• 대한의생명과학회지
• /
• 제23권3호
• /
• pp.194-200
• /
• 2017

Bone-resorbing osteoclasts play a major role in maintaining bone homeostasis with bone-forming osteoblasts. Although it has been reported that A2B adenosine receptor (A2BAR) regulates osteoclast differentiation, its effects on apoptosis or proliferation of osteoclasts have been less-defined. Here, we demonstrate that A2BAR stimulation regulates macrophage-colony stimulating factor (M-CSF)-mediated osteoclast proliferation. Stimulation with a specific agonist of A2BAR, BAY 60-6583, significantly reduced M-CSF-mediated osteoclast proliferation in a time- and dose-dependent manner. In addition, A2BAR stimulation induced both apoptosis of the cells and cell arrest in the G1 phase with a decrease of cell number in the G2/M phase. Stimulation with BAY 60-6583 inhibited the activation of Akt by M-CSF, whereas M-CSF-induced ERK1/2 activation was not affected. These results suggest that the inhibition of M-CSF-mediated Akt activation by A2BAR stimulation increases apoptotic response of osteoclasts and induces cell cycle arrest in the G1 phase, thus contributing to the down-regulation of osteoclast proliferation.

• Journal of Pharmaceutical Investigation
• /
• 제36권4호
• /
• pp.231-237
• /
• 2006

Hypoxia in solid tumors is known to contribute to intrinsic chemoresistance. Histocultures are in vitro 3 dimensional cultures of tumor tissues and maintain the characteristic microenvironment of human solid tumors in vivo including hypoxia and multicellular structure. In this study, we evaluated the pharmacodynamics of tirapazamine(TPZ), a hypoxia-selective cytotoxin, in human non small cell lung cancer(NSCLC) cells grown as monolayers and histocultures. Antiproliferative activity of TPZ was determined after various conditions of drug exposure, and cell cycle arrest and apoptosis were also measured using flow cytometry. In monolayers, hypoxia selectivity measured by hypoxic/normoxic cytotoxicity ratio was increased with longer exposure. Lower cytotoxicity of TPZ was observed in histocultures compared to monolayers, however, a similar level of cytotoxicity was obtained with longer exposure of 96 hr. TPZ induced $G_2/M$ arrest and apoptosis in both culture conditions, which were greatly enhanced under hypoxic condition. Our data clearly showed the different pharmacodynamics of TPZ in monolayers and histocultures. Antiproliferative activity of TPZ against human solid tumors can be improved with longer drug exposure by exploiting drug delivery systems or by combining angiogenesis inhibitors to maintain drug concentration in tumor tissues.

• Journal of Ginseng Research
• /
• 제35권1호
• /
• pp.45-51
• /
• 2011

Wild ginseng has been used as a traditional medicine for thousands of years and for increase physical strength in Korea, China and Japan. This study reports that cultivated wild ginseng (CWG) inhibits adipocyte differentiation of 3T3-L1 pre-adipocytes in a concentration-dependent manner. Inhibition of adipocyte differentiation is one possible anti-obesity strategy. CWG inhibits the expression of the adipocyte differentiation regulator peroxisome proliferators-activated receptor (PPAR)${\gamma}$ and CCAAT/enhancer-binding protein ${\alpha}$mRNA. It also inhibited the expression of PPAR${\gamma}$ and adiponectin at the protein level during the differentiation of pre-adipocytes into adipocytes. Additionally, CWG blocked the cell cycle at the sub-$G_1$ phase transition, causing cells to remain in the pre-adipocyte state. These results indicate that CWG inhibits adipocyte differentiation and adipogenesis through pre-adipocyte cell cycle arrest in cultured 3T3-L1 cells.

• 한국가축번식학회지
• /
• 제20권2호
• /
• pp.143-153
• /
• 1996

To improve the efficiency of nuclear transplantation in the rabbit, this study were evaluated the influence of celly cycle of donor nuclei on the in vitro developmental potential in the nuclear transplant embryos. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G1 phase of 32-cell stage. Synchronization of the cell cylce of blastomeres were induced, first, using an microtubules polymerization inhibitor, 0.5$\mu\textrm{g}$/ml colcemid for 10h to arrest blastomeres in metaphase, and secondly, using a DNA synthesis inhibitor, 0.1$\mu\textrm{g}$/ml aphidicolin for 1.5 to 2h to cleave to 32-cell stage and arrest them in G1 phase. The separated G1 phase blastomeres of 32-cell stage were injectied into enucleated recipient cytoplasms by micromanipulation. After culture until 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The nuclear transplant embryos were co-cultured for 120h. In vitro cultured embryos were monitored every 24h to assess for development rate. After in vitro cultue for 120h, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye for counting the number of blastomeres under a fluorescence microscopy. The cleavage rate of blastomeres from 16-cell stage stage rabbit embryos treated with colcemid for 10h or aphidicolin for 6h following colcemid for 10h were not significantly different. The electrofusion rate was similar by high in S and G1 phase donor nuclei as 80.6 and 79.1%, respectively. However, the nuclear transplant embryos using G1 phase donor nuclei were developed to blastocyst at high rate(60.3%) than those using S phase donor nuclei(26.0%). Moreover, the mean blastocyst stage were increased significantly(P<0.05) with the G1 phase donor nuclei(176.6 cells and 1.50 cycles), as compared with the S phase donor nuclei(136.6 cells and 1.42 cycles). These results show that the blastomeres of G1 phase were more successful as donor nuclei in the nuclear transplant procedure, compared with S phase.

