Root Bark of Paeonia suffruticosa Extract and Its Component Methyl Gallate Possess Peroxynitrite Scavenging Activity and Anti-inflammatory Properties through NF-κB Inhibition in LPS-treated Mice.

A peroxynitrite (ONOO-)-generating system induced by 3-morpholinosydnonimine, was used to evaluate the ONOO- scavenging properties of plants that have been widely used as traditional medicine in Korea for the treatment of several diseases. The most effective medicinal plants were Paeonia suffruticosa Andrew, followed in order by Lonicera japonica Thunb., Curcuma zedoaria (Christm.) Roscoe, and Pueraria thunbergiana Benth. In addition, root bark of P. suffruticosa was partitioned with organic solvents of different polarities, and the ethyl acetate (EtOAc) fraction showed the strongest ONOO- scavenging activity. Methyl gallate, a plant-derived phenolic compound identified from the EtOAc fraction, exerted strong ONOO- scavenging activity. The in vivo therapeutic potential of methyl gallate was investigated using lipopolysaccharide-treated mice. Oral administration of methyl gallate protected against acute renal injury and exhibited potential anti-inflammatory properties through an increase in antioxidant activity and decrease in nuclear factor-kappa B activity.

Tumor antigen PRAME is up-regulated by MZF1 in cooperation with DNA hypomethylation in melanoma cells.

Elevated expression of preferentially expressed antigen in melanoma (PRAME) has been implicated in disease progression in a variety of cancers. However, the mechanisms underlying the transcriptional regulation of PRAME remain largely unexplored. Initially, we observed that PRAME was elevated in proportion to the malignant potential of melanoma cells. From the in silico prediction of PRAME gene structure, we identified the putative myeloid zinc finger 1 (MZF1) binding sites, which overlap with a CpG-rich region located in the first intron. The transcription factor MZF1 increased PRAME expression via its direct binding to the intron DNA. Upon treatment with a DNA methylation inhibitor, 5-aza-2'-deoxycitidine (5-azaC), together with ectopic expression of MZF1, PRAME expression was significantly enhanced at both the protein and mRNA levels. More pronounced MZF1 binding to the PRAME DNA was observed in the presence of 5-azaC. DNA methylation was inversely correlated with PRAME expression in melanoma cells. Finally, we observed that MZF1, like PRAME, promotes the colony-forming ability in melanoma cells. Overall, our findings suggest that MZF1, via stimulation of PRAME expression, may be a potential prognostic and therapeutic target in melanoma.

Repression of LXRα by a novel member of additional sex comb-like family, ASXL3.

Among the members of the additional sex comb-like (ASXL) family, ASXL3 remains unexplored. Here, we showed that ASXL3 interacts with HP1α and LSD1, leading to transcriptional repression. We determined that ASXL3 depletion augments the ligand-induced transcriptional activities of LXRα and TRß, which were repressed by ASXL3 overexpression. The ligand-dependent interactions of ASXL3 with LXRα and TRß were demonstrated by the GST pull-down and immunoprecipitation analyses. We confirmed that ASXL3 suppresses the expression of LXRα target genes through its recruitment to the LXR-response elements. Finally, we observed that lipid accumulation in Hep3B cells is downregulated upon ASXL3 overexpression but upregulated upon ASXL3 depletion. Overall, our data suggest that ASXL3 is another corepressor of LXRα, promoting to the regulation of lipid homeostasis.

Quercetin potentiates apoptosis by inhibiting nuclear factor-kappaB signaling in H460 lung cancer cells.

