Ginsenoside Rg3 Inhibits the Growth of Osteosarcoma and Attenuates Metastasis through the Wnt/ß-Catenin and EMT Signaling Pathway.

Osteosarcoma (OS) is the most common primary malignant bone cancer. An increasing number of studies have demonstrated that ginsenoside Rg3 (Rg3), which is extracted from the roots of the traditional Chinese herb Panax ginseng, plays a tumor suppression role in various malignant tumors. In the present study, we aimed at investigating the role of Rg3 in the proliferation, migration, and invasion of OS and at exploring the underlying mechanisms. Cell viability and proliferation were observed by MTT assay and crystal violet staining. The migration and invasion of cells were measured by wound-healing assay and Transwell method. Signaling pathway screening was investigated using luciferase reporter gene assay. qRT-PCR and western blot were performed to measure the expression of molecules involved in cell epithelial-mesenchymal transition (EMT), and Wnt/ß-catenin pathway. Results suggested that Rg3 could not only inhibit proliferation but also hamper the migration and invasion of OS. qRT-PCR and western blot demonstrated that a reduced level of MMP2/MMP7/MMP9 was induced after Rg3 treatment. In addition, the expression levels of proteins related to EMT and the Wnt/ß-catenin pathway were downregulated. In summary, our data revealed that Rg3 could inhibit the proliferation, migration, and invasion of OS cells. This effect of Rg3 might be mediated by downregulating MMP2, MMP7, and MMP9 expression and suppressing EMT as well as the Wnt/ß-catenin pathway. Thus, Rg3 might be a potential agent for the treatment of OS.

Treatment with Rhodiola crenulata root extract ameliorates insulin resistance in fructose-fed rats by modulating sarcolemmal and intracellular fatty acid translocase/CD36 redistribution in skeletal muscle.

BACKGROUND: Rhodiola species have been used for asthenia, depression, fatigue, poor work performance and cardiovascular diseases, all of which may be associated with insulin resistance. To disclose the underlying mechanisms of action, the effect of Rhodiola crenulata root (RCR) on insulin resistance was investigated. METHODS: Male Sprague-Dawley rats were treated with liquid fructose in their drinking water over 18 weeks. The extract of RCR was co-administered (once daily by oral gavage) during the last 5 weeks. The indexes of lipid and glucose homeostasis were determined enzymatically and/or by ELISA. Gene expression was analyzed by Real-time PCR, Western blot and/or confocal immunofluorescence. RESULTS: RCR extract (50 mg/kg) suppressed fructose-induced hyperinsulinemia and the increases in the homeostasis model assessment of insulin resistance index and the adipose tissue insulin resistance index in rats. Additionally, this treatment had a trend to restore the ratios of glucose to insulin and non-esterified fatty acids (NEFA) to insulin. Mechanistically, RCR suppressed fructose-induced acceleration of the clearance of plasma NEFA during oral glucose tolerance test (OGTT), and decreased triglyceride content and Oil Red O staining area in the gastrocnemius. Furthermore, RCR restored fructose-induced sarcolemmal overexpression and intracellular less distribution of fatty acid translocase/CD36 that contributes to etiology of insulin resistance by facilitating fatty acid uptake. CONCLUSION: These results suggest that RCR ameliorates insulin resistance in fructose-fed rats by modulating sarcolemmal and intracellular CD36 redistribution in the skeletal muscle. Our findings may provide a better understanding of the traditional use of Rhodila species.

[Regulatory effect of ginsenoside Rh2 on HDAC1/2 activity and cyclin in human erythroleukemia K562 cells].

