Targeting Histone Methyltransferase DOT1L by a Novel Psammaplin A Analog Inhibits Growth and Metastasis of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most intractable cancer in women with a high risk of metastasis. While hyper-methylation of histone H3 catalyzed by disruptor of telomeric silencing 1-like (DOT1L), a specific methyltransferase for histone H3 at lysine residue 79 (H3K79), is reported as a potential target for TNBCs, early developed nucleoside-type DOT1L inhibitors are not sufficient for effective inhibition of growth and metastasis of TNBC cells. We found that TNBC cells had a high expression level of DOT1L and a low expression level of E-cadherin compared to normal breast epithelial cells and non-TNBC cells. Here, a novel psammaplin A analog (PsA-3091) exhibited a potent inhibitory effect of DOT1L-mediated H3K79 methylation. Consistently, PsA-3091 also significantly inhibited the proliferation, migration, and invasion of TNBC cells along with the augmented expression of E-cadherin and the suppression of N-cadherin, ZEB1, and vimentin expression. In an orthotopic mouse model, PsA-3091 effectively inhibited lung metastasis and tumor growth by the regulation of DOT1L activity and EMT biomarkers. Together, we report here a new template of DOT1L inhibitor and suggest that targeting DOT1L-mediated H3K79 methylation by a novel PsA analog may be a promising strategy for the treatment of metastatic breast cancer patients.

Effects of SHINBARO2 on Rat Models of Lumbar Spinal Stenosis.

Lumbar spinal stenosis (LSS) is a major cause of chronic low back pain; however, only a few therapies which have been used in clinics still have limited effects on functional recovery. SHINBARO2 is a refined traditional formulation for inflamed lesions and relieve pain of muscular skeletal disease. This study aimed at investigating the effects of SHINBARO2 on LSS and at determining its underlying molecular mechanism in rat models. The LSS rat models were set up by surgical operations in 6-week-old male Sprague-Dawley rats. SHINBARO2 was orally or intraperitoneally administered for 14 days. The motor and sensory ability of rats were evaluated using the activity cage and hot plate method. On the termination day, total vertebrae including the disc and spinal cord were excised for ex vivo study. SHINBARO2 improved locomotor functions and pain sensitivity in LSS rat models. Mechanism study suggested that SHINBARO2 inhibited the production of nitric oxide and prostaglandin E2 in tissues from LSS-induced rats. SHINBARO2 also suppressed the expression of proinflammatory cytokines including tumor necrosis factor-α and interleukin-1ß. The activation of NF-κB by LSS surgery was effectively reduced by SHINBARO2, which coincided with the inhibition of IκB degradation. In addition, brain-derived neurotrophic factor (BDNF), a potent promoter of neurite growth, and its downstream ERK signaling were also regulated by SHINBARO2. These findings suggest that the effect of SHINBARO2 might be associated in part with the anti-inflammation and pain control in LSS rat models.

Linarin inhibits radiation-induced cancer invasion by downregulating MMP-9 expression via the suppression of NF-κB activation in human non-small-cell lung cancer A549.

Radiotherapy is routinely used in the treatment of lung cancer patients. However, it often causes malignant effects, such as promoting cancer cell migration and invasion. Previous studies demonstrated that ionizing radiation (IR) promotes cancer cell invasion by stimulating the ß-catenin, IL-6, STAT3, and Bcl-XL signaling pathway or the PI3K, Akt, and NF-κB signaling pathway. Both Bcl-XL and NF-κB stimulate the secretion of matrix metalloproteases (MMPs), including MMP-2 and MMP-9. In the present study, linarin isolated from Chrysanthemum morifolium flowers significantly decreased the IR-induced cell migration and invasion at a concentration of 5 µM in A549 cells. This effect was mediated via MMP-9 downregulation and the suppression of NF-κB activation by inhibiting NF-κB and IκB-α phosphorylation. However, linarin did not affect the STAT3/Bcl-XL pathway or the stabilization of ß-catenin. Overall, these results suggest that linarin repressed the MMP-9-dependent invasion pathway by regulating NF-κB activity, thereby inhibiting IR-induced cancer metastasis.

