Structural dependence of antidiabetic effect of steviol glycosides and their metabolites on streptozotocin-induced diabetic mice.

BACKGROUND: Stevia has been proposed as a potential antidiabetic sweetener, mainly based on inconsistent results from stevioside or the plant extract, yet lacking relative experimental evidence from individual steviol glycosides (SGs) and their metabolites. RESULTS: The results systematically revealed that the typical SGs and their final metabolite (steviol) presented an antidiabetic effect on streptozotocin (STZ) diabetic mice in all assayed antidiabetic aspects. In general, the performance strength of the samples followed the sequence steviol > steviol glucosyl ester > steviolbioside > rubusoside > stevioside > rebaudioside A, which is opposite to their sweetness strength order, and generally in accordance with the glucosyl group numbers in their molecules. This may imply that the antidiabetic effect of the SGs might be achieved through steviol, which presented antidiabetic performance similar to that of metformin with a dose of 1/20 that of metformin. Moreover, the 18 F-fluorodeoxyglucose traced micro-PET experiment revealed that stevioside and steviol could increase the uptake of glucose in the myocardium and brain of the diabetic mice within 60 min, and decrease the accumulation of glucose in the liver and kidney. CONCLUSIONS: The SGs and steviol presented an antidiabetic effect on STZ diabetic mice in all assayed aspects, with an induction time to start the effect of the SGs. Stevioside and steviol could increase uptake of glucose in the myocardium and brain of the diabetic mice, and decrease accumulation of glucose in the liver and kidney. The performance strength of the SGs is generally in accordance with glucosyl group numbers in their molecules.

The natural sweetener metabolite steviol inhibits the proliferation of human osteosarcoma U2OS cell line.

Steviol is the colonic metabolite of the natural sweetener steviol glycosides. It does not diffuse to the blood and the half maximal inhibitory concentration of steviol is longer compared with that of current chemotherapy agents, including 5-fluorouracil and doxorubicin. The present study demonstrated that steviol inhibits the proliferation of the human osteosarcoma U2OS cell line in a dose- and time-dependent manner, and that the inhibition rate is comparative with that of doxorubicin and 5-fluorouracil. The mechanism of this anticancer activity is also investigated. The results indicated that steviol inhibits U2OS cells through inducing G1 phase cell cycle arrest, downregulating the ability of colony formation via a mitochondrial apoptotic pathway, which was indicated by an increase of the Bax/Bcl-2 ratio and activation of cyclin-dependent kinase inhibitor 1, tumor protein 53 and cyclin-dependent kinase; whereas a Survivin and Caspase 3-independent mechanism was involved. Considering that steviol appears minimally in the plasma during metabolism, and possesses a median lethal dose of 100-fold greater compared with that of 5-fluorouracil, it may become a potential chemotherapy agent.

Influence of substrates on the in vitro kinetics of steviol glucuronidation and interaction between steviol glycosides metabolites and UGT2B7.

Steviol glycosides, a natural sweetener, may perform bioactivities via steviol, their main metabolite in human digestion. The metabolising kinetics, i.e. glucuronidation kinetics and interaction between steviol glycosides or their metabolites and metabolising enzyme, are important for understanding the bioactivity and cytotoxicity. The present study investigated kinetics of steviol glucuronidation in human liver microsome and a recombinant human UDP-glucuronosyltransferases isomer, UGT2B7, along with molecular docking to analyse interaction between UGT2B7 and steviol or glucose. The active pocket of UGT2B7 is consisted of Arg352, Leu347, Lys343, Phe339, Tyr354, Lys355 and Leu353. The influence of stevioside, rebaudioside A, glucose and some chemotherapy reagents on the glucuronidation was also studied. The predicted hepatic clearence suggested that steviol could be classified as high-clearence drug. The steviol glycosides did not affect the glucuronidation of steviol notably.

Periostin promotes epithelial-mesenchymal transition via the MAPK/miR-381 axis in lung cancer.

