in volume with shrinking of the tunic, discoloration of the internal organs and necrosis of gill and hepatopancreas were observed.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.1
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• pp.125-131
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• 2015

Currently, taxol is mainly extracted from the bark of yews; however, this method can not meet its increasing demand on the market because yews grow very slowly and are a rare and endangered species belonging to first-level conservation plants. Recently, increasing efforts have been made to develop alternative means of taxol production; microbe fermentation would be a very promising method to increase the production scale of taxol. To determine the activities of the taxol extracted from endophytic fungus N. sylviforme HDFS4-26 in inhibiting the growth and causing the apoptosis of cancer cells, on comparison with the taxol extracted from the bark of yew, we used cellular morphology, cell counting kit (CCK-8) assay, staining (HO33258/PI and Giemsa), DNA agarose gel electrophoresis and flow cytometry (FCM) analyses to determine the apoptosis status of breast cancer MCF-7 cells, cervical cancer HeLa cells and ovarian cancer HO8910 cells. Our results showed that the fungal taxol inhibited the growth of MCF-7, HeLa and HO8910 cells in a dose-and time-dependent manner. IC50 values of fungal taxol for HeLa, MCF-7 and HO8910 cells were $0.1-1.0{\mu}g/ml$, $0.001-0.01{\mu}g/ml$ and $0.01-0.1{\mu}g/ml$, respectively. The fungal taxol induced these tumor cells to undergo apoptosis with typical apoptotic characteristics, including morphological changes for chromatin condensation, chromatin crescent formation, nucleus fragmentation, apoptotic body formation and G2/M cell cycle arrest. The fungal taxol at the $0.01-1.0{\mu}g/ml$ had significant effects of inducing apoptosis between 24-48 h, which was the same as that of taxol extracted from yews. This study offers important information and a new resource for the production of an important anticancer drug by endofungus fermentation.

• Journal of Korean Ophthalmic Optics Society
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• v.8 no.1
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• pp.65-71
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• 2003

The corneal epithelium is constantly being shed. The mechanism of corneal desquamation is not fully understood. Apoptosis, programmed cell death, may play a role. Apoptosis can be induced by a number of factors and different mechanisms. The study was performed to examine the apoptotic index induced in human corneal epithelial cells maintained in tissue culture by various apoptotic inducers. Various inducers, recombinant human cytokines($INF{\gamma}$, $TNF{\alpha}$, FASAb), actinomycin D. camptothecin, cycloheximide, dexamethasone and etoposide, were purchased from commercial suppliers. Inducers at manufacturer-recommended concentration were added to the corneal epithelial cells for 48 hours. Cell viability was measured using MTT assay. The cells were then assessed for the level of apoptosis. Morphologic changes and quantification of apoptotic cells were determined and counted under fluorescence microscope after inducers-treated human corneal epithelial (HCE) cells for 48 hours with Hoechst 33342 staining. Annexin V-FITC/PI staining and DePsipher assay. The expression of Fas protein was studied by immunocytochemistry. All inducers induced apoptosis in HCE cells in a dose dependent manner. Actinomycin D. camptothecin and etoposide induced apoptosis at lower than manufacturer-recommended concentration, while cytokines, cycloheximide and dexamethasone induced apoptosis at higher concentrations at the end of 48 hours. All inducers elicited typical apoptotic morphologic changes (chromatin condensation, nucleus fragmentations non-orange-red colored mitochondria) and expresses Fas protein highly. Apoptotic index of HCE cells by these inducers was different from the other cell lines. RNA synthesis inhibitor and topoisomerase inhibitors induced apoptosis at lower concentration than manufacturer-recommended concentration. Cytokines, cycloheximide and dexamethasone were able to produce apoptosis at 10 times higher concentrations. RNA synthesis inhibitor and topoisomerase inhibitors are more sensitive than intracellular receptor-activators in apoptotic induction of HCE cells.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.59-67
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• 2000

This study was investigated the developmental potential of bovine embryos following nuclear transfer with bovine fetal fibroblasts (BFF). BFF were isolated from a male 45-day-old-fetus. Non-starved BFF labeled with MitoTracker were transferred into perivitelline space of enucleated oocytes. BFF-oocyte units were fused by electric pulse, and then fused oocytes were activated with calcium ionophore A23187 and subsequently 6-dimethylaminopurine (6-DMAP). The resulting zygotes were placed into CRlaa bovine embryo culture medium. Transfer of the nucleus into enucleated oocyte led to premature chromosome condensation, swelling and pronucleus formation. Remodeled oocytes were developed to the mitotic and 2-cell stage at 18 to 26 h after nuclear transfer. The incidence of in vitro development to the blastocyst stages was 21% of fused oocytes. Mitochondria of BFF eliminated rapidly and were not detected at 8 h after fusion. These results suggest that BFF can be successfully reprogrammed in enucleated bovine oocytes, and that reconstructed embryos can develop to the blastocyst stage.

• Journal of Food Hygiene and Safety
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• v.31 no.4
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• pp.286-293
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• 2016

This study is an experiment for gastric protective effects of ursolic acid. In order to identify the effects of ursolic acid on gastrointestinal disorder, acute and chronic gastritis were also observed using HCl ethanol and indomethacin-induced gastric lesion models, respectively. As for gastric acid, it was also identified through proton pump ($H^+/K^+-ATPase$) inhibiting activity. In regards to protective factor for gastric damage, prostaglandin $E_2$ ($PGE_2$) was quantitatively analyzed. Antibacterial activity experiment was done on Helicobacter pylori (H.pylori), which is known to be the causing factor of chronic gastritis, gastric ulcer and gastric cancer. By making use of AGS cell, it was confirmed that ursolic acid was involved in apoptosis of gastric cancer cell through 4',6-diamidino-2-phenylindol (DAPI) staining and flow cytometry analysis. As a result, ursolic acid reduced gastric lesions caused by HCl ethanol and indomethacin. Ursolic acid inhibited acid secretion by inhibiting proton pump ($H^+/K^+-ATPase$), which is the gastric acid secreting enzyme involved at the final phase of gastric acid secretion. And ursolic acid was identified with gastric mucosa protection effects by increasing the concentration of $PGE_2$, a protective factor of gastric mucosa preservation. The antibacterial activity on H. pylori, which is aggressive factor in gastrointestinal disorder, ursolic acid showed inhibitory effects on H. pylori colonization. In the DAPI nuclear staining, unlike the control group, shape of the nucleus has deformed, and has been observed either shrinked cell or chromatin condensation phenomenon. In the Flow cytometry assay, confirmed the growth rate of apoptosis in a concentration-dependent manner.