A novel selenonucleoside suppresses tumor growth by targeting Skp2 degradation in paclitaxel-resistant prostate cancer.

Prostate cancer (PC) is the most common disease in men over age 50, and its prevalence rate has been gradually increasing since 1980. Taxane-derived anticancer agents are the primary agents used to treat metastatic prostate cancer patients; however, the side effects and acquired drug resistance limit the success of these therapies. Because there is no specific treatment for paclitaxel-resistant prostate cancer, it is necessary to develop new targets and therapeutic strategies to overcome the acquired resistance. In this study, the antitumor activity of a novel selenonucleoside (4'-selenofuranosyl-2,6-dichloropurine, LJ-2618), a third-generation nucleoside, and its plausible mechanisms of action in paclitaxel-resistant prostate cancer (PC-3-Pa) cells were investigated. The established PC-3-Pa cells exhibited over 100-fold resistance against paclitaxel compared to the paclitaxel-sensitive PC-3 cells. LJ-2618, however, effectively inhibited the proliferation of both cell lines with similar IC50 values in vitro. In PC-3-Pa cells, the activated PI3K/Akt signaling pathway was suppressed by LJ-2618 treatment. In addition, Skp2 was found to be over-expressed in paclitaxel-resistant cells, and the transfection of Skp2 siRNA recovered the sensitivity of paclitaxel in PC-3-Pa cells. Furthermore, LJ-2618 significantly down-regulated Skp2 expression in PC-3-Pa cells by promoting degradation and inducing destabilization of Skp2, which triggers G2/M cell cycle arrest. In a xenograft mouse model implanted with PC-3-Pa cells, LJ-2618 (3 or 10 mg/kg) effectively inhibited tumor growth with the enhancement of Skp2 degradation and induction of p27 expression in tumor tissues. These findings suggest that LJ-2618 may have potential for overcoming paclitaxel resistance via promoting Skp2 degradation and stabilizing p27 expression in PC-3-Pa cells. Therefore, the novel selenonucleoside LJ-2618 may lead to the development of a new treatment strategy for patients with paclitaxel-resistant, castration-resistant prostate cancer.

The Anti-Inflammatory Effects of Shinbaro3 Is Mediated by Downregulation of the TLR4 Signalling Pathway in LPS-Stimulated RAW 264.7 Macrophages.

Shinbaro3, a formulation derived from the hydrolysed roots of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, has been clinically used in the pharamacopuncture treatment of arthritis in Korea. In the present study, Shinbaro3 inhibited NO generation in LPS-induced RAW 264.7 cells in a dose-dependent manner. Shinbaro3 also downregulated the mRNA and protein expression of inflammatory mediators in a dose-dependent manner. Three mechanisms explaining the effects of Shinbaro3 in RAW 264.7 cells were identified as follows: (1) inhibition of the extracellular signal-regulated kinase 1 and 2 (ERK1/2), stress-activated protein kinase (SAPK)/c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways; (2) suppression of IκB kinase-α/ß (IKK-α/ß) phosphorylation and nuclear factor-kappa B (NF-κB) subunits in the NF-κB pathway, which are involved in MyD88-dependent signalling; and (3) downregulation of IFN-ß mRNA expression via inhibition of interferon regulatory factor 3 (IRF3) and Janus-activated kinase 1 (JAK1)/signal transducer and activator of transcription 1 (STAT1) phosphorylation, which is involved in TRIF-dependent signalling. Shinbaro3 exerted anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophage cells through modulation of the TLR4/MyD88 pathways, suggesting that Shinbaro3 is a novel anti-inflammatory therapeutic candidate in the field of pharmacopuncture.

Esculetin suppresses tumor growth and metastasis by targeting Axin2/E-cadherin axis in colorectal cancer.