The herbal flavonoid quercetin inhibits the growth of various cancer cells, but how it affects human cancer cells, particularly lung cancer cells, is unclear. We investigated the anticancer activity of quercetin and the underlying molecular mechanisms in non-small cell lung cancer (NSCLC) cells. Quercetin strongly inhibited cell proliferation, and increased sub-G1 and apoptotic cell populations regardless of p53 status. Quercetin-induced apoptosis was verified by caspase cleavage, Hoechst staining, trypan blue exclusion, and DNA fragmentation assays. Microarray analysis using H460 cells indicated that quercetin increased the expression of genes associated with death receptor signaling tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAILR), caspase-10, interleukin (IL) 1R DNA fragmentation faotor 45 (DFF45), tumor necrosis factor receptor (TNFR) 1, FAS, inhibitor of kappaBalpha (IκBα)) and cell cycle inhibition growth arrest and DNA-damage inducible 45 (GADD45), p21(Cip1)), but decreased the expression of genes involved in nuclear factor (NF)-kappaB activation (NF-κB, IKKα). Further validation assays confirmed that quercetin inhibited growth by suppressing NF-κB and by increasing the expression of death receptors and cell cycle inhibitors. Taken together, these findings suggest that quercetin may be useful in the prevention and therapy of NSCLC.

Negative regulation of adipogenesis by kaempferol, a component of Rhizoma Polygonati falcatum in 3T3-L1 cells.

Rhizoma Polygonati falcatum (RPF) has been used as a traditional herbal medicine in Asia, because of its anti-hyperglycemic, anti-triglycemic, and anti-tumor activity. In this study, we determined the anti-adipogenic potential of RPF extract and its component kaempferol in 3T3-L1 adipocytes, and the underlying molecular mechanism(s) using microarray analysis. Adipocyte differentiation of 3T3-L1 cells was significantly impaired by RPF extract and kaempferol as monitored by Oil Red O staining and quantitative measurement of lipid accumulation. Additionally, the mRNA expression of adipogenesis genes decreased on treatment with kaempferol. The role of kaempferol at the genome-wide level was further assessed by a microarray approach. Our analysis indicated that kaempferol decreased the expression of adipogenic transcription factors (Pparγ, Cebpß, Srebp1, Rxrß, Lxrß, Rorα) and genes involved in triglyceride biosynthesis (Gpd1, Agpat2, Dgat2), while increasing lipolysis-related genes, such as Tnfα, Lsr, and Cel. Finally, co-transfection assays using luciferase reporter gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis using peroxisome proliferator-activated receptor-γ (PPARγ) target genes indicated that kaempferol significantly repressed rosiglitazone-induced PPARγ transcriptional activity. Overall, our data suggests that kaempferol, a major component of RPF, may be beneficial in obesity, by reducing adipogenesis and balancing lipid homeostasis partly through the down-regulation of PPARγ.

Piperine, a component of black pepper, inhibits adipogenesis by antagonizing PPARγ activity in 3T3-L1 cells.

This study investigated the antiadipogenic activity of black pepper extract and its constituent piperine in 3T3-L1 preadipocytes as well as the underlying molecular mechanisms. Both black pepper extract and piperine, without affecting cytotoxicity, strongly inhibited the adipocyte differentiation of 3T3-L1 cells. The mRNA expression of the master adipogenic transcription factors, PPARγ, SREBP-1c, and C/EBPß, was markedly decreased. Intriguingly, mRNA levels of PPARγ target genes were also down-regulated. Moreover, a luciferase reporter assay indicated that pipierine significantly represses the rosiglitazone-induced PPARγ transcriptional activity. Finally, GST-pull down assays demonstrated that piperine disrupts the rosiglitazone-dependent interaction between PPARγ and coactivator CBP. Genome-wide analysis using microarray further supports the role of piperine in regulating genes associated with lipid metabolism. Overall, these results suggest that piperine, a major component of black pepper, attenuates fat cell differentiation by down-regulating PPARγ activity as well as suppressing PPARγ expression, thus leading to potential treatment for obesity-related diseases.

Lycii cortex radicis extract inhibits glioma tumor growth in vitro and in vivo through downregulation of the Akt/ERK pathway.

