• Journal of Life Science
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• v.19 no.6
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• pp.770-780
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• 2009

A new photosensitizer, 9-Hydroxypheophorbide-a (9-HpbD-a), was derived from Spirulina platensis. We conducted a series of experiments, in vitro and in vivo, to evaluate the anticancer effect and mechanism of photodynamic therapy using 9-HpbD-a and 660 nm diode lasers on a squamous carcinoma cell line. We studied the cytotoxic effects of pheophytin-a, 9-HpbD-a, 9-HpbD-a red and 660 nm diode lasers in a human head and neck cancer cell line (SNU-1041). Cell growth inhibition was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of 9-HpbD was higher than those of 9-HpbD-a red or pheophytin-a in PDT. We then tested the cytotoxic effects of 9-hydroxypheophorbide-a (9-HpbD-a) in vitro. The cultured SNU-I041 cells were treated with serial concentrations of 9-HpbD-a followed by various energy doses (0, 0.1, 0.5, 3.2 J/$cm^{2}$) and by various interval times (0, 3, 6, 9, 12 hr) until laser irradiation, then MTT assay was applied to measure the relative inhibitory effects of photodynamic therapy (PDT). Optimal laser irradiation time was 30 minutes and the cytotoxic effects according to incubation time after 9-HpbD-a treatment increased until 6 hours, after which it then showed no increase. To observe the cell death mechanism after PDT, SUN-I041 cells were stained by Hoechst 33342 and propidium iodide after PDT, and observed under transmission electron microscopy (TEM). The principal mechanism of PDT at a low dose of 9-HpbD-a was apoptosis, and at a high dose of 9-HpbD-a it was necrosis. PDT effects were also observed in a xenografted nude mouse model. Group I (no 9-HpbD-a, no laser irradiation) and Group II (9-HpbD-a injection only) showed no response (4/4, 100%), and Group III (laser irradiation only) showed recurrence (1/4,25%) or no response (3/4, 75 %). Group IV (9-HpbD-a + laser irradiation) showed complete response (10/16, 62.5%), recurrence (4/16, 25%) or no response (2/16, 12.5%). Group IV showed a significant remission rate compared to other groups (p<0.05). These results suggest that 9-HpbD-a is a promising photosensitizer for the future and that further studies on biodistribution, toxicity and mechanism of action would be needed to use 9-HpbD-a as a photosensitizer in the clinical setting.

• The Journal of Korean Obstetrics and Gynecology
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• v.15 no.4
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• pp.45-60
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• 2002

Mouse calvarial osteoblast cells were isolated and cultured. To examine whether the cells produce active gelatinases in culture medium or not,the cells were analyzed using by zymograsphic analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We show that mouse calvarial osteoblasts in culture constitutively synthesize progelatinase- A. Then, mouse osteoblasts, which were stimulated by PTH, $1,25(OH)_2D_3$, mononuclear cell conditioned medium (MCM) and IL-1 as bone resorption agent's, showed increased collagenolysis by producing the active gelatinase. However, treatment of indomethacin and dexamethasone significantly decreased those effects of collagenolysis in mouse osteoblastic cells. On the other hand, IL-1 in stimulating bone resorption was examined using fetal mouse long bone organ culture. IL-1 stimulated bone resorption and produced marked resorption when present simultaneously. Furthermore, when it was examined the effects of indomethacin and dexamethasone on the dose dependent responses of $IL-1{\alpha}$, indomethacin and dexametasone produced a rightward shift in the IL-1 dose response curve. The results of in vitro cytotoxicities showed that Taeyoungjon-Jahage water extracts(T.Y.J-J.H.G extracts) have no any cytotoxicities in concentrations of $1-200\;{\mu}g/ml$ and furthermore there is no any cytotoxicity even in concentration of $300\;{\mu}g/ml$ on mouse calvarial bone cells. T.Y.J.-J.H.G. extracts had protective activity against PTH (2 units/mI), or MCM (5%, v/v), or $rhIL-1{\alpha}$ (1 ng/mI) or $1,25(OH)_2D3$ (10 ng/ml) , $IL-1{\alpha}$ and $IL-1{\beta}-induced$ collagenolysis in the mouse calvarial cells. Pretreatment of the T.Y.J.-J.H.G.extracts for 1 h, which by itself had little effect on cell survival, did not enhance the collagenolysis, nor significantly reduced the collagenolysis by pretreatment. Furthermore. the medicinal extracts were shown to have the protective effects against collagenolysis induced by $IL-1{\alpha}$ and $IL-1{\beta}$. Pretreatment of the extracts for 1 h significantly reduced the collagenolysis. Interestingly, the T.Y.J.-J.H.G. extracts were shown to have the inhibiting effects against gelatinase enzyme and processing activity induced by the bone resortion agents of PTH, $1,25(OH)_2D_3$, $IL-1{\beta}$ and $IL-1{\alpha}$, with strong protective effect in pretreatment with the extracts. T.Y.J.-J.H.G. extracts were shown to have the inhibiting effects against $IL-1{\alpha}-$ and $IL-1{\beta}-stimulated$ bone resorption and the effect of the pretreatment with a various concentrations of the medicinal extracts were significant. The inhibition extent and phenomena of IL-1 stimulated bone resorption by nonsteroidal anti-inflammatory agents of indomethacin and dexamethasone were similar to those obtained by T.Y.J.-J.H.G. extracts treatment in the mouse calvarial tissue culture system. These results indicated that the T.Y.J.-J.H.G.-water extracts are highly stable and applicable to clinical uses in osteoporosis.