Colorectal cancer (CRC) is the most common malignant disease worldwide due to its metastasis via the epithelial-mesenchymal transition (EMT) process. E-cadherin and Wnt signaling are emerging as potential targets for suppressing the EMT. In this context, Axin2 has been recognized as a negative regulator that inhibits glycogen synthase kinase 3ß (GSK3ß)-mediated degradation of Snail1, a transcriptional repressor of E-cadherin. However, Axin2 can also impede Wnt signaling via ß-catenin degradation. Therefore, Axin2 may serve as either a promoter or suppressor of tumors, and the effects of its inhibition on the cell proliferation and metastasis of CRC require further elucidation. Here, esculetin (ES), a coumarin, was found to have the most potential effects on both ß-catenin-responsive transcriptional and E-cadherin promoter activities. ES also showed anti-proliferative and anti-invasive activities in CRC cells. Mechanistically, Axin2 suppression by ES contributed to E-cadherin-mediated Wnt signaling inhibition. Moreover, the ability of ES to inhibit tumor growth and metastasis via Axin2 suppression was further supported in an HCT116-implanted orthotopic mouse model. Collectively, these findings suggest that targeting the Axin2/E-cadherin axis by ES may be an attractive therapeutic strategy for the treatment of metastatic CRC.

Anti-inflammatory effect of Cortex Eucommiae via modulation of the toll-like receptor 4 pathway in lipopolysaccharide-stimulated RAW 264.7 macrophages.

ETHNOPHARMOCOLOGICAL RELEVANCE: Cortex Eucommiae (CE), the bark of Eucommia ulmoides Oliv., has been traditionally used for its kidney-tonifying and bone- and tendon-enhancing properties in Korea, China, and Japan. CE has been historically prescribed for inflammatory conditions such as arthritis of the knee and ankle. AIM OF THE STUDY: Although CE has recently been shown to suppress inflammation in scientific studies, whether this effect involves modulation of the toll-like receptor 4 (TLR-4) pathway is currently unknown. MATERIALS AND METHODS: The modulatory effect of CE on the TLR-4 pathway, both myeloid differentiation primary response gene 88 (Myd88)-dependent and independent, was investigated through real-time reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, and a reporter gene assay in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. RESULTS: CE dose-dependently inhibited nitric oxide production without significant cytotoxicity with an IC50 of 356.23µg/mL. In addition, CE down-regulated both LPS-induced mRNA and protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in a dose-dependent manner. CE suppressed LPS-induced activation of nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathways, which together comprise the Myd88-dependent TLR-4 pathway. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was also down-regulated by CE in a dose-dependent manner. CE additionally suppressed LPS-induced activation of interferon-ß (IFN-ß) and signal transducer and activator of transcription (STAT) pathway, which is associated with the Myd88-independent TLR-4 pathway. CONCLUSIONS: CE down-regulated both Myd88-dependent and independent TLR-4 pathways, thus exerting anti-inflammatory effects. These results suggest that CE may be used as a potential therapeutic agent against chronic inflammatory diseases.

Influence of Boiling Duration of GCSB-5 on Index Compound Content and Antioxidative and Anti-inflammatory Activity.

