• Radiation Oncology Journal
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• v.18 no.3
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• pp.214-219
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• 2000

Purpose : To Investigate the pathways of radiation induced apoptosls and the effect of cysteamine (${\beta}$-mercaptoethyiamine), as a radioprotector, on it. Materials and Methods : HL-50 ceils were assigned to control, irradiated, and cysteamlne (1 mM, 10mM) pretreated groups. Irradiation was given In a single fraction of 10 Gy (6 MV x-ray) and cysteamine was administered 1 hour before irradiation. The activities of caspase-8 were measured in control and irradiated group to evaluate its relation to the radiation Induced apoptosis. To evaluate the role of cysteamine In radiation Induced apoptosis, the number of viable cells, the expression and activity of caspase-3, and the expression of poly (ADP-ribose) polymerase (PARP) were measured and compared after irradiating the HL-60 celis with cysteamine pretreatment or not. Results : The intraceliular caspase-8 activity, known to be related to the death receptor induced apoptosis, was not affected by irradiation(p>0.05). The number of viable cells began to decrease from 6 hours after irradiation (p>0.05), but the number of viable cells In 1 mM cysteamine pretreated group was not decreased after irradiation and was similar to those in the control group. In caspase-3 analyses, known as apoptosis executioner, its expression was not different but its activity was Increased by irradiation(p>0.05). However, this Increase of activity was suppressed by the pretreatment of 1 mM cysteamine. The cleavage of PARP, thought to be resulted from caspase-3 activation, occurred after irradiation which was attenuated by the pretreatment of 1 mM cysteamine. Conclusion : These results show that radiation induced apoptotic process is somewhat different from death receptor induced one and the pretreatment of 1 mM cysteamine has a tendency to decrease the radiation-induced apoptosis in HL-60 cells.

• Radiation Oncology Journal
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• v.20 no.4
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• pp.367-374
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• 2002

Purpose : To investigate the growth inhibitory effects, and the underlying mechanism of human colon cancer cell (HT-29) death, induced by a new synthetic bile acid derivative (HS-1200). Materials and Methods : Human colon cancer cells (HT-29), in exponential growth phase, were treated with various concentrations of a new synthetic bile acid derivative (HS-1200). The growth inhibitory effects on HT-29 cells were examined using a frypan blue exclusion assay. The extent of apoptosis was determined using agarose gel electrophoresis, TUNEL assays and Hoechst staining. The apoptotic cell death was also confirmed by Western blotting of PARP, caspase-3 and DNA fragmentation factor (DFF) analysis. To investigate the involvement of mitochondria, we employed immunofluorescent staining of cytochrome c and mitochondrial membrane potential analyses. Results : The dose required for the half maximal inhibition $(IC_{50})$ of the HT-29 cell growth was $100\~150\;{\mu}M$ of HS-1200. Several changes, associated with the apoptosis of the HT-29 cells, were reveal by the agarose gel eletrophoresis, TUNEL assays and Hoechst staining, following their treatment with $100\;{\mu}M$ of HS-1200. HS-1200 treatment also induced caspase-3, PARP and DFF degradations, and the western blotting showed the processed caspase-3 p20, PARP p85 and DFF p30 and p11 cleaved products. Mitochondrial events were also demonstrated. The cytochrome c staining indicated that cytochrome c had been released from the mitochondria in the HS-1200 treated cells. The mitochondrial membrane potential $(\Delta\Psi_m)$ was also prominently decreased in the HS-1200 treated cells. Conclusion : These findings suggest that the HS-1200 - induced apoptosis of human colon cancer cells (HT-29) is mediated via caspase and mitochondrial pathways.

• Journal of Oral Medicine and Pain
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• v.31 no.1
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• pp.79-89
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• 2006