• The Journal of Korean Obstetrics and Gynecology
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• v.15 no.4
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• pp.61-75
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• 2002

Recombinant human $interleukin-1{\beta}$ $(rhIL-1{\beta})$ regulates several activities of the osteoblast cells derived from mouse calvarial bone explants in vitro. $rhIL-1{\beta}$ stimulated cellular proliferation and the synthesis of prostaglandin $E_2(PGE_2)$ and plasminogen activator activity in the cultured cells in a dose-dependent manner. However, the induction of osteocalcin synthesis and alkaine phosphatase activity in response to vitamine D, two characteristics of the osteoblast phenotype, were antagonized by $rhIL-1{\beta}$ over a similar dose range. This study supports the role of $IL-1{\beta}$ in the pathological modulation of bone cell metabolism, with regard to implication in the pathogenesis of osteoporosis by $IL-1{\beta}$. When the mouse calvarial bone cells were used, the bone resorption induced by $IL-1{\beta}$ was strongly inhibited by calcitonin treatment, indicating osteoclast-mediated bone resorption. On the other hand, the medicinal extracts of Taeyoungjon-Jahage (T.Y.J-J.H.G extracts) was tested for whether they could inhibit $IL-1{\beta}-induced$ $PGE_2$ production. Cell viability was not significantly affected by treatment with the indicated concentration of the extracts. The T.Y.J.-J.H.G. extracts were shown to have the inhibitory effects against the synthesis of $PGE_2$. We also examined the effect of the pretreatment with a various concentrations of the T.Y.J.-J.H.G. extracts then treated the $PGE_2-induction$ agents. Pretreatment of the T.Y.J.-J.H.G. extracts for 1 h, which by itself had little effect on cell survival, did not enhance the synthesis of $PGE_2$. Furthermore, the T.Y.J-J.H.G. extracts were shown to have the protective effects against plasminogen dependent fibrinolysis induced by the bone resorption agents of $IL-1{\beta}$. Pretreatment of the T.Y.J.-J.H.G. extracts for 1 h did not enhance the plasminogen dependent fibrinolysis. Finally, calcitonin showed the inhibitory activity the $IL-1{\beta}-stimulated$ bone resorption in the mouse calvarial bone cells having both of the osteoblast and osteoclast cells. Seemingly, pretreatment of the T.Y.J.-J.H.G. extracts for 1 h reduced the bone resorption. These results clearly indicated that calcitonin and T.Y.J.-J.H.G. extracts play key roles in inhibition of the osteoclast-mediated bone resorption.

• Korean Journal of Veterinary Research
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• v.32 no.1
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• pp.15-24
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• 1992

The effects of CC the ovulatory response, oocyte normality, ovarian steroidogenesis and subsequent embryo developmental potential were examined in PMSG-treated rats. On Days of 25~27 of age, immature female Sprague Dawley rats were treated with three different doses(0.05, 0.1 or 1.0mg /day) of clomiphene citrate or vehicle. The females subsequently received 4IU PMSG on Day 28 and/or 10IU hCG on Day 30, and were killed on Day 31. Some females given 0.1mg CC or vehicle with 4IU PMSG were then mated and killed on Days 2, 3, 4 and 5 of pregnancy. Compared to vehicle(control) group, by increasing the doses of CC, there were a significant decrease in the ovulatory response as judged by both the proportion of rats ovulating and the mean number of oocytes per rat and a marked reduction of ovarian weight. The increasing doses of CC substantially promoted the degeneration(%) of oocytes ovulating in a dose-dependent manner. The CC-mediated inhibitions of the ovulatory response and ovarian weight were oompletely overcome by a subsequent treatment of hCG. Increasing doses of CC resulted in a siginificant elevation of serum estradiol with the decreased levels of progesterone and androgens. The additive treatment with hCG was effective to reduce the elevation of estradiol and to increase the reduction of progesterone produced by high dose(1.0mg) of CC. The preimplantation embryos recovered from 0.1mg CC-treated pregnant rats demonstrated a progressive early loss from Day 3 of pregnancy with a significant increase in the percentage of degeneration during all periods examined, compared to controls. The rate of progressive embryo cleavage in the CC-treated rats were slower than that in controls from Day 3 of pregnancy. Additionally, the percentage of the cleaved embryos recovered from the CC-treated rats remained significantly lower consistently from Day 2 of pregnancy, compared to control regimen. These results demonstrate a possible mechanism of CC-mediated inhibition of ovulatory response in the rats which may include the attenuation or blockade of the endogenous secretion of gonadotropins and also suggest that its detrimental effects observed on oocyte normality and embryonic development may be caused by abnormal follicular steroidogenesis( especially elevated estradiol) preceding fertilization.