OBJECTIVE: To investigate the effects of the 20(S)-ginsenoside Rh2 [Rh2(S)]on cell proliferation, histone deacetylase 1 (HDAC1) and HDAC2 activity, and expression of cyclin in human erythroleukemia K562 cells. METHODS: The K562 cells were treated with Rh2(S) at various concentrations (10-80 µmol/L). Cell proliferation activity was detected by CCK-8 assay. Flow cytometry (FCM) was used to detect cell cycle and apoptotic changes. The HDAC activity of cells was measured by chemical colorimetry. The protein expressions of HDAC1, HDAC2, cyclin D1, CDK4, p16INK4A and p21 after 48 hour-treatment of Rh2 (S) (10, 20, 40, 60 µmol/L) were examined by Western blotting. RESULTS: The proliferation of K562 cells was inhibited by Rh2 (S) (20-80 µmol/L) in dose-and time-dependent manner. FCM analyses revealed that the number of the K562 cells treated with 60 µmol/L Rh2(S) was arrested in G0/G1 phase. The apoptosis rates of K562 cells were respectively (8.09±0.86)%, (9.44±0.53)% and (22.80±2.16)% after induced by 20, 40, 60 µmol/L Rh2(S), which showed statistically significant difference (P<0.05) compared with the control group (2.63±0.14)%. HDAC activity of the cells treated with Rh2(S) (40, 60 µmol/L) was reduced. Western blotting showed that the expressions of HDAC1, HDAC2, cyclin D1 and CDK4 decreased after induced by Rh2(S), and p16INK4A, p21 proteins were enhanced significantly. CONCLUSION: The Rh2(S) can inhibit the proliferation of K562 cells and induce its cycle arrest and apoptosis through inhibiting HDAC1 and HDAC2 activity, down-regulating the expression of cyclin D1 and activating p16INK4A and p21.

Ginger extract diminishes chronic fructose consumption-induced kidney injury through suppression of renal overexpression of proinflammatory cytokines in rats.

BACKGROUND: The metabolic syndrome is associated with an increased risk of development and progression of chronic kidney disease. Renal inflammation is well known to play an important role in the initiation and progression of tubulointerstitial injury of the kidneys. Ginger, one of the most commonly used spices and medicinal plants, has been demonstrated to improve diet-induced metabolic abnormalities. However, the efficacy of ginger on the metabolic syndrome-associated kidney injury remains unknown. This study aimed to investigate the impact of ginger on fructose consumption-induced adverse effects in the kidneys. METHODS: The fructose control rats were treated with 10% fructose in drinking water over 5 weeks. The fructose consumption in ginger-treated rats was adjusted to match that of fructose control group. The ethanolic extract of ginger was co-administered (once daily by oral gavage). The indexes of lipid and glucose homeostasis were determined enzymatically, by ELISA and/or histologically. Gene expression was analyzed by Real-Time PCR. RESULTS: In addition to improve hyperinsulinemia and hypertriglyceridemia, supplement with ginger extract (50 mg/kg) attenuated liquid fructose-induced kidney injury as characterized by focal cast formation, slough and dilation of tubular epithelial cells in the cortex of the kidneys in rats. Furthermore, ginger also diminished excessive renal interstitial collagen deposit. By Real-Time PCR, renal gene expression profiles revealed that ginger suppressed fructose-stimulated monocyte chemoattractant protein-1 and its receptor chemokine (C-C motif) receptor-2. In accord, overexpression of two important macrophage accumulation markers CD68 and F4/80 was downregulated. Moreover, overexpressed tumor necrosis factor-alpha, interleukin-6, transforming growth factor-beta1 and plasminogen activator inhibitor (PAI)-1 were downregulated. Ginger treatment also restored the downregulated ratio of urokinase-type plasminogen activator to PAI-1. CONCLUSIONS: The present results suggest that ginger supplement diminishes fructose-induced kidney injury through suppression of renal overexpression of macrophage-associated proinflammatory cytokines in rats. Our findings provide evidence supporting the protective effect of ginger on the metabolic syndrome-associated kidney injury.

Oleanolic Acid diminishes liquid fructose-induced Fatty liver in rats: role of modulation of hepatic sterol regulatory element-binding protein-1c-mediated expression of genes responsible for de novo Fatty Acid synthesis.

Oleanolic acid (OA), contained in more than 1620 plants and as an aglycone precursor for naturally occurred and synthesized triterpenoid saponins, is used in China for liver disorders in humans. However, the underlying liver-protecting mechanisms remain largely unknown. Here, we found that treatment of rats with OA (25 mg/kg/day, gavage, once daily) over 10 weeks diminished liquid fructose-induced excess hepatic triglyceride accumulation without effect on total energy intake. Attenuation of the increased vacuolization and Oil Red O staining area was evident on histological examination of liver in OA-treated rats. Hepatic gene expression profile demonstrated that OA suppressed fructose-stimulated overexpression of sterol regulatory element-binding protein-(SREBP-) 1/1c mRNA and nuclear protein. In accord, overexpression of SREBP-1c-responsive genes responsible for fatty acid synthesis was also downregulated. In contrast, overexpressed nuclear protein of carbohydrate response element-binding protein and its target genes liver pyruvate kinase and microsomal triglyceride transfer protein were not altered. Additionally, OA did not affect expression of peroxisome proliferator-activated receptor-gamma- and -alpha and their target genes. It is concluded that modulation of hepatic SREBP-1c-mediated expression of the genes responsible for de novo fatty acid synthesis plays a pivotal role in OA-elicited diminishment of fructose-induced fatty liver in rats.