Lycii cortex radicis (LCR) is a traditional Korean medicinal herb. The present study was undertaken to examine the effect of an LCR extract on glioma cell growth and to determine its molecular mechanism in U87MG human glioma cells. The LCR extract resulted in apoptotic cell death in a dose- and time-dependent manner. The LCR extract-induced cell death was associated with generation of reactive oxygen species (ROS). Western blot analysis showed that the LCR extract caused downregulation of Akt and ERK. The LCR-induced cell death was prevented by transfection with the constitutively active forms of Akt and MEK. Oral administration of LCR extracts in subcutaneous U87MG xenograft models reduced glioma tumor volume. Taken together, these findings suggest that the LCR extract results in human glioma cell death through mechanisms involving ROS generation, downregulation of Akt and ERK, and caspase activation in vitro and reduces glioma tumor growth in vivo. These data suggest that the LCR extract may serve as a potential therapeutic agent for malignant human glioblastomas.

Fructus ligustri lucidi extracts induce human glioma cell death through regulation of Akt/mTOR pathway in vitro and reduce glioma tumor growth in U87MG xenograft mouse model.

The present study was undertaken to examine the effect of Fructus ligustri lucidi (FLL) extracts on glioma cell growth and to determine the underlying mechanism by which FLL extracts exert anticancer properties in human U87MG glioma cells. The FLL extracts resulted in cell death in a dose- and time-dependent manner. Western blot analysis showed that treatment with FLL extracts caused down-regulation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Overexpression of Akt prevented the cell death induced by the FLL extracts. The FLL extracts caused a decrease in the expression of mammalian target of rapamycin (mTOR) and the FLL extract-induced cell death was increased by the mTOR inhibitor rapamycin. The FLL extracts decreased the expression of survivin. Oral administration of FLL extracts in subcutaneous U87MG xenograft models reduced the glioma tumor volume. These findings indicate that the FLL extracts resulted in glioma cell death through regulation of the Akt/mTOR/survivin pathway in vitro and inhibited glioma tumor growth in vivo. These data suggest that the FLL extracts may serve as a potential therapeutic agent for malignant human gliomas.

Mulberry fruit (Moris fructus) extracts induce human glioma cell death in vitro through ROS-dependent mitochondrial pathway and inhibits glioma tumor growth in vivo.

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruit extracts on cell viability in vitro in human glioma cells and the anticancer efficacy in vivo. This study was undertaken to examine the effect of mulberry fruit (Moris fructus; MF) extracts on cell viability in vitro and anticancer efficacy in vivo. Cell viability and cell death were estimated by MTT assay and trypan blue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with DiOC(6)(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Cell migration was estimated using the scratched wound model. In vivo anticancer efficacy of MF extracts was evaluated using a subcutaneously injected mouse tumor model. Changes in proliferation and apoptosis were estimated by immunohistochemistric analysis. MF extracts resulted in apoptotic cell death in a dose- and time-dependent manner. MF extracts increased ROS generation, and the MF extract-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in the MF extract-induced cell death. Western blot analysis showed that treatment of MF extracts caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). MF extracts induced depolarization of mitochondrial membrane potential, and its effect was inhibited by the antioxidants NAC and catalase. MF extracts induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by MF extracts, and caspase inhibitors prevented the MF extract-induced cell death. Treatment of MF extracts inhibited cell migration. Oral MF extracts administration in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed a decrease in PCNA-positive cells, an increase in TUNEL-positive cells, and caspase activation. From these data, we concluded that MF extracts reduce glioma tumor growth through inhibition of cell proliferation resulting from induction of apoptosis. These findings suggest that MF extracts result in human glioma cell death in vitro through ROS-dependent mitochondrial pathway and glioma tumor growth in vivo via reduction of tumor cell proliferation and induction of apoptosis.

Kaempferol induces cell death through ERK and Akt-dependent down-regulation of XIAP and survivin in human glioma cells.