The aim of this study is to know how the rat submandibular gland changes under various emotional stress condition, using molecular biological methods. Restraint and chronic unpredictable mild stress (CUMS) experiment is conducted on fifty one 7-week old Sprague-Dawley rats (restraint stress experiment: 21, CUMS: 30). The rats were sacrificed, the submandibular glands were excised immediately at certain time, and examined by the use of immunohistochemistry and western blotting. In CUMS experiment, sucrose preference test, water intake change, weight change were implemented at 1 week interval for the experimental period The results are as follows: 1. The number of clusterin-secreting cells of restraint stress group compared to control group showed significantly decreasing tendency in all experimental groups except for the 1st hour group (p<0.001 in the 9th, 24th, 72nd, 120th, and 168th hour group). 2. The number of clusterin-secreting cells of CUMS group compared to control group showed significantly increasing tendency in the 2nd week group (p<0.01), and significantly decreasing tendency in the 4th and 5th week group (p<0.001). 3. Sucrose preference test in CUMS experiment showed significant difference between the 5th week experimental group and control group (p<0.01). 4. Weight change in CUMS experiment showed significant difference between the 5th week experimental group and control group (p<0.01), but water intake change didn't show significant difference compared to control group. 5. In western blot analysis, clusterin expression was decreased on a gradual basis in due time compared to the control group in the restraint stress group. As for CUMS group (chronic unpredictable mild stress group), it was increased till the 2nd week and decreased till the 5th week after that, which is similar to immunohistochemical analysis result and the decreasing tendency of sucrose preference and weigh changes. Through the test, it was proved that expression of clusterin in saliva glands decreases after receiving either acute or chronic stress, indicating relation with depression caused by chronic stress. Unlike other data, however, apoptotic tendency was hardly found in tissues. Diverse possibilities could be suggested on that: first, the stress was not enough to expedite apoptosis; second, apoptosis-related protein was already being secreted though not detected with microscope; third, clusterin, a major secretion molecule of saliva, decreased with saliva's malfunction due to stress. In the respect, it will be necessary to examine proteins expressed in case of cell death or other heat-shock proteins at the same time, in order to see whether any cellular change or death is caused by decreasing clusterin under high stress, and whether the original state is restored as time goes by under mild stress, through longer-term tests using even higher acute stress.

• Tuberculosis and Respiratory Diseases
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• v.69 no.1
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• pp.16-23
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• 2010

Background: Most lung cancer patients receive systemic chemotherapy at an advanced stage disease. Cisplatin-based chemotherapy is the main regimen for treating advanced lung cancer. Recently, autophagy has become an important mechanism of cellular adaptation under starvation or cell oxidative stress. The purpose of this study was to determine whether or not autophagy can occurred in cisplatin-treated lung cancer cells. Methods: H460 cells were incubated with RPMI 1640 and treated in $5{\mu}M$ or $20{\mu}M$ cisplatin concentrations at specific time intervals. Cells surviving cisplatin treatment were measured and compared using an MTT cell viability assay to cells that underwent apoptosis with autophagy by nuclear staining, apoptotic or autophagic related proteins, and autophagic vacuoles. The development of acidic vascular organelles was using acridine orange staining and fluorescent expression of GFP-LC3 protein in its transfected cells was observed to evaluate autophagy. Results: Lung cancer cells treated with $5{\mu}M$ cisplatin-treated were less sensitive to cell death than $20{\mu}M$ cisplatin-treated cells in a time-dependent manner. Nuclear fragmentation at $5{\mu}M$ was not detected, even though it was discovered at $20{\mu}M$. Poly (ADP-ribose) polymerase cleavages were not detected in $5{\mu}M$ within 24 hours. Massive vacuolization in the cytoplasm of $5{\mu}M$ treated cells were observed. Acridine orange stain-positive cells was increased according in time-dependence manner. The autophagosome-incorporated LC3 II protein expression was increased in $5{\mu}M$ treated cells, but was not detected in $20{\mu}M$ treated cells. The expression of GFP-LC3 were increased in $5{\mu}M$ treated cells in a time-dependent manner. Conclusion: The induction of autophagy occurred in $5{\mu}M$ dose of cisplatin-treated lung cancer cells.

• Journal of Life Science
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• v.17 no.8 s.88
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• pp.1121-1128
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• 2007

The fruits of Zanthoxylum piperitum are known as having various physiology vitality, and the abstraction ingredient of the pericarp is also known as having strong antibiotic activities against various bacteria. Therefore, this study was carried out to estimate the effect of physiology vitality when the abstraction ingredient of Z. piperitum was added in soy sauce(Chopi-kanjang) and soybean paste(Chopi-doenjang). For the antibiotic activity against the pathogens of sitotoxism such as Staphylococcus aureus, Salmonella typhimurium, Vibrio parahemolyticus, Escherichia coli 0157:H7, the extracts of the Chopi-kanjang was added 1%, 2%, 4% pericarp of Z. piperitum in the manufacturing process of soy source. According to the results, the growth of E. coli 0157:H7 and V. parahemolyticus were respectively inhibited as 70% and 50% by the Chopi-kanjang added 2% of the ingredient. For the antibiotic effects of the aforementioned Chopi-kanjang against Sal. typhimurium and Sta. aureus, the growth of those pathogens was also inhibited between 40% and 60% according to the manufacturing period of Chopi-kanjang. It was confirmed that the antibiotic activity using the mixture of the abstraction ingredient and Chopi-doenjang was lower than those of Chopi-kanjang. In order to estimate the anticancer activity using by caspase-3 activity, the mixture of the abstraction ingredient of the pericarp of Z. piperitum and Chopi-kanjang was treated to leukemia cells. According to the results, the activities of caspase-3 using the mixture added 1%, 2% and 4% of the abstraction ingredient were respectively increased as much as 4, 12, 15 times comparing with the control which was treated with the soy source only. It could be that the mixture of the abstraction ingredient of the pericarp of Z. piperitum and soy source induced apoptosis, and the mixture of the abstraction ingredient and soybean paste had no effect on the activity of caspase-3. In order to find out the death of the aforementioned cells caused by necrosis or apoptosis, DNA fragmentation in the cell was examined. U-937 cells showed apoptotic DNA fragmentation in the incubation with Chopi-kanjang extract.