• Journal of Life Science
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• v.19 no.11
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• pp.1629-1636
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• 2009

Pine trees (Pinus densiflora Sieb. et Zacc.) have been used as a traditional health-promoting medicinal food in Korea. This research was performed to determine the antioxidative and antibacterial activities, tyrosinase, nitric oxide synthesis, angiotensin converting enzyme (ACE), and xanthine oxidase inhibition effects of the pine bud ethanol extract (PBE). Antioxidative activities of PBE were measured by using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging activity and superoxide dismutase-like activity (SODA). DPPH radical scavenging and SOD-like activities of PBE were remarkably increased in a dose-dependent manner, and were about 88.9% and 47.9% at 1 mg/ml and 10 mg/ml, respectively. The xanthine oxidase and angiotensin converting enzyme activities were inhibited about 71.9% and 60.8% at 1 mg/ml and $100{\mu}g/ml$ of PBE, respectively. The tyrosinase inhibitory activities of PBE were slightly increased in a dose-dependent manner. The PBE showed strong antimicrobial activities on Escherichia coli (E. coli) and Vibrio paraheamolyticus. Stimulation of the macrophages RAW264.7 cells with lipopolysaccharide (LPS) resulted in increased production of nitric oxide (NO) in the medium. However, NO synthesis was reduced up to 54% by addition of PBE at $200{\mu}g/ml$. These results revealed that pine buds have a strong antioxidative and anti-inflammatory activity, and exhibit angiotensin converting enzyme and xanthine oxidase inhibitory activities. This suggests that pine buds have the greatest property as a source for natural health products.

• Proceedings of the Ginseng society Conference
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• 2007.12a
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• pp.57-58
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• 2007

Purpose: Ginseng is a well-known medical plant used in traditional Oriental medicine. Korean red ginseng (KRG) has been known to have potent biological activities such as radical scavenging, vasodilating, anti-tumor and anti-diabetic activities. However, the mechanism of the beneficial effects of KRG on diabetes is yet to be elucidated. The present study was designed to investigate the anti-diabetic effect and mechanism of KRG extract in C57BL/KsJ db/db mice. Methods: The db/db mice were randomly divided into six groups: diabetic control group (DC), red ginseng extract low dose group (RGL, 100 mg/kg), red ginseng extract high dose group (RGH, 200 mg/kg), metformin group (MET, 300 mg/kg), glipizide group (GPZ, 15 mg/kg) and pioglitazone group (PIO, 30 mg/kg), and treated with drugs once per day for 10 weeks. During the experiment, body weight and blood glucose levels were measured once every week. At the end of treatment, we measured Hemoglobin A1c (HbA1c), blood glucose, insulin, triglyceride (TG), adiponectin, leptin, non-esterified fatty acid (NEFA). Morphological analyses of liver, pancreas and white adipose tissue were done by histological observation through hematoxylin-eosin staining. Pancreatic islet insulin and glucagon levels were detected by double-immunofluorescence staining. To elucidate an action of mechanism of KRG, DNA microarray analyses were performed, and western blot and RT-PCR were conducted for validation. Results: Compared to the DC group mice, body weight gain of PIO treated group mice showed 15.2% increase, but the other group mice did not showed significant differences. Compared to the DC group, fasting blood glucose levels were decreased by 19.8% in RGL, 18.3% in RGH, 67.7% in MET, 52.3% in GPZ, 56.9% in PIO-treated group. With decreased plasma glucose levels, the insulin resistance index of the RGL-treated group was reduced by 27.7% compared to the DC group. Insulin resistance values for positive drugs were all markedly decreased by 80.8%, 41.1% and 68.9%, compared to that of DC group. HbA1c levels in RGL, RGH, MET, GPZ and PIO-treated groups were also decreased by 11.0%, 6.4%, 18.9%, 16.1% and 27.9% compared to that of DC group, and these figure revealed a similar trend shown in plasma glucose levels. Plasma TG and NEFA levels were decreased by 18.8% and 16.8%, respectively, and plasma adiponectin and leptin levels were increased by 20.6% and 12.1%, respectively, in the RGL-treated group compared to those in DC group. Histological analysis of the liver of mice treated with KRG revealed a significantly decreased number of lipid droplets compared to the DC group. The control mice exhibited definitive loss and degeneration of islet, whereas mice treated with KRG preserved islet architecture. Compared to the DC group mice, KRG resulted in significant reduction of adipocytes. From the pancreatic islet double-immunofluorescence staining, we observed KRG has increased insulin production, but decreased glucagon production. KRG treatment resulted in stimulation of AMP-activated protein kinase (AMPK) phosphorylation in the db/db mice liver. To elucidate mechanism of action of KRG extract, microarray analysis was conducted in the liver tissue of mice treated with KRG extract, and results suggest that red ginseng affects on hepatic expression of genes responsible for glycolysis, gluconeogenesis and fatty acid oxidation. In summary, multiple administration of KRG showed the hypoglycemic activity and improved glucose tolerance. In addition, KRG increased glucose utilization and improved insulin sensitivity through inhibition of lipogenesis and activation of fatty acid $\beta$-oxidation in the liver tissue. In view of our present data, we may suggest that KRG could provide a solid basis for the development of new anti-diabetic drug.