The present study was undertaken to determine the molecular mechanism by which kaempferol induces cell death in human glioma cells. Kaempferol resulted in loss of cell viability and inhibition of proliferation in a dose- and time-dependent manner, which were largely attributed to cell death. Kaempferol caused an increase in reactive oxygen species (ROS) generation and the kaempferol-induced cell death was prevented by antioxidants, suggesting that ROS generation is involved in kaempferol-induced cell death. Kaempferol caused depolarization of mitochondrial membrane potential. Western blot analysis showed that kaempferol treatment caused a rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. The ERK inhibitor U0126 and the Akt inhibitor LY984002 increased the kaempferol-induced cell death and overexpression of MEK, the upstream kinase of ERK, and Akt prevented the cell death. The expression of anti-apoptotic proteins XIAP and survivin was down-regulated by kaempferol and its effect was prevented by overexpression of MEK and Akt. Kaempferol induced activation of caspase-3 and kaempferol-induced cell death was prevented by caspase inhibitors. Taken together, these findings suggest that kaempferol results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of XIAP and survivin regulating by ERK and Akt.

Zingerone as an antioxidant against peroxynitrite.

Peroxynitrite (ONOO(-)), formed from the reaction of superoxide ((*)O(2)(-)) and nitric oxide ((*)NO), induces cellular and tissue injury, resulting in several human diseases such as stroke, Alzheimer's disease, and atherosclerosis. Due to the lack of endogenous enzymes responsible for ONOO(-) scavenging activity, finding a specific ONOO(-) scavenger is of considerable importance. In this study we examined the scavenging effects of zingerone from ginger against ONOO(-), intracellular RS (reactive species), and ONOO(-). The data show that zingerone can efficiently scavenge native ONOO(-) as well as ONOO(-) derived from the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1). Zingerone inhibited the formation of ONOO(-)-mediated tyrosine nitration through electron donation, nitration of bovine serum albumin (BSA) by ONOO(-), and intracellular RS and ONOO(-). The present study suggests that zingerone has an efficient ONOO(-) scavenging ability, which may be a potent ONOO(-) scavenger for the protection of the cellular defense activity against ONOO(-)- involved diseases.

Effects of Bambusae concretio Salicea (Chunchukhwang) on amyloid beta-induced cell toxicity and antioxidative enzymes in cultured rat neuronal astrocytes.

Bambusae concretio Salicea (BCS; plant family name: Phyllostachys bambusoides Siebold et Zuccarinii) is a medicinal plant used in Korea for the treatment of various symptoms accompanying hypertension and cerebrovascular disorders. Previously, it was shown that BCS is an effective protectant against oxidative glutamate toxicity in the murine neuroblastoma cells and human neuroblastoma cells. Treatment with BCS increased the secretion of the non-amyloidogenic amyloid precursor protein fragment, and decreased the secretion of amyloid-beta (Abeta) peptides from neuronal cells [Jeong, J.C., Seo, Y.J., Kim, H.M., Lee, Y.C., Kim, C.H., 2003. Inhibitory effects of Bombusae concretio Salicea on neuronal secretion of Alzheimer's beta-amyloid peptides, a neuro-degenerative peptide. Neurochemical Research 28, 1785-1792.]. To further examine the pharmacological activity of BCS, we studied the protective effect of the water extracts on Abeta25-35 peptide-induced neuronal death by microscopic observation and lactate dehydrogenase (LDH) assay, and action on antioxidative enzymes using cultured astrocyte cells. Ten microM Abeta25-35-induced cell death was protected by the application of water extract of BCS in a dose-dependent manner, and concentrations of 1-10 microg/ml had a significant effect compared to exposure to Abeta25-35 only. When antioxidative enzyme activities such as catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were assayed after Abeta25-35 treatment, the enzymes were decreased in a similar fashion. However, those activities were enhanced by BCS treatment and this may have resulted from the potentiation of antioxidative ability by BCS. The ability of BCS to reduce cellular cytotoxicity induced by 10 microM Abeta25-35 suggests that BCS may be a protective agent for free radical generating compounds such as Abeta25-35, and that Abeta25-35 is not only a potent lipid peroxide inducer, but also causes changes in antioxidative enzymes. From the results, it was concluded that BCS has a protective effect on Abeta-induced neuronal death in cultured astrocyte cells through the inhibition of lipid peroxidation and protection of antioxidative enzymes.