• Journal of Chest Surgery
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• v.41 no.4
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• pp.447-456
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• 2008

Background: Caspase-3 is a cysteine protease that plays a major role in the process of apoptotic cell death. The dysregulated expression of c-myc contributes to the tumorigenesis in a variety of human cancers. The aim of this study was to investigate the expressions of caspase-3 and c-myc and their significances as prognosis markers in patients with completely resected non-small cell lung cancer (NSCLC). Material and Method: A total 130 consecutive patients who had undergone complete resection without pre-operative radio-therapy or chemotherapy between May 1996 and December 2003 for NSCLC were retrospectively reviewed. The median follow-up period of the patients was 50 months (range: $3{\sim}128$ months). The expressions of caspase-3 and c-myc were immuno-histochemically examined, and these were correlated with the clinico-pathologic data. Result: The prevalence of caspase-3 and c-myc expressions in the patients was 68% (88/130) and 59% (77/130), respectively. Significant association was found between the frequency of the expressions of caspase-3 and c-myc (p=0.025). The caspase-3 and c-myc expressions were not significantly associated with the prognosis in all the patients. However, according to stages, a positive caspase-3 expression was significantly correlated with a favorable prognosis for patients with stage IIIa disease (median survival period: 35 months vs. 10 months, p=0.021). Multivariate analysis showed the pathologic stage to be significantly correlated with a good prognosis in all the patients (p=0.024), and with a positive caspase-3 expression, well differentiated tumor and negative neuronal invasion in the patients with stage llla disease (p=0.005, p=0.003, p=0.004, respectively). Conclusion: Caspase-3 and c-myc were frequently expressed in NSCLC, suggesting its possible involvement in tumor development. The caspase-3 expression, as determined with performing immunohistochemical staining, may be a favorable prognostic indicator in patients with completely resected NSCLC an advanced stage (IIIa).

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.113-123
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• 1996

It has been well known that polyamines ensure the stability of chromatin structure and the fidelity of DNA transcription. This study was carried out to evaluate the effect of polyamines on the apoptosis of mouse thymocytes induced by dexamethasone and polyamine synthesis inhibitors. 1) In the histological death findings of thymocytes double-stained with acridine orange and ethidium bromide, the apoptotic and the necrotic fractions (AF; NF) in the control group were $9.4{\pm}4.2%$ and $4.5{\pm}5.3%$, respectively. Dexamethasone $(3\;{\times}\;10^{-8}\;M:\;DX)$ in creased AF upto $52.0{\pm}8.1%$ and did not change NF, but A23187 $(5\;{\times}\;10^{-7}\;M:\;A2)$ increased AF and NF upto $45.0{\pm}8.9%$ and $20.5{\pm}10.6%$, respectively. 2) The thymocyte viability was significantly reduced by DX, DHEA $(1\;{\times}\;10^{-4}\;M)$, A2, DFMO $(1\;{\times}\;10^{-4}\;M)$, and $MGBG\;(1\;{\times}\;10^{-4}\;M)$, respectively. It was, however, little affected by $aminoguanidine\;(1\;{\times}\;10^{-4}\;M:\;AG)$, $putrescine\;(1\;{\times}\;10^{-5}\;M:\;PT)$, $spermidine\;(1\;{\times}\;10^{-5}\;M:\;SD)$, and $spermine\;(1\;{\times}\;10^{-5}\;M:\;SM)$. 3) The genomic DNA of mouse thymocyte was markedly fragmented by DX and A2, respectively, and to a lesser extent, by DHEA, but was little affected by MGBG, DFMO, AG, and each of polyamines. 4) The DX induced reduction of thymocyte viability was moderately attenuated by DHEA, but little affected by DFMO, MGBC, and AG. However, SM significantly attenuated the viability reduction induced by A2 as well as DX. 5) The thymocyte viability reduction by MGBG and DFMO was significantly attenuated by only SM among three polyamines applied in this study. 6) The thymocyte viability redution by combined treatments of DX with DFMO and MGBG, respectively, was significantly attenuated by SM, and moderately by PT. But the viability reduction by combined treatment of DX with AG or DHEA was not affected by polyamines. These results suggest that polyamines, particularly spermine, might play the inhibitory role in thymocyte apoptosis and the inhibitory effect can be ascribed in part to the increase of polyamine uptake by thymocytes pretreated with DFMO and MGBG.