• Journal of Life Science
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• v.21 no.9
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• pp.1346-1353
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• 2011

Sophora tonkinensis Gapnep has been used as a traditional herbal medicine in oriental regions since ancient times. In this study, the effect and mechanism of the MeOH extract of Sophora tonkinensis Gapnep (STME) on adipocite differentiation and adipogenesis in 3T3-L1 preadipocites were investigated. Treatment with STME in the concentration range of 0-200 ${\mu}g$/ml significantly inhibited the differentiation of 3T3-L1 preadipocites in a dose-dependent manner, as determined by a decrease in intracellular lipid droplets and lipid contents measured by Oil Red O staining. In association with the inhibitory effect of lipid accumulation, the expressions of the proteins concerned with adipogenesis in 3T3-L1 preadipocites were also investigated. Treatment with STME reduced the expressions of peroxisome proliferator-activated receptor ${\gamma}$ (PPAR${\gamma}$), cytidine-cytidine-adenosine-adenosine-thymine (CCAAT)/enhancer-binding proteins ${\alpha}$ and ${\beta}$ (C/EBP${\alpha}$ and C/EBP${\beta}$) and sterol regulatory element binding protein (SREBP), which are adipocyte specific markers. In flow cytometry analysis, the inhibitory effect of differentiation was caused by G1 arrest and following mitotic clonal expansion cease. Therefore, we also investigated the alteration of G1 phase arrest-related proteins. As a result, the expression of p21 protein was significantly increased, while the expressions of Cdk2, E2F-1 and phospho-Rb were reduced in a dose-dependent manner in STME treated 3T3-L1 cells. According to these results, STME might inhibit differentiation through G1 arrest in 3T3-L1 preadipocytes adipogenesis, and further studies, which are in progress, have to be completed to identify the active compounds.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.4
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• pp.844-852
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• 2006

This study was to evaluate the effect of Yeongyupaedog-san (YGPDS) on mouse Thl and Th2 cells' differentiation and ovalbumin (OVA)-induced allergic inflammation. The proliferation of mouse CD4 T cells and the secretion of Th1/Th2 cytokines under the influence of YGPDS extract were measured as well as the amount of ${\beta}-hexosaminidase$ in RBL-2H3 cells and the levels of $TNF-{\alpha}$ and 1L-6 secretion in Raw264.7 cells. BALB/c mice were orally administered with YGPDS extract and simultaneously inoculated with OVA to induce allergic reaction and measure the level of total IgE, OVA-specific IgE and the production of IFN- g, IL-4, IL-5 by the spleen cells. When mouse CD4 T cell were stimulated with anti-CD3 and anti-CD28 for 48 hours in various concentrations of YGPDS extract, it increased proliferation of CD4 cells by 11% in $100\;{\mu}g/^{ml}$ concentration but it showed an inhibition by 37% at $200\;{\mu}g/^{ml}$ CD4 T cells under Th1/Th2 polarizing conditions for 3 days with YGPDS resulted in mild decrease of IFN- g in Thl cells and significant decrease of IL-4 in Th2 cells at $500\;{\mu}g/^{ml}\;and\;100\;{\mu}g/^{ml}$ by 18% and 21%, respectively. YGPDS extract had a dose-dependent inhibitory effect on antigen-induced release of ${\beta}-hexosaminidase$ in RBL-2H3 cells. Treatment of YGPDS extract on LPS stimulated Raw 264.7 cells showed dose-dependent decrease in TNF-n production. Oral administration of YGPDS extract on OVA-induced allergic mice showed an inhibitory effect on the levels of total serum IgE and OVA-specific IgE by 25% and 34% , respectively. Culture of spleen cells with OVA resulted in significant increase of IFN- g by 44% and significant decrease of IL-4 and IL-5 by 56%, and 24%, respectively. The results show that YGPDS does not strongly induce mouse T cells to transform into Thl or Th2 but it has an anti-allergic effect in vitro, and that it also corrects the unbalance between the reactions of Th cells in allergic diseases.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.6
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• pp.1467-1476
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• 2006