Periostin (POSTN, PN, or osteoblast-specific factor OSF-2) is a multifunctional cytokine that signals between the cell and the extracellular matrix. Periostin plays an important role in tumor development and is involved in carcinoma cell epithelial-mesenchymal transition (EMT), whereby mature epithelial cells undergo phenotypic morphological changes and become invasive, motile cells. Here, we discuss the molecular mechanisms involved in periostin-induced promotion of EMT in lung cancer cells. Online TCGA datasets demonstrate the prognostic relevance of periostin in lung cancer; a higher periostin level correlates with poor overall survival. Similarly, our IHC results show that high periostin expression is positively correlated with the EMT markers Snail and Twist, as well as stage of lung cancer. We found that recombinant periostin induces the EMT phenotype in lung cancer cells through the p38/ERK pathway, while pretreatment with chemical inhibitors prevented periostin-induced EMT induction. Moreover, we found that periostin regulates EMT by repressing microRNA-381 (miR-381) expression, which targets both Snail and Twist. Using the miR-381 mimic, we dramatically reversed periostin-induced Snail and Twist expression. Furthermore, periostin knockdown dramatically affected EMT markers and cell migration potential. The role of periostin in lung cancer progression is elucidated by the in vivo mouse model. Our findings indicate that changes in periostin expression in lung cancer may serve as a therapeutic target for the treatment of lung cancer metastasis.

ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma.

BACKGROUND: Activating transcription factor 2 (ATF2) is a basic helix-loop-helix transcription factor, which has been shown to participate in the pathobiology of numerous cancers. However, the role of ATF2 in renal cell carcinoma (RCC) remains unclear. METHODS: ATF2 knockdown and overexpression studies were performed in RCC cells to evaluate changes in cell viability, cell cycle, apoptosis, migration and invasion. Xenograft models were used to examine the tumorigenic and metastatic capability of RCC cells upon ATF2 suppression. The expression of ATF2 in human RCC samples was determined using immunohistochemistry on a tissue microarray. RESULTS: ATF2 knockdown in RCC cells reduced their proliferative and metastatic potentials, whereas ATF2 overexpression enhanced these properties. Mechanistic studies revealed that the transcription of CyclinB1, CyclinD1, Snail and Vimentin was directly regulated by ATF2 in RCC cells. Moreover, ATF2 was shown to be highly expressed in RCC tissues, especially in tumors with metastases. High expression of ATF2 correlated with aggressive clinico-pathological characteristics and predicted poor prognosis of RCC patients. CONCLUSIONS: ATF2 exerts an oncogenic role in RCC and could serve as an important prognostic biomarker.

Qinggan Huoxue Recipe suppresses epithelial-to-mesenchymal transition in alcoholic liver fibrosis through TGF-ß1/Smad signaling pathway.