BACKGROUND: GCSB-5, an herbal drug composition with an anti-inflammatory effect, is prepared by boiling, which is the most common herbal extraction method in traditional Korean medicine. Several parameters are involved in the process, i.e., extractant type, herb-to-extractant ratio, extraction temperature and pressure, and total boiling time. OBJECTIVES: The aim of this study was to examine the influence of boiling time on index compound amount and the antioxidative and anti-inflammatory activities of GCSB-5. MATERIALS AND METHODS: Different samples of GCSB-5 were obtained by decocting for 30, 60, 90, 120, 150, and 240 min. Each sample was tested for hydrogen ion concentration (pH), total soluble solid content (TSSC), marker compound profiles, and antioxidative and anti-inflammatory activity. RESULTS: pH was found to decrease while TSSC increased with extended decoction. Marker compound contents for GCSB-5 (acanthoside D for Acanthopanax sessiliflorus Seem, 20-hydroxyecdysone for Achyranthes japonica Nakai, and pinoresinol diglucoside for Eucommia ulmoides Oliver) remained relatively constant regardless of the length of boiling. Total D-glucose amount increased with longer boiling. The antioxidative and anti-inflammatory potentials of GCSB-5 were not substantially affected by decoction duration. CONCLUSION: Biological characteristics and marker compound content of GCSB-5 were not altered significantly in prolonged boiling. SUMMARY: Longer boiling duration of GCSB-5 did not increase yield in a time-dependent manner, but yields of 210 and 240 min samples were significantly higherHydrogen ion concentration of GCSB-5 samples decreased while total soluble solid content and D-glucose concentration levels increased with boiling durationAlthough concentrations of some index compounds increased with extended boiling duration of GCSB-5, increase was small and not in a direct proportional relationshipAntioxidative and anti-inflammatory properties of GCSB-5 were not substantially affected by decoction duration. Abbreviations used: CAM: Complementary and alternative medicine; KIOM: Korea Institute of Oriental Medicine; KMD: Korean medicine doctor; TSSC: Total soluble solid content; pH: Hydrogen ion concentration; HPLC: High-performance liquid chromatography; NO: Nitric oxide; NO2: Nitric dioxide; LPS: Lipopolysaccharide; DMSO: Dimethyl sulfoxide.

Anti-Osteoporotic Activity of Harpagoside by Upregulation of the BMP2 and Wnt Signaling Pathways in Osteoblasts and Suppression of Differentiation in Osteoclasts.

Harpagoside (1) is an iridoid glycoside isolated from the radix of Harpagophytum procumbens var. sublobatum, commonly called Devil's claw. The anti-osteoporotic effect of 1 was investigated in both in vitro cell cultures and in vivo using an ovariectomized (OVX) mouse model. Compound 1 induced bone formation by stimulating osteoblast proliferation, alkaline phosphatase activity, and mineralization in osteoblastic MC3T3-E1 cells. Treatment with 1 increased the mRNA and protein expression of bone formation biomarkers through regulation of the BMP2 and Wnt signaling pathway in MC3T3-E1 cells. Compound 1 also suppressed the RANKL-induced osteoclastogenesis of cultured mouse bone marrow cells. Oral administration of 1 restored the OVX-induced destruction of trabecular bone. The bone mineral density of the femur was also increased significantly by 1. The elevated serum levels of osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase in the OVX mice were decreased by treatment with 1. These findings suggest that compound 1 may protect against bone loss induced by OVX in mice by regulating stimulation of osteoblast differentiation and inhibition of osteoclast resorption. Therefore, harpagoside (1) is a potential candidate for management of postmenopausal osteoporosis.

In Vitro and In Vivo Anti-Allergic and Anti-Inflammatory Effects of eBV, a Newly Developed Derivative of Bee Venom, through Modulation of IRF3 Signaling Pathway in a Carrageenan-Induced Edema Model.

BACKGROUND: Bee venom (BV), a type of toxin extracted from honeybees (Apis mellifera), has been empirically and widely used to treat inflammatory diseases throughout Asia. Essential BV (eBV) was developed by removing phospholipase A2 (PLA2) and histamine to lower occurrence of allergic reaction. This study investigated the anti-allergic and anti-inflammatory activities of eBV in vitro and in vivo and its underlying mechanism of action. METHODS: The anti-inflammatory potential of eBV was assessed in vivo using a carrageenan-induced paw edema model. To further investigate the mechanism by which eBV exerts anti-allergic and anti-inflammatory effects, compound 48/80-stimulated RBL-2H3 cells and lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells were studied in vitro. RESULTS: Release of ß-hexosaminidase and histamine was increased by eBV in a dose-dependent manner, but these levels were lower in eBV compared to original BV at the same concentration. In addition, eBV suppressed compound 48/80-induced expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RBL-2H3 cells. eBV was also shown to suppress nitric oxide (NO) production by down-regulating mRNA expression and subsequent protein expression of inflammatory mediators in LPS-induced RAW 264.7 cells. Phosphorylation of activators and signal transducers of transcription 1/interferon regulatory factor 3 (STAT1/IRF3) was attenuated by eBV treatment. eBV significantly inhibited carrageenan-induced acute edema in vivo. Serum levels of prostaglandin E2 (PGE2), TNF-α, and IL-1ß were also down-regulated by eBV. CONCLUSIONS: These results demonstrate that eBV inhibits allergic and inflammatory response by reducing inflammatory mediator production via regulation of the STAT1/IRF3 signaling pathway, suggesting that eBV is a feasible candidate for regulation of allergic-inflammatory response in complementary and alternative medicine.