• Asian Pacific Journal of Cancer Prevention
• /
• 제16권7호
• /
• pp.2719-2724
• /
• 2015

Objective: The aim of this study was to investigate the clinical significance of annexin a1 (ANXA1) and provide molecular evidence to support that decreased ANXA1 expression could enhance cancer migration and invasion in pancreatic ductal adenocarcinoma (PDAC). Materials and Methods: Immunohistochemistry of a tissue microarray with 162 surgically resected PDAC specimens was performed to examine the expression of ANXA1. We also investigated the relationship between ANXA1 expression and clinicopathological factors and prognosis of PDAC patients. We further studied the role of ANXA1 in PDAC cell proliferation, migration and invasion by cell proliferation assay, migration assay and matrigel invasion assay with reduced ANXA1 expression by RNAi. Western blotting was used to detect matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression. We also detected MMP-9 enzyme activity by gelatin zymography. Results: Decreased expression of ANXA1 was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage of PDAC patients (p<0.05). Moreover, decreased expression of ANXA1 was correlated with poor survival (p<0.05). Furthermore, we found that ANXA1 knockdown inhibited cell proliferation, induced G1 phase cell cycle arrest, increased PDAC cell migration and invasion capacity compared with controls. In addition, Western blotting showed that ANXA1 knockdown increased the MMP-9 protein level and decreased TIMP-1 expression. Gelatin zymography showed that MMP-9 enzyme activity was also elevated. Conclusions: Negative ANXA1 expression is a most unfavorable prognostic factor for PDAC patients. ANXA1 knockdown inhibits cell proliferation by inducing G1 phase cell cycle arrest and increases migration and invasion of PDAC cells through up-regulating MMP-9 expression and activity, implying that ANXA1 may serve as a promising prognostic biomarker and therapeutic target for PDAC.

• Biomolecules & Therapeutics
• /
• 제26권5호
• /
• pp.494-502
• /
• 2018

Breast cancer is currently the most prevalent cancer in women, and its incidence increases every year. Azole antifungal drugs were recently found to have antitumor efficacy in several cancer types. They contain an imidazole (clotrimazole and ketoconazole) or a triazole (fluconazole and itraconazole) ring. Using human breast adenocarcinoma cells (MCF-7 and MDA-MB-231), we evaluated the effects of azole drugs on cell proliferation, apoptosis, cell cycle, migration, and invasion, and investigated the underlying mechanisms. Clotrimazole and ketoconazole inhibited the proliferation of both cell lines while fluconazole and itraconazole did not. In addition, clotrimazole and ketoconazole inhibited the motility of MDA-MB-231 cells and induced $G_1$-phase arrest in MCF-7 and MDA-MB-231 cells, as determined by cell cycle analysis and immunoblot data. Moreover, Transwell invasion and gelatin zymography assays revealed that clotrimazole and ketoconazole suppressed invasiveness through the inhibition of matrix metalloproteinase 9 in MDA-MB-231 cells, although no significant changes in invasiveness were observed in MCF-7 cells. There were no significant changes in any of the observed parameters with fluconazole or itraconazole treatment in either breast cancer cell line. Taken together, imidazole antifungal drugs showed strong antitumor activity in breast cancer cells through induction of apoptosis and $G_1$ arrest in both MCF-7 and MDA-MB-231 cells and suppression of invasiveness via matrix metalloproteinase 9 inhibition in MDA-MB-231 cells. Imidazole drugs have well-established pharmacokinetic profiles and known toxicity, which can make these generic drugs strong candidates for repositioning as antitumor therapies.

• Archives of Pharmacal Research
• /
• 제26권12호
• /
• pp.1055-1060
• /
• 2003

Lysophosphatidic acid (LPA) is a well-known mitogen in various cell types. However, we found that LPA inhibits melanocyte proliferation. Thus, we further investigated the possible signaling pathways involved in melanocyte growth inhibition. We first examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by LPA. The activations of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) were observed in concert with the inhibition of melanocyte proliferation by LPA, whereas p38 MAP kinase and Akt were not influenced by LPA. However, the specific inhibition of the ERK or JNK pathways by PD98059 or D-JNKI1, respectively, did not restore the antiproliferative effect. We next examined changes in the expression of cell cycle related proteins. LPA decreased cyclin $D_1 and cyclin D_2$ levels but increased $p21^{WAF1/CIP1}$ (p21) and $p27^{KIP1}$ (p27) levels, which are known inhibitors of cyclin-dependent kinase. Flow cytometric analysis showed the inhibition of DNA synthesis by a reduction in the S phase and an increase in the $G_0/G_1$ phase of the cell cycle. Our results suggest that LPA induces cell cycle arrest by regulating the expressions of cell cycle related proteins.