Effects of Drynariae rhizoma on the proliferation of human bone cells and the immunomodulatory activity.

Drynariae Rhizoma (DR), a traditional Korea medicine, which is known for its effect to strengthen myoskeletal systems, frequently appears as the main ingredient in prescriptions for bone injuries. However, it is unclear how it pharmacologically contributes to the reformation of bone. In this study, the effect of DR on bone cells was investigated in vitro for the first time. The human osteoprecursor cells (OPC-1) were incubated in the medium with different concentrations of DR and the cell proliferation was studied. When the concentration of DR was < or = 120 microg ml(-1), the proliferation of OPC-1 was enhanced. However, the proliferation of OPC-1 was inhibited by DR with the concentrations of > 250 microg ml(-1). Under most treatments, the cells presented very pale expression for cyclooxygenase-2 (Cox-2) protein; slightly intensified band showed at the highest DR concentration, 120 microg ml(-1) during the course of culture. On the other hand, we investigated the immunomodulatory activity of DR on cellular and humoral immunity. When different doses of ethanolic and water extracts of DR was administered to mice, it was dose-dependently potentiated the delayed-type hypersensitivity reaction induced by both sheep red blood cells (SRBC) and oxazolone. It significantly enhanced the production of circulating antibody titre in mice in response to SRBC. But, DR did not any effect on macrophage phagocytosis. Prolonged administration of DR significantly ameliorated the total white blood cell count and also restored the immunosuppressive effects induced by cyclophosphamide. The present investigation reveals that DR possesses immunomodulatory activity. From the results, it was concluded that DR directly stimulated the proliferation, alkaline phosphatase activity, protein secretion and particularly type I collagen synthesis of OPC-1 at dose-dependent manner, and stimulated both the cellular and the humoral immunity.

Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

Pharmacological factors are needed to prevent bone loss that occurs with increasing age. The chemical compounds that act on bone metabolism in herbal medicines, however, are poorly understood. Effects of traditional Korean medicine, Drynariae Rhizoma [Drynaria fortunei (kunze) J. Sm] extract (DR), on the osteoblastic proliferation and differentiation were investigated. The effect of DR, a natural phyto herb, on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. DR dose-dependently increased DNA synthesis (significant at 50-150 microg/ml). DR increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (50-150 microg/ml). Antiestrogen tamoxifen eleminated the stimulation of proliferation and ALP activity of MC3T3-E1, which were induced by DR. DR at concentrations ranged from 30-100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that DR directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and DR is effective for bone anti-resorptive action in bone cells.

Inhibitory activity of Drynariae rhizoma extracts on cathepsin having bone resorption activity.

Effects of traditional Korean (Hanbang) medicine, Drynariae rhizoma (DR), on the protease activity of bone loss-initiation in rats and mice were investigated. Ethanol extracts-DR (EE-DR) and water extracts-DR (WE-DR) were identified as potent inhibitor of cathepsins K and L. The original WE-DR inhibits cathepsins K and L with IC50 values of 3.7 microg/ml and 4.5 microg/ml, respectively. EE-DR was more potent than that of WE-DR, because the inhibitions of cathepsin K and L increased to 0.5 microg/ml and 0.8 microg/ml, respectively. The EE-DR was proved to be the most potent. EE-DR was found to be a potent inhibitor of cathepsins K with a Ki value of 5.0 microg/ml for cathepsin K. The activity was increased by 10-fold when the assay is performed in the presence of glutathione at pH 7.0, which favors the formation of a GSH thiolate anion. Thus, it is suggested that this increase in potency is probably due to an enhanced chemical reactivity of the extract mixtures toward the thiolate of the active site of the enzyme. WE-DR exhibited time-dependet inhibition which allowed us to determine the association and dissociation rate constants with cathepsin K. Finally, EE-DR inhibits bone resorption in an in vitro assay involving mouse osteoclasts and bovine bone with an IC50 value of 70 microg/ml. WE-DR represents a new herbal formulation inhibiting cathepsin K and L activity and proteolysis of bone collagen. These results strongly suggest that DR is effective for preventing the development of bone loss induced by cathepsin K. This result also suggested that the DR is effective for bone resorptive action in bone cells.