Safety of essential bee venom pharmacopuncture as assessed in a randomized controlled double-blind trial.

ETHNOPHARMACOLOGICAL RELEVANCE: While bee venom (BV) pharmacopuncture use is common in Asia, frequent occurrence of allergic reactions during the treatment process is burdensome for both practitioner and patient. AIM OF THE STUDY: This study compared efficacy and safety in isolated and purified essential BV (eBV) pharmacopuncture filtered for phospholipase A2 (PLA2) and histamine sections, and original BV to the aim of promoting safe BV pharmacopuncture use. MATERIALS AND METHODS: In in vitro, we examined the effect of BV and eBV on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages, and clinically, 20 healthy adults aged 20-40 years were randomly allocated and administered eBV 0.2mL and BV pharmacopuncture 0.2mL on left and right forearm, respectively, and physician, participant, and outcome assessor were blinded to treatment allocation. Local pain, swelling, itching, redness, wheals, and adverse reactions were recorded by timepoint. RESULTS: eBV and BV exhibited similar inhibitory effects on NO production. Also, in comparison between eBV and BV pharmacopuncture administration areas on each forearm, eBV displayed significantly lower local pain at 24h post-administration (P=0.0062), and less swelling at 30min (P=0.0198), 2 (P=0.0028), 24 (P=0.0068), and 48h post-administration (P=0.0253). eBV also showed significantly less itching at 24 (P=0.0119), 48 (P=0.0082), and 96h (P=0.0141), while redness was significantly less at 30min (P=0.0090), 6 (P=0.0005), and 24h (P<0.0001). Time-by-treatment interactions were statistically significant for itching and redness (P<0.001, and P<0.001, respectively), and all original BV pharmacopuncture administered regions showed a tendency toward more severe itching and redness in later measurements. CONCLUSIONS: eBV and BV displayed comparable anti-inflammatory effects, and eBV pharmacopuncture presented less local allergic reactions.

Antitumor Activity of Spicatoside A by Modulation of Autophagy and Apoptosis in Human Colorectal Cancer Cells.

The antitumor activity of spicatoside A (1), a steroidal saponin isolated from the tuber of Liriope platyphylla, and its underlying mechanisms were investigated in HCT116 human colorectal cancer cells. Compound 1 induced autophagy and apoptotic cell death and inhibited tumor growth in a nude mouse xenograft model implanted with HCT116 cells. Treatment with 1 for 24 h enhanced the formation of acidic vesicular organelles in the cytoplasm, indicating the induction of the onset of autophagy. This event was associated with the regulation of autophagic markers including microtubule-associated protein 1 light chain 3 (LC3)-II, p62, beclin 1, lysosomal-associated membrane protein 1 (LAMP 1), and cathepsin D by inhibiting the PI3K/Akt/mTOR signaling pathway, regulating mitogen-activated protein kinase (MAPK) signaling, and increasing p53 levels. However, a prolonged exposure to 1 resulted in apoptosis characterized by the accumulation of a sub-G1 cell population and an annexin V/propidium iodide (PI)-positive cell population. Apoptosis induced by 1 was associated with the regulation of apoptotic proteins including Bcl-2, Bax, and Bid, the release of cytochrome c into the cytosol, and the accumulation of cleaved poly-ADP-ribose polymerase (PARP). Further study revealed that cleavage of beclin 1 by caspases plays a critical role in the 1-mediated switch from autophagy to apoptosis. Taken together, these findings highlight the significance of 1 in the modulation of crosstalk between autophagy and apoptosis, as well as the potential use of 1 as a novel candidate in the treatment of human colorectal cancer cells.