• 한방안이비인후피부과학회지
• /
• 제27권4호
• /
• pp.158-176
• /
• 2014

Purpose : The purpose of this study is to investigate the effect of ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts on cell death in pancreatic cancer cells. Method : Human-derived pancreatic cancer cell line, MIA PaCa-2 cells were treated by Prescription A with various concentrations and the cytotoxicity was determined by MTT assay. To investigate the effects of Prescription A on pancreatic cancer cells, the staining of Annexin V/PI, cell cycle arrest, nuclear chromatin condensation and the production of reactive oxygen species (ROS) were examined. The effect of Prescription A's effective components, ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts on cell death were also observed. Results : The viability of MIA PaCa-2 cells treated with Prescription A were decreased in a dose dependent manner. Prescription A induced cell death in MIA PaCa-2 cells as shown by result of Annexin V/PI double staining, chromatin condensation and cell cycle arrest. In addition, production of ROS was increased by Prescription A treatment, suggesting that ROS induced by Prescription A mediated cell death. Furthermore, Prescription A's effective components, ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts were also induced apoptosis of MIA PaCa-2 cells through ROS production. Conclusion : These results suggest that Prescription A's effective components, ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts induced death of MIA PaCa-2 through ROS production.

• 생명과학회지
• /
• 제29권6호
• /
• pp.679-687
• /
• 2019

본 연구에서는 인체 폐암 세포인 A549를 사용하여 Litsea populifolia 에탄올 추출물(EELP)의 항산화 및 항암활성과 그 분자적 기전에 관하여 연구하였다. 먼저 EELP의 DPPH 라디칼 소거활성을 측정한 결과, $IC_{50}$가 $11.71{\mu}g/ml$로 유의적인 항산화활성을 보였다. 또한 EELP가 인체폐암세포주인 A549와 정상 폐세포인 IMR90의 세포증식에 미치는 영향을 알아본 결과, 정상세포의 생존율에는 거의 영향을 끼치지 않은 반면, EELP 농도의존적으로 A549 세포의 성장이 저해되었으며, 세포 주기 변화를 분석한 결과 EELP에 의해 A549 세포의 강력한 G1 arrest가 유도되는 것을 확인하였다. EELP에 의해 유도되는 G1 arrest는 세포주기 조절 인자인 Cyclin D1, Cyclin E, Cyclin-dependent kinase인 CDK2와 CDK6의 mRNA 발현 감소와 더불어 단백질 발현 감소와 연관되어 있었다. 또한 EELP 처리에 의한 CDK/Cyclin complex의 발현 저해는 DNA 손상에 의해 활성화되는 CHK2의 활성화 형태인 p-CHK2의 발현 증가에 따른 p53 인산화에 따른 활성화와 CDK 활성화 효소인 CDC25A 탈인산화효소의 인산화에 따른 저해에 의해 나타나는 결과로 사료된다. 이러한 결과들로부터 EELP는 두가지 경로인 p53-의존성과 p53-비의존성(ATM/CHK2/CDC25A/CDK2) 경로를 통해 A549의 G1 arrest를 유도하여 세포 증식을 억제하는 것으로 사료된다. 본 연구결과는 EELP가 폐암에 대한 새로운 항암활성 소재로서 사용될 수 있는 가능성을 시사하며, 또한 EELP의 세포주기 조절에 의한 항암기전을 이해하고 향후 지속적 연구를 하는 데 있어서 귀중한 기초자료로 사용될 수 있을 것이다.

• IMMUNE NETWORK
• /
• 제9권6호
• /
• pp.236-242
• /
• 2009

Background: Melanoma is the most fatal form of skin cancer due to its rapid metastasis. Recently, several studies reported that selenium can induce apoptosis in melanoma cells. However, the precise mechanism remains to be elucidated. In this study, we investigated the effect of selenium on cell proliferation in murine melanoma and on tumor growth and metastasis in C57BL/6 mice. Methods: Cell proliferation was measured by MTT assay in selenium-treated melanoma cells. Cell cycle distribution was analysized by staining DNA with propidum iodide (PI). mRNA and protein expression related to cell cycle arrest was measured by reverse transcription PCR and western blot. Tumor growth and metastasis was measured by in vivo model. Results: Selenium was suppressed the proliferation of melanoma cells in a dose dependent manner. The growth inhibition of melanoma by selenium was associated with an arrest of cell cycle distribution at G0/G1 stage. The mRNA and protein level of CDK2/CDK4 was suppressed by treatment with selenium in a time-dependent manner. In vivo, tumor growth was not suppressed by selenium; however tumor metastasis was suppressed by selenium in mouse model. Conclusion: These results suggest that selenium might be a potent agent to inhibit proliferative activity of melanoma cells.