• Journal of Life Science
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• v.21 no.3
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• pp.451-458
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• 2011

Cordycepin (3'-deoxyadenosine), a nucleoside derivative isolated from Cordyceps militaris, is reported to have antitumor effects. However, neither its molecular mechanism nor its molecular targets are well understood. In the present study, molecular mechanisms for the anti-tumor effects of cordycepin were investigated in human prostate cancer PC-3 cells. The MTT assay was used to detect cell viability. Annexin V/FITC assay, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and $Ca^{2+}$ flux were used to assess for the presence of apoptosis. Western blot analysis was used to detect protein expression. Treatment of cordycepin resulted in significantly decreased cell viability of PC-3 cells in a dose- and time-dependent manner. A dose-dependent apoptotic cell death was also measured by flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled cordycepin treatment resulted in significant mitochondrial dysfunction, ROS production, and elevation of $Ca^{2+}$ concentrations. These phenomena were followed activation of caspase-3, subsequently leading to PARP cleavage and cell apoptosis. Taken together, cordycepin induces apoptosis in PC-3 cells through regulation of a mitochondrial mediated pathway.

• Journal of Life Science
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• v.24 no.1
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• pp.92-97
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• 2014

Cordycepin, an active component originally isolated from the traditional medicine Cordyceps militaris, is a derivative of the nucleoside adenosine, which has been shown to possess a number of pharmacological properties, including antioxidant and anti-inflammatory activities, immunological stimulation, and antitumor effects. This study was conducted on cultured human prostate carcinoma LNCap cells to elucidate the possible mechanisms by which cordycepin exerts its anticancer activity, which, until now, has remained poorly understood. Cordycepin treatment of LNCap cells resulted in dose-dependent inhibition of cell growth and the induction of apoptotic cell death as detected by an MTT assay, cleavage of poly ADP-ribose polymerase, and annexin V-FITC staining. Flow cytometric analysis revealed that cordycepin resulted in G2/M arrest in cell cycle progression and downregulation of cyclin B1 and cyclin A expression in a concentration-dependent manner. Moreover, the incubation of cells with cordycepin caused a striking induction in the expression of the cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1 without affecting the expression of the tumor suppressor p53. It also resulted in a significant increase in the binding of CDK2 and CDC2 to p21. These findings suggest that cordycepin-induced G2/M arrest and apoptosis in human prostate carcinoma cells is mediated through p53-independent upregulation of the CDK inhibitor p21.

• Journal of Life Science
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• v.27 no.7
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• pp.834-839
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• 2017

Propolis is a natural product collected from plants by honey bees product used extensively in traditional medicine for its antioxidant, anti-inflammatory, immunomodulatory and anti-cancer effects. Propolis exhibits a broad spectrum of biological activities because it is a complex mixture of natural substances. Ovarian cancer is the second most common newly diagnosed cancer from all cancers among women in Korea and the leading cause of death from gynecological malignancies. While most ovarian cancer patients initially respond to surgical debulking and chemotherapy, patients later succumb to the disease. Thus, there is an urgent need to test novel therapeutic agents to counteract the high mortality rate associated with ovarian cancer. In this study, we investigated the anti-cancer properties and the active mechanism of Australian propolis in human epithelial ovarian cancer A2780 cells. Our data revealed that propolis showed a cytotoxic activity in a dose-dependent manner. Flow cytometric analysis for cell cycle arrest and apoptosis using propidium iodide staning and annexin V-FITC indicated that propolis could induce cycle arrest in the G0/G1 phase and apoptosis in a dose-dependent manner on human epithelial ovarian cancer cells. These results suggest that the Australian propolis is potential alternative agent on ovarian cancer prevention and treatment.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.36 no.3
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• pp.215-219
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• 2010

In this study, we investigated effects of Monegy (mixture of Sophorae Radix, Panax ginseng, Salvia miltiorrhiza BUNGE) on epilate-induced hair-loss in dorsal region of C57/BL6 mice and external structure of human hair. For morphological and histological analysis in scalp of epilate-induced hair-loss animal model, we utilized several microscopic techniques, such as confocal laser scanning microscopy (CLSM) and LAS 4000. Confocal analysis showed the distribution of FITC-conjugated Monegy and penetration depth compared with normal and control group. Furthermore, when Monegy was topically administrated onto a C57BL6 mouse, it penetrated very well. The fluorescence intensity was increased upto 205 and 113 folds compared to normal and control group, respectively. Also, area of fluorescence was increased to upto 255 to 127 folds compared to normal and control group. Broad scale area of fluorescence in dermis region was observed in the Monegy-treated mice. Furthermore, Monegy induced upto 75% hair repair against depilation. It might be promoted via the induction of growth factors in hair follicle.