Effects of intra-articular SHINBARO treatment on monosodium iodoacetate-induced osteoarthritis in rats.

BACKGROUND: SHINBARO is a refined herbal formulation used to treat inflamed lesions and bone diseases. This study aimed to investigate the anti-osteoarthritic activities of intra-articular administration of SHINBARO and determine its underlying molecular mechanism in a monosodium iodoacetate (MIA)-induced osteoarthritis rat model. METHODS: Male Sprague-Dawley rats received a single intra-articular injection of MIA into the infrapatellar ligament of the right knee. Subsequently, the rats were treated with normal saline, SHINBARO, and diclofenac once daily for 21 days. Rats treated with normal saline, but not MIA, comprised the control group. Histological changes in the femur of the MIA-induced osteoarthritis rat model were observed by micro-computed tomography scanning and staining with hematoxylin and eosin, and safranin-O fast green. Serum levels of PGE2 and anti-type II collagen antibodies in the MIA-induced osteoarthritis rat model were measured using commercial kits. Protein levels of inflammatory enzymes (iNOS, COX-2), pro-inflammatory cytokines (TNF-α, IL-1ß), and inflammatory mediators (NF-κB, IκB) in cartilaginous tissues were determined by western blot analysis. RESULTS: Intra-articular administration of SHINBARO (IAS) at 20 mg/kg remarkably restrained the decrease in bone volume/total volume, being 28 % (P = 0.0001) higher than that in the vehicle-treated MIA group. IAS (2, 10, and 20 mg/kg) treatment significantly recovered the mean number of objects values with increased percentage changes of 13.5 % (P = 0.147), 27.5 % (P = 0.028), and 44.5 % (P = 0.031), respectively, compared with the vehicle-treated MIA group. The serum level of PGE2 in the IAS group at 20 mg/kg was markedly inhibited by 60.6 % (P = 0.0007) compared with the vehicle-treated MIA group, and the anti-collagen type II antibody level in the IAS group was reduced in a dose-dependent manner. IAS (20 mg/kg) effectively suppressed the induction of inflammation-mediated enzymes (iNOS and COX-2) and pro-inflammatory cytokines (TNF-α and IL-1ß). IAS treatment also downregulated the NF-κB level and increased the IκB-α level in the MIA- induced osteoarthritis rat model. CONCLUSION: SHINBARO inhibited PGE2 and anti-type II collagen antibody production and modulated the balance of inflammatory enzymes, mediators, and cytokines in the MIA-induced osteoarthritis rat model.

Anti-tumoral effect of arsenic compound, sodium metaarsenite (KML001), in non-Hodgkin's lymphoma: an in vitro and in vivo study.

Arsenic compounds have been used in traditional medicine for several centuries. KML001 (sodium metaarsenite; NaAsO2) is an orally bio-available arsenic compound with potential anti-cancer activity. However, the effect of KML001 has not been studied in lymphoid neoplasms. The aim of this study is to evaluate the anti-proliferative effect of KML001 in non-Hodgkin's lymphoma and to compare its efficacy with As2O3. KML001 inhibited cellular proliferation in all tested lymphoma cell lines as well as JurkatR cells (adriamycin-resistant Jurkat cells) in a dose-dependent manner, while As2O3 was not effective. Cell cycle regulatory protein studies have suggested that KML001 induces G1 arrest via p27-induced inhibition of the kinase activities of CDK2, 4, and 6. Treatment of KML001 induced apoptosis in Jurkat and JurkatR cells. The apoptotic process was associated with down-regulation of Bcl-2 (antiapoptotic molecule), up-regulation of Bax (proapoptotic molecule), and inhibition of caspase-3, -8, and -9. In addition, cell signaling including the STAT, PI3K/Akt, MAPK, and NF-κB signal pathways were inhibited in KML001-treated Jurkat and JurkatR cells. Furthermore, targeting the telomere by KML001 was observed in the Jurkat and JurkatR cells. The In vivo anti-tumoral activity of KML001 was confirmed in a xenograft murine model. Interestingly, partial responses were seen in two lymphoma patients treated with 10 mg/day (follicular lymphoma for 16 weeks and mantle cell lymphoma for 24 weeks) without severe toxicities. These findings suggest that KML001 may be a candidate agent for the treatment of de novo, refractory, and relapsed non-Hodgkin's lymphoma patients.

Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

ETHNOPHARMACOLOGICAL RELEVANCE: Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. MATERIAL AND METHODS: Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. RESULTS: Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. CONCLUSION: Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease.

Effects of JSOG-6 on protection against bone loss in ovariectomized mice through regulation of osteoblast differentiation and osteoclast formation.

BACKGROUND: JSOG-6 is used as a traditional medicine to relieve the symptoms associated with inflammation, rheumatism, and osteoporosis in Korea. In the present study, we investigated the effects of JSOG-6 on bone loss prevention both in in vitro and in vivo as well as its underlying mechanism of action. METHODS: Protection against bone loss was assessed in an ovariectomized (OVX) mouse model. Bone microarchitecture was measured using a micro-computed tomography to detect the parameters of three-dimensional structure of a trabecular bone. Serum biomarkers were also evaluated in an OVX-induced model. Osteoclasts derived from mouse bone marrow cells (BMCs) and osteoblastic MC3T3-E1 cells were also employed to investigate the mechanism of action. RESULTS: Oral administration of JSOG-6 significantly increased the bone mineral density (BMD) of the femur in OVX mice in vivo. Especially, the reduced Tb.No (trabecular bone number) in the OVX group was significantly recovered by JSOG-6 treatment. The serum levels of alkaline phosphatase (ALP), osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase, biomarkers of bone resorption, were significantly elevated in OVX mice, but JSOG-6 effectively inhibited the increase in OVX mice. JSOG-6 was also found to enhance the osteoblastic differentiation and maturation with the increase of the density and ALP activity, a marker of osteoblastic differentiation, as well as calcium deposition, a marker of osteoblastic maturation in MC3T3-E1 cells. The effects of JSOG-6 on osteoblastic differentiation were also associated in part with the increase of ALP and OPN mRNA expressions and the decrease of RANKL mRNA expression in MC3T3-E1 cells. CONCLUSIONS: The findings demonstrate that JSOG-6 induced protection against bone loss in OVX mice, and its anti-osteoporotic property might be, in part, a function of the stimulation of osteoblast differentiation and the inhibition of osteoclast formation. These findings suggest that JSOG-6 might be an applicable therapeutic traditional medicine for the regulation of the osteoporotic response.

Suppression of inducible nitric oxide synthase expression by nyasol and broussonin A, two phenolic compounds from Anemarrhena asphodeloides, through NF-κB transcriptional regulation in vitro and in vivo.

Anemarrhena asphodeloides is widely used in traditional Chinese medicine, and is known to possess antidiabetic and anti-inflammatory properties. Because inducible nitric oxide synthase (iNOS) plays an important role in inflammation, we investigated the inhibitory effects of two known phenolic compounds, nyasol (1) and broussonin A (2), from A. asphodeloides, on iNOS and its plausible mechanism of action. Compounds 1 and 2 exhibited inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Compounds 1 and 2 also suppressed the expressions of iNOS protein and mRNA. Moreover, compounds 1 and 2 suppressed the expression of inflammatory cytokines such as interleukin-1ß (IL-1ß) and interferon-ß (IFN-ß). They also inhibited the transcriptional activity of NF-κB and degradation of IκB-α, as well as the activation of Akt and ERK in LPS-stimulated RAW 264.7 cells. In in vivo animal model, compounds 1 and 2 significantly inhibited TPA-induced mouse ear edema. These results suggest that 1 and 2 suppress LPS-stimulated iNOS expression at the transcriptional level through modulating NF-κB and down-regulation of the Akt and ERK signaling pathways. Taken together, these findings indicate that the suppressive effects of 1 and 2 on iNOS expression might provide one possible mechanism for their anti-inflammatory activities.

