• Journal of Acupuncture Research
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• v.19 no.2
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• pp.78-91
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• 2002

Objective : This study was designed to investigate the anti-cancer effects of bee venom on melanoma in C57BL mice. Materials and Methods : For the induction of melanoma, C57BL mice were treated by DMBA(7, 12-dimethylbenz[a]anthracene). Each group of C57BL mouse was treated with DMBA $50{\mu}g$, $75{\mu}g$, $100{\mu}g$ respectively once a week for 15 weeks. Tumor generation in each group of 10 mice was observed. Cumulative curves were showed in the density and frequency of skin tumor generation. To know the effects of pre-treatment of bee venom on tumor generation by DMBA treatment(frequency of tumor generation), Each group of C57BL mouse was pretreated and treated with bee venom $5{\mu}{\ell}$, $25{\mu}{\ell}$, $50{\mu}{\ell}$ respectively once a week for 3 weeks, whereafter each mouse was treated with DMBA $100{\mu}g$ once a week for 15 weeks. Results and Conclusion (1) There was chemotherapeutic effect, but not chemopreventive effect. (2) Cpp32 activity was increased by $50{\mu}{\ell}$ bee venom treatment. (3) Bee venom treatment inhibited expression of cell-cycle regulating, growth-promoting genes such as c-Jun, c-Fos, and Cyclin Dl, and increased tumor suppressors p53 and p21/Wafl. (4) Bee venom treatment activated expression of a representative apoptosis-inducing gene Bax.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.5
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• pp.299-304
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• 2005

Cardiac hypertrophy contributes an increased risk to major cerebrovascular events. However, the molecular mechanisms underlying cerebrovascular dysfunction during cardiac hypertrophy have not yet been characterized. In the present study, we examined the molecular mechanism of isoproterenol (ISO)-evoked activation of Ras/Raf/MAPK pathways as well as PKA activity in cerebral artery of rabbits, and we also studied whether the activations of these signaling pathways were altered in cerebral artery, during ISO-induced cardiac hypertrophy compared to heart itself. The results show that the mRNA level of c-fos (not c-jun and c-myc) in heart and these genes in cerebral artery were considerably increased during cardiac hypertrophy. These results that the PKA activity and activations of Ras/Raf/ERK cascade as well as c-fos expression in rabbit heart during cardiac hypertrophy were consistent with previous reports. Interestingly, however, we also showed a novel finding that the decreased PKA activity might have differential effects on Ras and Raf expression in cerebral artery during cardiac hypertrophy. In conclusion, there are differences in molecular mechanisms between heart and cerebral artery during cardiac hypertrophy when stimulated with β2 adrenoreceptor (AR), suggesting a possible mechanism underlying cerebrovascular dysfunction during cardiac hypertrophy.

• International Journal of Oral Biology
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• v.36 no.4
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• pp.187-194
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• 2011

Periodontitis is an inflammatory disorder of the periodontium, characterized by destruction of the tooth supporting tissues including alveolar bone and mediated by various pro-inflammatory mediators. Here, we demonstrated that HP08-0106, composed of four crude drugs-Gardenia jasminoides Grandiflora, Angelica gigas Nakai, Rehmannia glutinosa, and Schizonepeta tenuifolia in a weight ratio of 2:2:1:2, perturbs inflammatory responses, osteoclast formation in LPS-induced RAW 264.7 cells and alveolar bone resorption in ligature-induced periodontitis. HP08-0106 decreased the protein level of iNOS and COX2 as well as the secreted level of IL-$1{\beta}$, indicating that HP08-0106 has antiinflammatory effects. HP08-0106 also inhibited the expression of genes associated with osteoclastogenesis including c-Fos, MMP-9 and TRAP. Moreover, HP08-0106 exhibited a protective effect from alveolar bone loss in ligature-induced periodontitis animal models. Our results strongly suggest that HP08-0106 represent an important therapeutic tool to treat inflammatory disorders associated with bone loss such as periodontitis.

• Journal of Acupuncture Research
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• v.17 no.2
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• pp.169-186
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [3H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, [$^3H$]thymidine release assay, and flow cytometric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and $Bcl-X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment. 5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of $G_1$ phase was increased by a dose-dependant manner. 6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.

• Molecules and Cells
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• v.38 no.3
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• pp.279-284
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• 2015

Obatoclax, a pan-Bcl2 inhibitor, shows antitumor activities in various solid malignancies. Bcl2-deficient mice have shown the importance of Bcl2 in osteoclasts, as the bone mass of the mice was increased by the induced apoptosis of osteoclasts. Despite the importance of Bcl2, the effects of obatoclax on the proliferation and differentiation of osteoclast precursors have not been studied extensively. Here, we describe the anti-proliferative effects of obatoclax on osteoclast precursors and its negative role on fusion of the cells. Stimulation with low doses of obatoclax significantly suppressed the proliferation of osteoclast precursors in a dose-dependent manner while the apoptosis was markedly increased. Its stimulation was sufficient to block the activation of ERK MAP kinase by RANKL. The same was true when PD98059, an ERK inhibitor, was administered to osteoclast precursors. The activation of JNK1/2 and p38 MAP kinase, necessary for osteoclast differentiation, by RANKL was not affected by obatoclax. Interestingly, whereas the number of TRAP-positive mononuclear cells was increased by both obatoclax and PD98059, fused, multinucleated cells larger than $100{\pm}m$ in diameter containing more than 20 nuclei were completely reduced. Consistently, obatoclax failed to regulate the expression of osteoclast marker genes, including c-Fos, TRAP, RANK and CtsK. Instead, the expression of DC-STAMP and Atp6v0d2, genes that regulate osteoclast fusion, by RANKL was significantly abrogated by both obatoclax and PD98059. Taken together, these results suggest that obatoclax down-regulates the proliferation and fusion of osteoclast precursors through the inhibition of the ERK1/2 MAP kinase pathway.