AIM: To investigate the mechanism by which Qinggan Huoxue Recipe (QGHXR) inhibits epithelial-to-mesenchymal transition (EMT) in rats with alcoholic liver fibrosis (ALF). METHODS: A total of 75 male SD rats were used to induce ALF. Serum biochemical indicators, including alanine aminotransferase, aspartate aminotransferase, laminin and hyaluronidase, were measured. Liver histopathological changes were evaluated using hematoxylin-eosin and Sirius red staining. EMT was examined by analyzing the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and fibronectin using RT-PCR and Western blot. The inhibitory effect of QGHXR on EMT markers, as well as its effect on molecules associated with the transforming growth factor (TGF)-ß1/Smad signaling pathway, including TGF-ß1, Smad3, snail, occludin, ZO-1 and claudin, was also examined. RESULTS: Compared with normal control rats, ALF rats exhibited a decrease in E-cadherin levels (mRNA: ALF 0.16 ± 0.05 vs control 1.00 ± 0.08; protein: ALF 0.09 ± 0.05 vs control 0.70 ± 0.17, P < 0.01) and an increase in vimentin and fibronectin levels (mRNA: 11.43 ± 0.39 vs 1.00 ± 0.19 and 9.91 ± 0.34 vs 1.00 ± 0.44, respectively, P < 0.01; protein: 1.13 ± 0.42 vs 0.09 ± 0.03 and 1.16 ± 0.43 vs 0.09 ± 0.00, respectively, P < 0.01). This indicates that EMT occurred in ALF rats. In addition, the TGF-ß1/Smad signaling pathway was activated in ALF rats, as evidenced by the increase in TGF-ß1 and snail levels (mRNA: 1.76 ± 0.12 vs 1.00 ± 0.05 and 6.98 ± 0.41 vs 1.00 ± 0.10, respectively, P < 0.01; protein: 1.43 ± 0.05 vs 0.12 ± 0.03 and 1.07 ± 0.29 vs 0.07 ± 0.02, respectively, P < 0.01) and the decrease in Smad3 levels (mRNA: 0.05 ± 0.01 vs 1.00 ± 0.12, P < 0.01; protein: 0.06 ± 0.05 vs 0.89 ± 0.12, P < 0.01). Furthermore, levels of the tight junction markers occludin, ZO-1 and claudin decreased in ALF rats compared with healthy control rats (mRNA: 0.60 ± 0.09 vs 1.00 ± 0.12, 0.11 ± 0.00 vs 1.00 ± 0.12 and 0.60 ± 0.01 vs 1.00 ± 0.08, respectively, P < 0.01; protein: 0.05 ± 0.01 vs 0.87 ± 0.40, 0.09 ± 0.05 vs 0.89 ± 0.18 and 0.04 ± 0.03 vs 0.95 ± 0.21, respectively, P < 0.01). In ALF rats treated with QGHXR, E-cadherin levels increased (mRNA: QGHXR 0.67 ± 0.04 vs ALF model 0.16 ± 0.05, P < 0.01; protein: QGHXR 0.66 ± 0.21 vs ALF model 0.09 ± 0.05, P < 0.01), and vimentin and fibronectin levels decreased (mRNA: 6.57 ± 1.05 vs 11.43 ± 0.39 and 1.45 ± 1.51 vs 9.91 ± 0.34, respectively, P < 0.01; protein: 0.09 ± 0.03 vs 1.13 ± 0.42 and 0.10 ± 0.01 vs 1.16 ± 0.43, respectively, P < 0.01). In addition, QGHXR inhibited the expression of TGF-ß1 and increased the expression of Smad3 (mRNA: 1.03 ± 0.11 vs 1.76 ± 0.12, 0.70 ± 0.10 vs 0.05 ± 0.01, respectively, P < 0.05 and P < 0.01; protein: 0.12 ± 0.03 vs 1.43 ± 0.05 and 0.88 ± 0.20 vs 0.06 ± 0.05, respectively, P < 0.01). QGHXR treatment also reduced the levels of the EMT-inducing transcription factor snail (mRNA: 2.28 ± 0.33 vs 6.98 ± 0.41, P < 0.01; protein: 0.08 ± 0.02 vs 1.07 ± 0.29, P < 0.01) and increased the occludin, ZO-1 and claudin levels (mRNA: 0.73 ± 0.05 vs 0.60 ± 0.09, 0.57 ± 0.04 vs 0.11 ± 0.00 and 0.68 ± 0.03 vs 0.60 ± 0.01, respectively, P < 0.01, P < 0.01 and P < 0.05; protein: 0.92 ± 0.50 vs 0.05 ± 0.01, 0.94 ± 0.22 vs 0.09 ± 0.05 and 0.94 ± 0.29 vs 0.04 ± 0.03, respectively, P < 0.01). The effects of QGR and HXR on the TGF-ß1/Smad signaling pathway were similar to that of QGHXR; however, the QGR- and HXR-induced changes in vimentin mRNA levels, the QGR-induced changes in fibronectin mRNA levels and the HXR-induced changes in snail and TGF-ß1 mRNA levels were not significant. CONCLUSION: Qinggan Huoxue Recipe inhibits EMT in ALF rats by modulating the TGF-ß1/Smad signaling pathway, suggesting that the mechanism underlying the amelioration of ALF induced by QGHXR is associated with this pathway.