• Radiation Oncology Journal
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• v.18 no.1
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• pp.51-58
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• 2000

Purpose :The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph-positive KS62 leukemia cell line was investigated. Materials and Methods :K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2×106 cells/mL. The cells were irradiated with 10 Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37$^{\circ}C$ for 0$\~$48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bel-2, bel-X$_{L}$ and bax protein levels. Results :Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electro-phoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bel-2 or bel-X$_{L}$ anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30$\~$40$\%$ at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. Conclusion : We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210$^{bcr/abl}$ failed to enhance the radiation induced apoptosis in KS62 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is attributable to bel-2 family. It is plausible that the relationship between cell cycle delays and cell death is essential for drug development based on molecular targeting designed to modify radiation-induced apoptosis.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.65-70
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• 2006

This study investigated apoptosis and in vitro development of parthenogenetic preimplantation porcine embryos. In vitro matured oocytes for $42{\sim}44h$ were used. Apoptotic cell death was analyzed by using a terminal deoxynucleatidyl transferase mediated deoxyuridine 5-triphosphate nick-end tabling (TUNEL) assay. In experiment 1, oocytes were activated with two electric pulses (CH) of 1.2 kV/cm for $30{\mu}sec$ (E), E + 6-dimethylaminopurine (6-DMAP) or E + cycloheximide (CH) and cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$. In experiment 2, oocytes were activated by E and cultured in PZM-3 or NCSU-23 under a gas atmosphere of 20% $O_2$ ($5%\;CO_2$, in air) or 5% $O_2$ $(5%\;CO_2,\;5%\;O_2\;90%\;N_2)\;at\;38.5^{\circ}C$. Oocytes activated with E+6-DMAP or E+CH showed higher blastocyst rates (36.3% and 32.5%) compared to E alone (27.7%). The frequency of apoptosis according to treatments were 5.3%, 7.7% and 7.1% respectively. Oocytes activated with E alone showed lower (P<0.05) frequency of apoptosis compared to other groups. In experiment 2, parthenotes cultured in PZM-3 showed slightly higher blastocyte rates (28.2% and 29.7%) compared to NCSU-23 (22.6% and 24.4%) regardless of atmosphere. Blastocysts generated in PZM-3 showed lower (P<0.05) apoptosis rate under 20% $O_2$ (9.2% vs 16.9%), whereas those in NCSU-23 had slightly lower apoptosis rate under 5% $O_2$ (14.0% vs 18.4%). This result represents that activation method and culture condition could affect the frequency of apoptosis as well as in vitro developmental rate.

• Journal of Life Science
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• v.23 no.5
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• pp.689-697
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• 2013

In the United States, about 40,000 new cases of oral cancer are diagnosed each year and nearly 7,800 patients died from it in 2012. Omega-3 polyunsaturated fatty acids have been found to have anticancer effects in a variety of cancer cell lines and animal models, but their effect in oral cancer remains unclear. This study was designed to examine the effect of docosahexaenoic acid (DHA, a kind of omega-3 fatty acid) on oral cancer cells and the molecular mechanism of its action. We found that exposure of squamous cell carcinoma-4 (SCC-4) and squamous cell carcinoma-9 (SCC-9) human oral cancer cells to DHA induced growth inhibition in a dose- and time-dependent manner. Meanwhile, in addition to the elevated levels of apoptotic markers, such as cleaved PARP, subG1 portion and TUNEL-positive nuclei, DHA led to autophagic vesicle formation and an increase in autophagic flux, indicating the involvement of both apoptosis and autophagy in the inhibitory effects of DHA on oral cancer cells. Further experiments revealed that the apoptosis and autophagy induced by DHA were linked to inhibition of mammalian target of rapamycin (mTOR) signaling by AKT inhibition and AMP-activated protein kinase (AMPK) activation in SCC-9 cells. Together, our results suggest that DHA induces apoptosis- and autophagy-associated cell death through the AMPK/AKT/mTOR signaling pathway in oral cancer cells. Thus, utilization of omega-3 fatty acids may represent a promising therapeutic approach for chemoprevention and treatment of human oral cancer.