Drynariae Rhizoma promotes osteoblast differentiation and mineralization in MC3T3-E1 cells through regulation of bone morphogenetic protein-2, alkaline phosphatase, type I collagen and collagenase-1.

In a previous study (Jeong et al., 2003, Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. International Immunopharmacology 3, 1685-1697), treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extract prevented the intracellular maturation of cathepsin K and thus, it was considered that DR is a pro-drug of a potent bone resorption inhibitor. To further clarify the role of DR in ossification, we investigated the effects of DR on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, the bone effect of DR is studied. We assessed the effects of DR on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. DR enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the DR was observed at relatively low doses (significant at 50-150 microg/ml and maximal at 150 microg/ml). Northern blot analysis showed that the DR (100 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. DR (60 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that DR has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.

Inhibitory effects of Bombusae concretio Salicea on neuronal secretion of Alzheimer's beta-amyloid peptides, a neurodegenerative peptide.

Alzheimer's disease (AD) is characterized by the age-related deposition of beta-amyloid (A beta) 40/42 peptide aggregates in vulnerable brain regions. Multiple levels of evidence implicate a central role for A beta in the pathophysiology of AD. A beta is generated by the regulated cleavage of a = 700 amino acid A beta precursor protein (betaAPP). Full-length betaAPP can undergo proteolytic cleavage either within the A beta domain to generate secreted sbetaAPP alpha or at the N-terminal and C-terminal domain(s) of A beta to generate amyloidogenic A beta peptides. Several epidemiological studies have reported that estrogen replacement therapy protects against the development of AD in postmenopausal women. The aim of this study was to elucidate the antioxidant neuroprotective mechanism of Bombusae concretio Salicea (BC). BC was effective protectants against oxidative glutamate toxicity in the murine neuroblastoma cells (N2a) and human neuroblastoma cells (SK-N-MC). BC exhibited similar protective properties against oxidative glutamate toxicity and H2O2 toxicity. BC exhibited an antioxidant activity at approximately 20 microg/ml. BC of 5 microg/ml was ineffective in preventing the oxidative modification of LDL. The half-maximal effective concentration for BC was 16 microg/ml. These results suggested that BC supplementation in elderly men may be protective in the treatment of Alzheimer's disease (AD). We report here that treatment with BC increases the secretion of the nonamyloidogenic APP fragment, sbetaAPP alpha and decreases the secretion of A beta peptides from N2a cells and rat primary cerebrocortical neurons. These results raise the possibility that BC supplementation in elderly men may be protective in the treatment of AD.

Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts.

In the traditional Korean medicine, Drynariae Rhizoma (DR) [Drynaria fortunei (kunze) J. Sm] has been reported as a good enhancer for bone healing. In this experiment, we investigate the effects of DR on bone resorption using the bone cells culture. Different concentrations of crude extract of DR were added to mouse bone cells culture. The mitochondria activity of the bone cells after exposure was determined by colorimetric MTT assay. It was demonstrated that DR has potential effects on the bone cells culture without any cytotoxicity. The most effective concentration of DR on bone cells was 100 micro g/ml. On the other hand, cathepsin K (Cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. In this study, Mouse long bone cells including osteoclasts and osteoblast were treated with the PI3-kinase inhibitor, wortmannin (WT), and a specific inhibitor of protein kinase C (PKC), calphostin C. Although WT prevented the osteoclast-mediated intracellular processing of Cat K, calphostin C did not. Similarly, treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extracts prevented the intracellular maturation of Cat K, suggesting that DR may disrupt the intracellular trafficking of pro Cat K. This is similar to that of WT. Since secreted proenzymes have the potential to reenter the cell via mannose-6-phosphate (M6P) receptor, to prevent this possibility, we tested WT and DR in the absence or presence of M6P. Inhibition of Cat K processing by WT or DR was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of WT and DR. DR dose-dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of Cat K processing.