Kinetic Curve Type Assessment for Classification of Breast Lesions Using Dynamic Contrast-Enhanced MR Imaging.

OBJECTIVE: The aim of this study was to employ a kinetic model with dynamic contrast enhancement-magnetic resonance imaging to develop an approach that can efficiently distinguish malignant from benign lesions. MATERIALS AND METHODS: A total of 43 patients with 46 lesions who underwent breast dynamic contrast enhancement-magnetic resonance imaging were included in this retrospective study. The distribution of malignant to benign lesions was 31/15 based on histological results. This study integrated a single-compartment kinetic model and dynamic contrast enhancement-magnetic resonance imaging to generate a kinetic modeling curve for improving the accuracy of diagnosis of breast lesions. Kinetic modeling curves of all different lesions were analyzed by three experienced radiologists and classified into one of three given types. Receiver operating characteristic and Kappa statistics were used for the qualitative method. The findings of the three radiologists based on the time-signal intensity curve and the kinetic curve were compared. RESULTS: An average sensitivity of 82%, a specificity of 65%, an area under the receiver operating characteristic curve of 0.76, and a positive predictive value of 82% and negative predictive value of 63% was shown with the kinetic model (p = 0.017, 0.052, 0.068), as compared to an average sensitivity of 80%, a specificity of 55%, an area under the receiver operating characteristic of 0.69, and a positive predictive value of 79% and negative predictive value of 57% with the time-signal intensity curve method (p = 0.003, 0.004, 0.008). The diagnostic consistency of the three radiologists was shown by the κ-value, 0.857 (p<0.001) with the method based on the time-signal intensity curve and 0.826 (p<0.001) with the method of the kinetic model. CONCLUSIONS: According to the statistic results based on the 46 lesions, the kinetic modeling curve method showed higher sensitivity, specificity, positive and negative predictive values as compared with the time-signal intensity curve method in lesion classification.

Inhibitor or promoter? The performance of polysaccharides from Ganoderma lucidum on human tumor cells with different p53 statuses.

Polysaccharides from Ganoderma lucidum (GLPs) have been taken as effective supplements by both healthy people and cancer patients for many years. However, this short survey indicates that instead of inhibiting cancer cell growth, both submerge-cultured intracellular GLP and fruiting body GLP can stimulate the growth of human carcinoma cell lines lacking functional p53, such as HCT-116 p53(-/-), Saos-2, H1299, HL-60, MDA-MB-157. Conversely, the two GLPs inhibit all other assayed cells with functional p53. These results could be an alert since mutational inactivation of the tumor suppressor protein p53 is the most frequent genetic alteration found in human tumors.

Production of a bioactive sweetener steviolbioside via specific hydrolyzing ester linkage of stevioside with a ß-galactosidase.

A ß-galactosidase from Kluyveromyces lactis was found to specifically catalyze hydrolysis of the glycosyl ester linkage of stevioside to yield steviolbioside, a rare sweetener that also exists in Stevia rebaudiana leaves. In a packed bed reactor, a reaction coupling separation was realized and a production yield of steviolbioside reached 90% in 6 h. The hydrolysis product steviolbioside presented higher cytoxicity on human normal cells (hepatocytes cell L02 and intestinal epithelial cell T84) than stevioside did. Comparing to the typical chemotherapy agent, 5-fluorouracil (5-FU), steviolbioside presents much lower cytotoxicity on all assayed human normal cells; it presented notable inhibition on human hepatocarcinoma cell Hep3B, human breast cancer cell MDA-MB-231 and human pancreatic cancer cell BxPC-3. The remarkable inhibition on MDA-MB-231 cells makes steviolbioside a potential remedy for human breast cancer, when steviolbioside is served as a natural sweetener.