Suppression of inflammatory responses by handelin, a guaianolide dimer from Chrysanthemum boreale, via downregulation of NF-κB signaling and pro-inflammatory cytokine production.

The anti-inflammatory activity of handelin (1), a guaianolide dimer from Chrysanthemum boreale flowers, was evaluated in vivo, and the effects on mediators nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) and the nuclear factor-κB (NF-κB) and ERK/JNK signaling pathways were investigated in vitro. Compound 1 inhibited lipopolysaccharide (LPS)-induced production of NO and PGE2 in cultured mouse macrophage RAW 264.7 cells. The suppression of NO and PGE2 production by 1 was correlated with the downregulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compound 1 also suppressed the induction of pro-inflammatory cytokines TNF-α and IL-1ß in LPS-stimulated RAW 264.7 cells. To further clarify the transcriptional regulatory pathway in the expression of iNOS and COX-2 by 1, the role of NF-κB was determined in RAW 264.7 cells. Compound 1 inhibits the binding activity of NF-κB into the nuclear proteins. The transcriptional activity of NF-κB stimulated with LPS was also suppressed by 1, which coincided with the inhibition of IκB degradation. Compound 1 also suppressed the activation of mitogen-activated protein kinases, including ERK and JNK signaling. In addition, the LPS-stimulated upregulation of miRNA-155 expression was suppressed by 1. The oral administration of 1 inhibited acute inflammation in carrageenan-induced paw and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear edema models. The serum level of IL-1ß was also inhibited by 1 in a carrageenan-induced paw edema model. These findings suggest that the suppression of NF-κB activation and pro-inflammatory cytokine production may be a plausible mechanism of action for the anti-inflammatory activity of handelin.

Induction of Cell Cycle Arrest and Apoptosis by Physcion, an Anthraquinone Isolated From Rhubarb (Rhizomes of Rheum tanguticum), in MDA-MB-231 Human Breast Cancer Cells.

BACKGROUND: Physcion is an anthraquinone from rhubarb (rhizomes of Rheum tanguticum) and has been reported to have anti-inflammatory, hepatoprotective, antifungal, and anti-cancer activities. However, the growth inhibitory activity against human cancer cells and the underlying molecular mechanisms have been poorly determined. This study was designed to investigate the anti-proliferative activity of physcion by induction of cell cycle arrest and apoptosis in human MDA-MB-231 triple negative breast cancer cell line. METHODS: MDA-MB-231 cells were treated with physcion, and the anti-proliferative activity was evaluated by the sulforhodamine B assay. The mechanisms of action for the growth inhibitory activity of physcion were evaluated by flow cytometry for cell cycle distribution, and by Western blot for the assessment of potential target proteins. RESULTS: Physcion showed a significant anti-proliferative activity against MDA-MB-231 human breast cancer cells. Flow cytometric analysis indicated that physcion markedly induced the accumulation of cells in the G0/G1 phase and the increase of cell population in the sub-G1 phase. The G0/G1 cell cycle arrest by physcion was associated with the down-regulation of Cyclin D1, Cyclin A, CDK4, CDK2, c-Myc and phosphorylated Rb protein expressions. The increase of sub-G1 peak by physcion was closely correlated with the induction of apoptosis, which was confirmed by the induction of cleaved poly-(adenosine diphosphate ribose) polymerase, activation of Caspases, and suppression of Bid and Bcl-2 expression. CONCLUSIONS: The induction of G0/G1 cell cycle arrest and apoptosis might be one of the plausible mechanisms of actions for the anti-proliferative activity of physcion in human breast cancer cells.