• Journal of Korean Medicine Rehabilitation
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• v.34 no.2
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• pp.15-28
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• 2024

Objectives We used the D-galactose (D-gal) induced C2C12 myoblast senescence model to investigate whether ethanol extract of Perilla. fructescens leaves (EEPF) could delay cellular senescence and regulate related mechanisms. Methods C2C12 myogenic cells were cultured in an incubator under 37 ℃ and 5% CO₂ conditions. EEPF, dried perilla leaves were pulverized and extracted at 1:10 (v/v) at 50 ℃ for 4 hours. Cell counting kit-8 and western blot analysis was performed. Annexin V-FITC apoptosis detection kit and DAPI staining was applied. Catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde analysis kits were used. To measure the level of reactive oxygen species generation, staining and flow cytometry was used. To analyze the mitochondrial activity, membrane potential changes were measured using JC-1. 𝛽-gal activity was analyzed using SA-𝛽-gal staining solution, and DNA damage was analyzed by using 𝛾-H2AX. Quantikine ELISA kit was used to analyze inflammatory cytokine production. Results According to the results of this study, EEPF significantly alleviated the decrease in cell viability in C2C12 cells treated with D-gal and suppressed the decrease in the expression of proliferating cell nuclear antigen. EEPF also markedly blocked D-gal-induced C2C12 cell apoptosis and restored reduced activity of CAT, GSH-Px, T-AOC, SOD. In addition, EEPF suppressed the decrease in 𝛽-galactosidase activity, the induction of DNA damage and the increase in expression of senescence-associated secretory phenotype proteins such as p16, p53 and p21 in D-gal-treated C2C12 cells. Furthermore, EEPF significantly attenuated D-gal-induced production and expression of inflammatory cytokines such as interleukin (IL)-6 and IL-18. Conclusions The results of this study indicate that EEPF can be used as a potential candidate for the prevention and treatment of muscle aging.

• Development and Reproduction
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• v.4 no.2
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• pp.161-173
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• 2000

The aim of this study was to assess the effect of the frequency of the L$N_2$ infusion on the ultrastructure, metabolism and development of the embryo after freezing and thawing by computerized cell freezer. Two-cell embryos of ICR mouse were randomly allocated into fresh (control), high-frequency freezing (group 1) and low-frequency freezing (group 2). For fresh and frozen-thawed intact 2-cell embryos, total ceil number in the blastocyst was counted by fluorescent microscope after Hoechst 33258 staining. Relative amount of $H_2O$$_2$ was measured by DCHFDA. Intracellular location and membrane potential of the mitochondria were evaluated by staining with rhodamine 123 and JC-1. The structure of actin filament was also evaluated by confocal microscope. DNA fragmentation was assessed by TUNEL method after development into blastocyst. The survival rate of intact embryo was higher in group 1 than group 2 (50.7％ vs. 34.6％ respectively, p<0.05). The blastocyst developmental rate was significantly low in group 2 (86.7％, 76.7％ vs. 44.0％ for control, group 1 and group 2 respectively, p<0.05). Total cell number in the blastocyst was also significantly lower in group 2 than control (79.5$\pm$12.9, 71.6$\pm$8.0, and 62.5$\pm$4.7 for control, group 1 and group 2 respectively, p<0.05). The relative amount of $H_2O$$_2$ was higher in group 2 than other groups (15.3$\pm$3.0, 16.6$\pm$1.6 vs. 23.4$\pm$1.8, p<0.05). After JC-1 staining, relative intensity of mitochondria with high membrane potential was significantly lower in group 2 than control and group 1 (17.2$\pm$3.8, 17.4$\pm$1.3 vs. 13.2$\pm$2.0, p<0.05). In group 2, partial deletion and aggregation of the actin filament was found. DNA fragmentation rate was also hieher for group 2 versus other groups (30.8％, 36.0％ vs. 65.6％, p<0.05). The frequency of the L$N_2$ infusion is an important factor for the development of frozen-thawed mouse embryo. High-frequency infusion may prevent damages of cytoskeleton and mitochondria in the embryo probably by preventing the temperature fluctuation during dehydration phase. We speculate that the application of high-frequency infusion method in human embryo may be promising.