Improvement of liquid fructose-induced adipose tissue insulin resistance by ginger treatment in rats is associated with suppression of adipose macrophage-related proinflammatory cytokines.

Adipose tissue insulin resistance (Adipo-IR) results in excessive release of free fatty acids from adipose tissue, which plays a key role in the development of "lipotoxicity." Therefore, amelioration of Adipo-IR may benefit the treatment of other metabolic abnormalities. Here we found that treatment with the alcoholic extract of ginger (50 mg/kg/day, by oral gavage) for five weeks attenuated liquid fructose-induced hyperinsulinemia and an increase in the homeostasis model assessment of insulin resistance (HOMA-IR) index in rats. More importantly, ginger reversed the increases in the Adipo-IR index and plasma nonesterified fatty acid concentrations during the oral glucose tolerance test assessment. Adipose gene/protein expression profiles revealed that ginger treatment suppressed CD68 and F4/80, two important macrophage accumulation markers. Consistently, the macrophage-associated cytokines tissue necrosis factor alpha and interleukin-6 were also downregulated. In contrast, insulin receptor substrate (IRS)-1, but not IRS-2, was upregulated. Moreover, monocyte chemotactic protein (MCP)-1 and its receptor chemokine (C-C motif) receptor-2 were also suppressed. Thus these results suggest that amelioration of fructose-induced Adipo-IR by ginger treatment in rats is associated with suppression of adipose macrophage-related proinflammatory cytokines.

Treatment with ginger ameliorates fructose-induced Fatty liver and hypertriglyceridemia in rats: modulation of the hepatic carbohydrate response element-binding protein-mediated pathway.

Ginger has been demonstrated to improve lipid derangements. However, its underlying triglyceride-lowering mechanisms remain unclear. Fructose overconsumption is associated with increase in hepatic de novo lipogenesis, thereby resulting in lipid derangements. Here we found that coadministration of the alcoholic extract of ginger (50 mg/kg/day, oral gavage, once daily) over 5 weeks reversed liquid fructose-induced increase in plasma triglyceride and glucose concentrations and hepatic triglyceride content in rats. Plasma nonesterified fatty acid concentration was also decreased. Attenuation of the increased vacuolization and Oil Red O staining area was evident on histological examination of liver in ginger-treated rats. However, ginger treatment did not affect chow intake and body weight. Further, ginger treatment suppressed fructose-stimulated overexpression of carbohydrate response element-binding protein (ChREBP) at the mRNA and protein levels in the liver. Consequently, hepatic expression of the ChREBP-targeted lipogenic genes responsible for fatty acid biosynthesis was also downregulated. In contrast, expression of neither peroxisome proliferator-activated receptor- (PPAR-) alpha and its downstream genes, nor PPAR-gamma and sterol regulatory element-binding protein 1c was altered. Thus the present findings suggest that in rats, amelioration of fructose-induced fatty liver and hypertriglyceridemia by ginger treatment involves modulation of the hepatic ChREBP-mediated pathway.

BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential.

Promoting osteogenic differentiation and efficacious bone regeneration have the potential to revolutionize the treatment of orthopaedic and musculoskeletal disorders. Mesenchymal Stem Cells (MSCs) are bone marrow progenitor cells that have the capacity to differentiate along osteogenic, chondrogenic, myogenic, and adipogenic lineages. Differentiation along these lineages is a tightly controlled process that is in part regulated by the Bone Morphogenetic Proteins (BMPs). BMPs 2 and 7 have been approved for clinical use because their osteoinductive properties act as an adjunctive treatment to surgeries where bone healing is compromised. BMP-9 is one of the least studied BMPs, and recent in vitro and in vivo studies have identified BMP-9 as a potent inducer of osteogenic differentiation in MSCs. BMP-9 exhibits significant molecular cross-talk with the Wnt/ ß-catenin and other signaling pathways, and adenoviral expression of BMP-9 in MSCs increases the expression of osteogenic markers and induces trabecular bone and osteiod matrix formation. Furthermore, BMP-9 has been shown to act synergistically in bone formation with other signaling pathways, including Wnt/ ß-catenin, IGF, and retinoid signaling pathways. These results suggest that BMP-9 should be explored as an effective bone regeneration agent, especially in combination with adjuvant therapies, for clinical applications such as large segmental bony defects, non-union fractures, and/or spinal fusions.