• Journal of the East Asian Society of Dietary Life
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• v.24 no.6
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• pp.739-747
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• 2014

The aim of the study was to determine the effects of Ixeris dentata extract (IDE) on anticancer activity in human breast cancer MDA-MB-231 cells at both cellular and molecular levels. The cells were cultured in the presence of 0, 20, 30 and $40{\mu}g/mL$ Ixeris dentata extract for 24 hours, respectively. At the end of culture, cytochemical analyses for MTT activity, trypan blue dye exclusion, Annexin V-FITC Apoptosis, and radical oxygen species (ROS) were conducted. RT-PCR was also performed to determine whether or not alterations in cell viability affect the Bax/Bcl-2 ratio. MTT assay showed that relative cell viability decreased in a dose-dependent manner (p<0.05). Reduction of cell viability matched well with increased cell membrane permeability as determined by trypan blue dye exclusion test (p<0.05). The rates of intracellular ROS also increased in a similar manner to those of TB-stained cells. There was an associated shift of apoptotic cells from early to late apoptosis between the 30 and $40{\mu}g/mL$. Bax/Bcl-2 ratio significantly increased along with significant decreases in Bcl-2 expression between 30 and $40{\mu}g/mL$ groups (p<0.05). In conclusion, anticancer activity of Ixeris dentata extract is modulated by a reduction in cell viability along with increased membrane permeability, leading to ROS accumulation within cells, and subsequently cell death through an apoptotic pathway that involves Bax and Bcl-2 in human breast cancer MDA-MB-231 cells.

• Herbal Formula Science
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• v.23 no.2
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• pp.199-208
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• 2015

Objectives : In the present study, we investigated the effects of ethanol extract of Scutellaria baicalensis (EESB) on the progression of cell cycle in human renal cell carcinoma Caki-1 cells. Methods : The effects of EESB on cell growth and apoptosis induction were evaluated by trypan blue dye exclusion assay and flow cytometry, respectively. The mRNA and protein levels were determined by Western blot analysis and reverse transcription-polymerase chain reaction, respectively. Results : It was found that EESB treatment on Caki-1 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by Annexin V-FITC staining. The flow cytometric analysis indicated that EESB resulted in G2/M arrest in cell cycle progression which was associated with the down-regulation of cyclin A expression. Our results also revealed that treatment with EESB increased the mRNA and proteins expression of tumor suppressor p53 and cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/CIP1), without any noticeable changes in cyclin B1, Cdk2 and Cdc2. In addition, the incubation of cells with EESB resulted in a significant increase in the binding of p21 and Cdk2 and Cdc2. These findings suggest that EESB-induced G2/M arrest and apoptosis in Caki-1 cells is mediated through the p53-mediated upregulation of Cdk inhibitor p21. Conclusions : Taken together, these findings suggest that EESB may be a potential chemotherapeutic agent and further studies will be needed to identify the biological active compounds that confer the anti-cancer activity of S. baicalensis.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.9-14
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• 2000

Objective: The sperm acrosome reaction is a $Ca^{2+}$-dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies suggested a role of $Ca^{2+}$ channels in acrosome reactions. This study was conducted to investigate the T-type calcium channel is operated in acrosome reaction of human spermatozoa. Method: Human semen samples were obtained from healthy donors with normal criteria. The spermatozoa were divided into five groups: Group 1 were non-treated as a control; Group 2 where spermatozoa were exposed to 5 ${\mu}M$ $Ca^{2+}$ A23187 $(Ca^{2+}i)$; Group 3 where spermatozoa were exposed 5 ${\mu}M$ $Ca^{2+}i$ and mibefradil; Group 4 where spermatozoa were exposed 5 ${\mu}M$ $Ca^{2+}i$ and nifedipine, and Group 5 where spermatozoa were treated with 5 ${\mu}M$ $Ca^{2+}i$ and both of mibefradil and nifedipine. Spermatozoa in all groups were retrieved after incubation for 15 and 30 minutes at $37^{\circ}C$. After staining with PSA-FITC, fluorescence was observed under a fluorescence microscope, and AR was evaluated on a total>100 spermatozoa/side. Result and Conclusion: We observed on acrosome reaction inhibition rate in human spermatozoa the various of concentration of mibefradil, nifedipine. Maximum response was noted with 1.0 ${\mu}M$ mibefradil and the decrease of acrosome reaction inhibition rate 45%. Nifedipine in acrosome reaction inhibition rate was only about 25%. The $Ca^{2+}i$-induced AR of spermatozoa was significantly suppressed by mibefradil. Incidence of the suppression was depending on concentration of mibefradil. Results from the present study suggest that the human spermatozoa possess T-type channel. The observation that reversible inhibitor of T channels in male germ cells provides a new mechanism of contraceptive action.