• Korean Journal of Head & Neck Oncology
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• v.10 no.2
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• pp.200-205
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• 1994

Proliferating cell nuclear antgen(PCNA) plays an important role in DNA synthesis in nucleoli and is highly conserved non-histone nuclear protein composed of 261 amino acid. and is considered to correlated with the cells proliferative state, because it is synthesized particulary during the proliferative period of late Gland S-phase. Therefore, PCNA index meaningfully increases in the active or proliferative kinetic cells. By the use of recently developed monoclonal antibodies against PCNA, the immunohistochemical staining methods can make possible. These staining methods are the useful and productive one for ascertaining the cell's proliferating abillity. Moreover, immunohistochemical staining method with a antiPCNA antibody has particulrar advantages as follows. By means of these methods, we can stain the tissue that was already fixed in formalin or paraffin wax. We can see with naked eye that which cell is, where is differentiated through a microscope. Lastly, it maintains the whole tissue architecture and makes a search for the correlation. As we have seen above, the immunohistochemical staining methods for PCNA have been studied as an impotant factor that can find the cell proliferative kinetics in malignancy and biologic behavior of tumors. To investigate of the proliferative activity in thyroid nodule, Authors evaluated cell proliferative activity by immunostaing for PNCA in 45 pathologically confirmed solitary thyroid nodule. The results were as follows. 1) The benign nodules were 25 cases(Adenomatous Goiter: 20 cases, Follicular adenoma: 5 cases) and malignant nodules were 20 cases(Papillary Ca : 14 cases, Follicular Ca : 4 cases, Anaplastic Ca : 2 cases). 2) The Most prevalent age groups were 4th decade(11 cases), and the next group was 5th decade. 3) The average PCNA labelling indices were as follows. Adenomatous goiter(I6.9%), Follicular adenoma(37.6%), papillary Ca(26.3%), Follicular Ca(8.8%) and Anaplastic Ca(86.7%). There were no significant differences in benign(20.4) and malignant nodules (28.8%) except anaplastic Ca(p=0.3226). 4) When the average tumor size 2cm in papillary Ca, the PCNA indices were 26.0% (below 2cm) : 26.6% (above 2cm) (p=0.9642). The PCNA incidies were 23.9% (with lymphatic spread) : 28.7% (without lymphatic spread) (p=0.7056). There were no signlficant differences in the above cases. In conclusion, there were no significant differences in cell proliferative activity by staining for PCNA between benign and malignat nodules except anaplastic Ca.

• International Journal of Oral Biology
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• v.33 no.1
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• pp.25-31
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• 2008

Inhibition of proteasome activity may reduce many types of cancer, so it's pathway is effective in cancer as well as in clinical fields. Here the author has carried out experiment targeting on the elevation of apoptosis in oral cancer cells by combination of proteasome inhibitor, lactacystin, and DNA replication inhibitor, etoposide. The growth of KB cells was measured by MTT methods and apoptosis was analyzed by DNA fragmentation and Hochest nucleus staining. The proteasome activity was analyzed by fluorescent tagged peptide and cellular protein expression was detected by Western hybridization. Though lactacystin and etoposide inhibited KB cell growth alone, but low combined doses inhibited cell growth more strongly and induced apoptosis. The proteasome activity was also seriously inhibited by the combination of both chemicals. Tumor suppressor proteins and apoptosis inducing proteins were highly increased under the combination of both chemicals. From above studies we can conclude that proteasome inhibitors may be used for the treatment of oral cancer and proteasome inhibitors with DNA replication inhibitors may be effective in clinical trials of oral cancer.

• Toxicological Research
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• v.13 no.1_2
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• pp.39-48
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• 1997

Cyclophosphamide(25, 50 or 100 mg/kg), orally administered to male Sprague-Dawley rats, caused a time- and dose-dependent thymic atrophy. In the light microscopic examination of the atrophic thymus, thymocytes with condensed or fragmented nucleus were multifocally observed in the cortical region, started to increase 8 hr after CPA treatment and reached to the maximal level at 16 hr, although such cells were not seen after 48 hr when the severe depletion of thymocytes were marked. In agarose gel electrophoresis to analyze the DNA changes, DNA extracted from atrophic thymus showed a oligonucleosomal laddering at the corresponding time to morphological changes. In an additional supportive experiment, thymocytes showing morphological changes, nuclear condensation or apoptotic body, exhibited a positive reaction to immunoperoxidase staining using in situ apoptosis detection kit. Separately, agarose gel electrophoresis of DNA from bone .marrow cells was performed to investigate the involvement of bone marrow cells in the process of thymocyte apoptosis. Although DNA laddering was slightly increased 2 and 4 hr after treatment, no clear correlation was inferred. Taken togather, it is concluded that thymocytes showing morphological changes in thymic atrophy induced by cyclophosphamide administration represent an apoptosis having biochemical nature of programmed cell death.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.209-214
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• 2008

VBNC (Viable but nonculturable) state is an adaptive response of cells in adverse environments, which lead cell not grow on routine nutrient agar. In this study, we induced VBNC in Escherichia coli using copper and verify the characterization of it. After treatment of copper, we didn't detect any cells via plate cultivation, namely, colony forming unit (CFU) was zero. However, we identified the existence of VBNC by staining live cells with Live/Dead BacLight bacterial viability kit and counting them through flow cytometry. Then we isolated genomic DNA and RNA from VBNC-induced cells and analyzed the stability of them. Degradation of RNA is more severe than that of DNA and RNA is degraded as specific fragments. In addition, we showed the morphology of VBNC cell by Bio-Transmission Electron Microscope (Bio-TEM). VBNC cell showed impaired periplasmic space and inner and outer membrane were separated and the amount of cytosol were significantly decreased.