Therapeutic Implications of PPARgamma in Human Osteosarcoma.

Osteosarcoma (OS) is the most common nonhematologic malignancy of bone in children and adults. Although dysregulation of tumor suppressor genes and oncogenes, such as Rb, p53, and the genes critical to cell cycle control, genetic stability, and apoptosis have been identified in OS, consensus genetic changes that lead to OS development are poorly understood. Disruption of the osteogenic differentiation pathway may be at least in part responsible for OS tumorigenesis. Current OS management involves chemotherapy and surgery. Peroxisome proliferator-activated receptor (PPAR) agonists and/or retinoids can inhibit OS proliferation and induce apoptosis and may inhibit OS growth by promoting osteoblastic terminal differentiation. Thus, safe and effective PPAR agonists and/or retinoid derivatives can be then used as adjuvant therapeutic drugs for OS therapy. Furthermore, these agents have the potential to be used as chemopreventive agents for the OS patients who undergo the resection of the primary bone tumors in order to prevent local recurrence and/or distal pulmonary metastasis.

Total saponins of Panax ginseng induces K562 cell differentiation by promoting internalization of the erythropoietin receptor.

Ginseng is a commonly used herbal medicine with a wide range of therapeutic benefits. Total saponins of Panax ginseng (TSPG) is one of the main effective components of ginseng. Our previous studies have shown that TSPG could promote the production of normal blood cells and inhibition of the leukemia cell proliferation. However, whether ginseng can induce the differentiation of leukemia cells is still unclear. This study was to examine the effect of TSPG or the combination of erythropoietin (EPO) and TSPG on the erythroid differentiation of K562 cells, and their corresponding mechanisms regarding erythropoietin receptor (EPOR) expression. Under light and electron microscopes, the TSPG- or TSPG + EPO-treated K562 cells showed a tendency to undergo erythroid differentiation; early and intermediate erythroblast-like cells were observed. Hemoglobin and HIR2 expressions were significantly increased. As determined by Western blotting analysis, the EPOR protein level in the K562 cytoplasmic membrane was significantly decreased after TSPG treatment, while its cytoplasm level increased in a dose-dependent manner. However, the total cellular EPOR level was unchanged. These results indicate that TSPG-induced erythroid differentiation of K562 cells may be accompanied by the internalization of EPOR. Thus, our study suggests that treatment with a combination of TSPG and EPO may induce erythroid differentiation of K562 cells at least in part through induction of EPOR internalization.

Microarray expression profiling of Yersinia pestis in response to berberine.

Coptis chinensis Franch. is a natural herb widely used in China for prevention and treatment of infectious diseases. Plague is a deadly disease caused by Yersinia pestis. Coptis chinensis Franch. is considered the therapeutic agent of choice against plague rather than conventional antibiotics because of its low cost and low toxicity. Berberine is the major constituent of a Coptis chinensis Franch. extract. In the present study, DNA microarray was used to investigate the transcription of Y. pestis in response to berberine. The minimal inhibition concentration (MIC) of berberine to Y. pestis was determined with the liquid dilution method. The gene expression profile of Y. pestis was performed by exposing Y. pestis to berberine at a concentration of 10 x MIC for 30 min. Total RNA was extracted and purified from Y. pestis, reverse-transcribed to cDNA, and then labeled with Cy-dye probes. The labeled probes were hybridized to the microarray. The results were obtained by a laser scanner and analyzed with SAM software. A total of 360 genes were differentially expressed in response to berberine: 333 genes were upregulated, and 27 were downregulated. The upregulation of genes that encode proteins involved in metabolism was a remarkable change. In addition to a number of genes of unknown encoding or unassigned functions, genes encoding cellular envelope and transport/binding functions represented the majority of the altered genes. A number of genes related to iron uptake were induced. This study revealed global transcriptional changes of Y. pestis in response to berberine, hence providing insights into the mechanisms of Coptis chinensis Franch. against Y. pestis.