• KSBB Journal
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• v.19 no.2
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• pp.118-124
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• 2004

Melanocytes synthesize melanin within discrete organelle termed melanosomes which are transferred to the surrounding keratinocytes and can be produced in varying sizes, numbers and densities. Skin whitening products have become increasingly popular in the past few years. The most successful natural skin whitening agents are: Arbutin, Vitamin C, Kojic acid, Mulberry, which are all tyrosinase inhibitors. In this work, melanoston, a melanogenesis inhibitor isolated from yeast was studied to understand its mechanism of melanogenesis inhibition. It was found that melanoston was not a tyrosinase inhibitor, while when melanoston was applied to the B16 melanoma cell culture media, the intracellular tyrosinase activity was decreased by more than 30％, When B16 melanoma was stimulated with ${\alpha}$-MSH, cell morphololgy was dramatically changed to have lots of dendrites on the cell membrane surface. On the other hand, B16 was treated with ${\alpha}$-MSH and melanoston, simultaneously, the change of cell morphology was not so great. This inhibition effect of melanoston was found to be related to the inhibition of intracellular activation and transportation of tyrosinase, which was observed by immunostaining of B16 melanoma using anti-tyrosinase antibody. From these results, melanoston was regarded as an inhibitor to the differentiation of melanoma cells.

• Molecules and Cells
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• v.37 no.11
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• pp.785-794
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• 2014

Mitophagy, a cellular process that selectively targets dysfunctional mitochondria for degradation, is currently a hot topic in research into the pathogenesis and treatment of many human diseases. Considering that hypoxia causes mitochondrial dysfunction, which results in cell death, we speculated that selective activation of mitophagy might promote cell survival under hypoxic conditions. In the present study, we introduced the Regulator of calcineurin 1-1L (Rcan1-1L) to initiate the mitophagy pathway and aimed to evaluate the effect of Rcan1-1L-induced mitophagy on cell survival under hypoxic conditions. Recombinant adenovirus vectors carrying Rcan1-1L were transfected into human umbilical vein endothelial cells and human adult cardiac myocytes. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay and Trypan blue exclusion assay, Rcan1-1L overexpression was found to markedly reverse cell growth inhibition induced by hypoxia. Additionally, Rcan1-1L overexpression inhibited cell apoptosis under hypoxic conditions, as detected by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay. Meanwhile, the mitochondria-mediated cell apoptotic pathway was inhibited by Rcan1-1L. In contrast, knockdown of Rcan1-1L accelerated hypoxia-induced cell apoptosis. Moreover, Rcan1-1L overexpression significantly reduced mitochondrial mass, decreased depolarized mitochondria, and downregulated ATP and reactive oxygen species production. We further delineated that the loss of mitochondrial mass was due to the activation of mitophagy induced by Rcan1-1L. Rcan1-1L overexpression activated autophagy flux and promoted translocation of the specific mitophagy receptor Parkin into mitochondria from the cytosol, whereas inhibition of autophagy flux resulted in the accumulation of Parkin-loaded mitochondria. Finally, we demonstrated that mitochondrial 1permeability transition pore opening was significantly increased by Rcan1-1L overexpression, which suggested that Rcan1-1L might evoke mitophagy through regulating mitochondrial permeability transition pores. Taken together, we provide evidence that Rcan1-1L overexpression induces mitophagy, which in turn contributes to cell survival under hypoxic conditions, revealing for the first time that Rcan1-1L-induced mitophagy may be used for cardioprotection.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.51-59
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• 2003

This study was carried out to evaluate the effect of different freezing and thawing rates on the viability, motility and acrosomal changes of frozen canine spermatozoa. The ejaculated semen was extended with Tris-egg yolk buffer containing 8% glycerol and equilibrated for 60 min after cooled to 4$^{\circ}C$ for 58 min. The straws were cryopreserved gradually by slow-cooling at different distance(6, 10 and 17 cm, respectively) from the liquid nitrogen (L$N_2$) to achieve temperature rate of 3, 8.9 and 19$^{\circ}C$ /min. Thawing of the straws was performed in a water bath fur 2 min at 37$^{\circ}C$ and 55$^{\circ}C$ , respectively. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Concentration of the ejaculated fresh semen was normal range of 3.44 $\times$ 10$^{8}$ /ml. Freezing temperature were reduced to -110, -70 and -35$^{\circ}C$, as higher distance from liquid nitrogen, 6, 10 and 17 cm, respectively. Freezing at 3$^{\circ}C$/min in distance of 17 cm from liquid nitrogen yielded better motility, viability and rate of intact acrosome than 8.9 or 19$^{\circ}C$/min and the optimal thawing was 37$^{\circ}C$ for 2 min.