• International Journal of Oral Biology
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• v.39 no.3
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• pp.159-167
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• 2014

Curcumin is a widely used flavoring agent in food, and it has been reported to inhibit cell growth, to induce apoptosis, and to have antitumor activity in many cancers. Cisplatin is one of the most potent known anticancer agents and shows significant clinical activity against a variety of solid tumors. This study was undertaken to investigate the synergistic apoptotic effects of co-treatment with curcumin and cisplatin on human tongue SCC25 cells. To investigate whether the co-treatment efficiently reduced the viability of the SCC25 cells compared with the two treatments separately, an MTT assay was conducted. The induction and the augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining, and an analysis of DNA hypoploidy. Western blot, MMP and immunofluorescence tests were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following the co-treatment. In this study, following the co-treatment with curcumin and cisplatin, the SCC25 cells showed several forms of apoptotic manifestation, such as nuclear condensation, DNA fragmentation, reduction of MMP, increased levels of Bax, decreased levels of Bcl-2, and decreased DNA content. In addition, they showed a release of cytochrome c into the cytosol, translocation of AIF and DFF40 (CAD) to the nuclei, and activation of caspase-7, caspase-3, PARP, and DFF45 (ICAD). In contrast, separate treatments of $5{\mu}M$ of curcumin or $4{\mu}g/ml$ of cisplatin, for 24 hours, did not induce apoptosis. Therefore, our data suggest that combination therapy with curcumin and cisplatin could be considered as a novel therapeutic strategy for human oral squamous cell carcinoma.

• Korean journal of applied entomology
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• v.10 no.2
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• pp.103-107
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• 1971

In this report, an experiment has been conducted to test the quantitative changes of nucleic acids in the nuclei of the epidermal cell of the galls, caused by Eriophyes kuko Kishida on the leaf of Lycium chinense Mill by means o( microspectrophotometric techniques, the two-wave-length methods. And the sizes of the epidermal cells and nuclei have been measured. The experimental results were summarized as follows: 1) It has been found that as the gall grows, the diameter of the epidermal cells and their nuclei increased and they were larger than those of tile healthy ones. 2) Microspectrophotometric measurement of nuclei by the 'Two-wave-length method' after staining with Feulgen reagent showed no changes in DNA content in the early stage of the gall. As the gall matured, however, DNA content of the gall increased more than that of the healthy leaf. 3) RNA-measurement of nuclei stained with Azur-B in DNase treated epidermal cells of the gall revealed that temporary increase in RNA content occurred in early to middle stages after the gall formation. As the gall matured, however, RNA content of the gall decreased more as against that of the healthy leaf.

• BMB Reports
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• v.47 no.10
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• pp.575-580
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• 2014

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced ${\gamma}H2AX$ foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated ${\beta}$-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.5507-5512
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• 2013

Objective: To investigate the correlation between ERCC1 expression levels in tumor tissue and peripheral blood lymphocytes (PBL) from patients with gastric cancer and assess the relationship between PBL DNA repair rate (DRR) and the efficacy of platinum chemotherapy. Methods: A total of 53 patients with gastric cancer receiving surgery and 20 controls were studied. ERCC1 protein expression in tumour tissue and PBL were determined by immunohistochemical staining. The PBL DRRs of 47 advanced patients and 20 controls were estimated by comet assay. Results: The positive expression rates of ERCC1 were 67. 9%, 56. 6% and 10.0% in tumour tissues, PBLs of gastric cancer patients, and PBLs of the control group. PBL ERCC1 expression correlated with that in tissue (${\chi}^2$=15. 463, p=0.000). Pearson contingency coefficient=0.475). DRRs of cancer patients by tail length (TL) (Z=4. 662, p=0.000) and tail moment (TM) (Z=3. 827, p=0.000) were significantly lower than that of control group. When TL was applied as an indicator, the correlation between DRR and chemotherapy efficacy was significant (Spearman rank correlation r=0.327, p=0.032). Patients with low levels of DRR in PBL presented better short-term efficacy of chemotherapy than those with high levels of DRR. Conclusions: The ERCC1 expression in PBLs may indirectly reflect ERCC1 expression in gastric cancer tissues. Compared with non-cancer populations, patients with gastric cancer may have lower DNA repair capacity. DRR in PBL may predict the short-term efficacy of platinum-based chemotherapy for patients with advanced gastric cancer.