• Molecules and Cells
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• v.40 no.10
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• pp.752-760
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• 2017

A2B adenosine receptor (A2BAR) is known to be the regulator of bone homeostasis, but its regulatory mechanisms in osteoclast formation are less well-defined. Here, we demonstrate the effect of A2BAR stimulation on osteoclast differentiation and activity by RANKL. A2BAR was expressed in bone marrow-derived monocyte/macrophage (BMM) and RANKL increased A2BAR expression during osteoclastogenesis. A2BAR stimulation with its specific agonist BAY 60-6583 was sufficient to inhibit the activation of ERK1/2, p38 MAP kinases and $NF-{\kappa}B$ by RANKL as well as it abrogated cell-cell fusion in the late stage of osteoclast differentiation. Stimulation of A2BAR suppressed the expression of osteoclast marker genes, such as c-Fos, TRAP, Cathepsin-K and NFATc1, induced by RANKL, and transcriptional activity of NFATc1 was also inhibited by stimulation of A2BAR. A2BAR stimulation caused a notable reduction in the expression of Atp6v0d2 and DC-STAMP related to cell-cell fusion of osteoclasts. Especially, a decrease in bone resorption activity through suppression of actin ring formation by A2BAR stimulation was observed. Taken together, these results suggest that A2BAR stimulation inhibits the activation of ERK1/2, p38 and $NF-{\kappa}B$ by RANKL, which suppresses the induction of osteoclast marker genes, thus contributing to the decrease in osteoclast cell-cell fusion and bone resorption activity.

• Molecules and Cells
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• v.43 no.1
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• pp.34-47
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• 2020

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbβ, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Rev-erbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo. Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.6047-6053
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• 2015

Background: Hepatocellular carcinoma is one of the most common cancers worldwide. Its prevalence is increasing in many countries. Plant products can be used to protect against cancer due to natural anticancer and chemopreventive constituents. Strobilanthes crispus is one of plants with potential chemopreventive ability. Objective: This study aimed to evaluate the anticancer effects of Strobilanthes crispus juice on hepatocellular carcinoma cells. Materials and Methods: MTT assays, flow cytometry, comet assays and the reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the effects of juice on DNA damage and cancer cell numbers. Results: This juice induced apoptosis after exposure of the HepG2 cell line for 72 h. High percentages of apoptotic cell death and DNA damage were seen at the juice concentrations above 0.1%. It was found that the juice was not toxic for normal cells. In addition, juice exposure increased the expression level of c-myc gene and reduced the expression level of c-fos and c-erbB2 genes in HepG2 cells. The cytotoxic effects of juice on abnormal cells were in dose dependent. Conclusions: It was concluded that the Strobilanthes crispus juice may have chemopreventive effects on hepatocellular carcinoma cells.

• Journal of Dental Anesthesia and Pain Medicine
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• v.18 no.1
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• pp.9-17
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• 2018

Background: The structure and function of bone tissue is maintained through a constant remodeling process, which is maintained by the balance between osteoblasts and osteoclasts. The failure of bone remodeling can lead to pathological conditions of bone structure and function. Remifentanil is currently used as a narcotic analgesic agent in general anesthesia and sedation. However, the effect of remifentanil on osteoclasts has not been studied. Therefore, we investigated the effect of remifentanil on pre-osteoclast (pre-OCs) differentiation and the mechanism of osteoclast differentiation in the absence of specific stimulus. Methods: Pre-OCs were obtained by culturing bone marrow-derived macrophages (BMMs) in osteoclastogenic medium for 2 days and then treated with various concentration of remifentanil. The mRNA expression of NFATc1 and c-fos was examined by using real-time PCR. We also examined the effect of remifentanil on the osteoclast-specific genes TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. Finally, we examined the influence of remifentanil on the migration of pre-OCs by using the Boyden chamber assay. Results: Remifentanil increased pre-OC differentiation and osteoclast size, but did not affect the mRNA expression of NFATc1 and c-fos or significantly affect the expression of TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. However, remifentanil increased the migration of pre-OCs. Conclusions: This study suggested that remifentanil promotes the differentiation of pre-OCs and induces maturation, such as increasing osteoclast size. In addition, the increase in osteoclast size was mediated by the enhancement of pre-OC migration and cell fusion.

• Molecules and Cells
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• v.40 no.10
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• pp.706-713
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• 2017

Osteoclasts are bone-resorbing cells that are derived from hematopoietic precursor cells and require macrophage-colony stimulating factor and receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) for their survival, proliferation, differentiation, and activation. The binding of RANKL to its receptor RANK triggers osteoclast precursors to differentiate into osteoclasts. This process depends on RANKL-RANK signaling, which is temporally regulated by various adaptor proteins and kinases. Here we summarize the current understanding of the mechanisms that regulate RANK signaling during osteoclastogenesis. In the early stage, RANK signaling is mediated by recruiting adaptor molecules such as tumor necrosis factor receptorassociated factor 6 (TRAF6), which leads to the activation of mitogen-activated protein kinases (MAPKs), and the transcription factors nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and activator protein-1 (AP-1). Activated NF-${\kappa}B$ induces the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is the key osteoclastogenesis regulator. In the intermediate stage of signaling, the co-stimulatory signal induces $Ca^{2+}$ oscillation via activated phospholipase $C{\gamma}2$ ($PLC{\gamma}2$) together with c-Fos/AP-1, wherein $Ca^{2+}$ signaling facilitates the robust production of NFATc1. In the late stage of osteoclastogenesis, NFATc1 translocates into the nucleus where it induces numerous osteoclast-specific target genes that are responsible for cell fusion and function.