Danshensu inhibits acetaldehyde-induced proliferation and activation of hepatic stellate cell-T6.

OBJECTIVE: To evaluate the effects of danshensu, the main component of the extract of Chinese medicine Salvia miltiorrhiza, on the proliferation and activation of hepatic stellate cells (HSCs). METHODS: The activation of HSC-T6 was induced by exposure to acetaldehyde. In the meantime, different doses of danshensu were added to the culture medium. After 24 h of treatment with danshensu in acetaldehyde, the viability of HSC-T6 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cell cycle was determined through flow cytometry, and the gene transcription levels of plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-ß1 (TGF-ß1), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP-2) were analyzed by real-time quantitative polymerase chain reaction. RESULTS: The proliferation of HSCs induced by 200 µmol/L acetaldehyde could be significantly inhibited by danshensu, and the percentage of HSCs in S phase was significantly increased as compared with the control cells (P<0.05), which were respectively evidenced by MTT assay and flow cytometry. Danshensu down-regulated the mRNA expression of TGF-ß1 and PAI-1 and up-regulated the uPA transcription level (P<0.01), while the transcription level of MMP-2 was not significantly affected in HSC-T6. CONCLUSION: Danshensu can inhibit the proliferation and activation of HSC-T6, as well as regulate some cytokines involved in extracellular matrix accumulation, which offers a potential therapeutic alternative for liver fibrosis.

[Regulatory effects of Qinggan Huoxue Recipe on matrix metalloproteinases of alcoholic liver fibrosis rats].

OBJECTIVE: To study the action mechanism of Qinggan Huoxue Recipe (QGHXR) and its disassembled recipes for treatment of alcoholic liver fibrosis (ALF) by observing their regulation on the expressions of matrix metalloproteinases (MMPs) and type 1 tissue inhibitor of metalloproteinase (TIMP-1). METHODS: 130 male SD rats were randomly divided into the blank control group (n=10), the CCI4 group (n=10), and the modeling group (n=110). Rats in the modeling group were intervened by complex factors dominated as alcohol. Eight weeks later they were randomly divided into 4 subgroups, i.e., the model group (n=25), the QGHXR group (n= 15), the Qinggan Recipe (QGR) group (n=15), and the Huoxue Recipe (HXR) group (n=15). Eight model rats were selected for pathological analysis to monitor the development of the modeling at the 4th, 8th, and 10th week of the experiment. The rest rats died during the modeling. Corresponding medicines were given to these treatment groups (at the dose of 4.75 g/kg, 1.50 g/kg, and 3.25 g/kg). All rats were killed at the end of the 12th week. The protein and mRNA expressions of MMP-2, MMP-9, and TIMP-1 were detected using Western blotting, fluorescent quantitative polymerase chain reaction, and immunofluorescence method. RESULTS: Compared with the blank control group, the expressions of MMP-2, MMP-9, and TIMP-1 significantly increased in the model group (1.81 +/- 0.28 versus 0.53 +/- 0.04, 1.60 +/- 0.16 versus 0.45 +/- 0.05, 1.20 +/- 0.02 versus 0.35 +/- 0.07, P < 0.01). Compared with the model group, QGHXR and its disassembled recipes all could decrease the protein and mRNA expressions of TIMP-1 (0. 56 +/- 0.05, 0.67 +/- 0.02, 0.70 +/- 0.02 versus 1.20 +/- 0.02, P < 0.05), increase the expressions of MMP-2 and MMP-9 (4.18 +/- 0.53, 2.70 +/- 0.40, 2.38 +/- 0.22 versus 1.81 +/- 0.28, 3.31 +/- 0.06, 2.56 +/- 0.20, 1.87 +/- 0.05 versus 1.60 +/- 0.16, P < 0.05, P < 0.01). QGHXR was superior to its disassembled recipes (P < 0.05, P < 0.01). CONCLUSION: The action mechanisms of QGHXR and its disassembled recipes might possibly be correlated with regulating MMPs.