This study was to evaluate the effect of Bulhwangeumjeonggi-san (BS) on mouse Th1 and Th2 cells' differentiation and ovalbumin (OVA)-induced allergic inflammation. The proliferation of mouse CD4 T cells and the secretion of Th1/Th2 cytokines under the influence of BS extract were measured as well as the amount ${\beta}$-hexosaminidase in RBL-wH3 cells and the levels of TNF-${\alpha}$ and IL-6 secretion in Raw264.7 cells. BALB/c mice were orally administered with BS extract and simultaneously inoculated with OVA to induce allergic reaction and measure the level of total lgE, OVA-specific lgE and the production of IFN- g, IL-4, IL-5 by the spleen cells. When mouse CD4- T cell were stimulated with anti-CD3 and anti-CD28 for 48 hours in various concentrations of BS extract, it increased proliferation of CD4 cells by 14% in 50 ${\mu}g/m{\ell}$ concentration but it showed an inhibition in higher concentrations. CD4 T cells under Th1/Th2 polarizing conditions for 3 days with BS resulted in mild decrease of IFN- g in Th1 cells and mild increase of IL-4 in Th2 cell at 50 ${\mu}g/m{\ell}$ but the level of IL-4 decreased by 18% at 100 ${\mu}g/m{\ell}$. BS extract had a dose-dependent inhibitory effect on antigen-induced release of ${\beta}$-hexosaminidase in RBL-2H3 cells. Treatment of BS extract on LPS stimulated Raw 264.7 cells showed dose-dependent decrease in TNF-${\alpha}$ production. Oral administration of BS extract on OVA-induced allergic mice showed an inhibitory effect on the levels of total serum lgE and OVA-specific lgE by 50% and 55%, respectively. Culture of spleen cells with OVA resulted in significant increase of IFN- g by 25% and significant decrease of IL-4 and IL-5 by 53%, and 38%, respectively. The results show that BS does not strongly induce mouse T cells to transform into Th1 or Th2 but it has an anti-allergic effect in vitro, and that it also corrects the unbalance between the reactions of Th cells in allergic diseases.

• Journal of Life Science
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• v.26 no.2
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• pp.147-154
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• 2016

Osteosarcoma (OS) is the most common and malignant bone tumors. Although many types of resection surgery and experimental agents were developed, median survival and clinical prognosis are poorly investigated. Recently, several researches have reported that Eucheuma cottonii has potent as protective effects of coal dust-induced lung damage via inhibition of malondialdehyde (MDA) and oxidative stress in bronchoalveolar lavage fluids (BALF). However, anti-cancer effects and specific molecular mechanism of extract from Eucheuma cottonii (EE) has not been clearly studied yet. This study evaluated that anti-cancer potential of EE in human osteosarcoma Saos-2 cells. EE indicated cytotoxicity on Saos-2 cells in a dose-dependent manner. Morphological degradation and nucleic condensation were also observed under the EE treatment. However, it did not significantly affect on non-cancerous kidney HEK-293 cells under the same concentration which is shown cytotoxicity on Saos-2 cells. The phosphorylation of Fas-Associated Death Domain (FADD) and expression of cleaved caspase-8, -7 and -3 were upregulated in a dose-dependent manner. In immunofluorescence staining, expression level of Fas and cleaved PARP were upregulated by EE treatment. Furthermore, treatment of EE induces upregulation of sub G1 phase by flow cytometry analysis. The results demonstrated that EE has a therapeutic potential against osteosarcoma via FADD mediated caspase cascade apoptosis signal pathway.