[Effects of Qinggan Huoxue Recipe and its separated recipes on urokinase-type plasminogen activator and plasminogen activator inhibitor-1 fibrinolytic system in rats with alcoholic liver fibrosis].

OBJECTIVE: To study the action mechanisms of Qinggan Huoxue Recipe (QGHXR), a compound traditional Chinese herbal medicine, and its separated recipes by observing their effects on expressions of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in rats with alcoholic liver fibrosis (ALF). METHODS: A total of 150 SD rats were divided into three groups: blank group, carbon tetrachloride (C(4)) group and ALF-inducing group. Rats in the ALF-inducing group were administered with a mixture diet (56% alcohol 10 mL/kg, corn oil 2 mL/kg, pyrazole 25 mg/kg) once daily and intraperitoneally injected with 0.3 mL/kg 25% solution of C(4) in olive oil twice weekly. The C(4) group was intraperitoneally injected with equal volume of C(4) and olive oil as the ALF-inducing group and ingested normal saline (12 mL/kg per day). The blank group was intraperitoneally injected with and ingested saline in equal volumes of the above. At the end of the eighth week, the survived rats in the ALF-inducing group were divided into four subgroups: untreated group, QGHXR group, Qinggan Recipe (QGR) group and Huoxue Recipe (HXR) group. The three treated groups were given corresponding drugs respectively (4.75, 1.5, 3.25 g/kg). The blank group, CCl(4) group and untreated group were given normal saline in equal volume (5 mL/kg per day). All rats were anaesthetized and killed at the end of the twelfth week. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were analyzed. Pathological changes in liver tissues were observed by hematoxylin and eosin staining and Masson staining. The expressions of uPA and PAI-1 were evaluated with Western blotting, immunofluorescence, and real-time polymerase chain reaction. RESULTS: There existed obvious liver fibrosis in liver tissues in the untreated group as compared with the blank group (P<0.01), and the activities of ALT and AST and the expressions of uPA and PAI-1 also increased in the untreated group. QGHXR and its separated recipes could improve the degree of liver fibrosis (P<0.01). QGHXR and its separated recipes could degrade the activity of ALT as compared with the untreated group; QGHXR and its separated recipes advanced the expression of uPA, and decreased the expression of PAI-1 significantly as compared with the untreated group. The effect of QGHXR was the best among the three recipes. CONCLUSION: QGHXR and its separated recipes may improve ALF in rats by decreasing the expression of PAI-1 and advance the expression of uPA. The effect of QGHXR is the best among them.

Chemical characteristics of three medicinal plants of the Panax genus determined by HPLC-ELSD.

The morphological appearance and main ingredients of three Chinese medicines (CMs), P. ginseng, P. quinquefolius, and P. notoginseng of the Panax genus, are similar. However, their pharmacological activities are obviously different. To ensure their safety and efficacy, chemical characteristics of the three CMs were determined using pressurized liquid extraction and HPLC-evaporative light scattering detection. Twelve major saponins, namely notoginsenoside R1, pseudo-ginsenoside F11, ginsenosides Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, Rb3, Rd, and Rg3 were also quantitatively compared among the three CMs. The contents of total investigated saponins varied considerably, by up to 4-14-fold, between the highest (P. notoginseng, 82.8-136.5 mg/g) and the lowest values (P. ginseng, 10.0-21.1 mg/g). Hierarchical clustering analysis based on the characteristics of 11 investigated saponins (except ginsenoside Rb3) and notoginsenoside R1, pseudo-ginsenoside F11, and the ratio of ginsenoside Rg1/Rb1 and Rg1/Re showed that 56 tested samples were divided into three main clusters in accordance with the three Panax species. Similarity evaluation of chromatograms was also performed using "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A)". The results showed that a high degree of similarity existed within individual clusters, but a low degree between the clusters, which could be used for quality control of the three CMs.