Hongsheng Tan

Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4

Cardiac progenitor cells derived from adult heart have emerged as one of the most promising stem cell types for cardiac protection and repair. Exosomes are known to mediate cell–cell communication by transporting cell-derived proteins and nucleic acids, including various microRNAs (miRNAs). Here we investigated the cardiac progenitor cell (CPC)-derived exosomal miRNAs on protecting myocardium under oxidative stress. Sca1+CPCs-derived exosomes were purified from conditional medium, and identified by nanoparticle trafficking analysis (NTA), transmission electron microscopy and western blotting using CD63, CD9 and Alix as markers. Exosomes production was measured by NTA, the result showed that oxidative stress-induced CPCs secrete more exosomes compared with normal condition. Although six apoptosis-related miRNAs could be detected in two different treatment-derived exosomes, only miR-21 was significantly upregulated in oxidative stress-induced exosomes compared with normal exosomes. The same oxidative stress could cause low miR-21 and high cleaved caspase-3 expression in H9C2 cardiac cells. But the cleaved caspase-3 was significantly decreased when miR-21 was overexpressed by transfecting miR-21 mimic. Furthermore, miR-21 mimic or inhibitor transfection and luciferase activity assay confirmed that programmed cell death 4 (PDCD4) was a target gene of miR-21, and miR-21/PDCD4 axis has an important role in anti-apoptotic effect of H9C2 cell. Western blotting and Annexin V/PI results demonstrated that exosomes pre-treated H9C2 exhibited increased miR-21 whereas decreased PDCD4, and had more resistant potential to the apoptosis induced by the oxidative stress, compared with non-treated cells. These findings revealed that CPC-derived exosomal miR-21 had an inhibiting role in the apoptosis pathway through downregulating PDCD4. Restored miR-21/PDCD4 pathway using CPC-derived exosomes could protect myocardial cells against oxidative stress-related apoptosis. Therefore, exosomes could be used as a new therapeutic vehicle for ischemic cardiac disease.

Bioassay-Guided Isolation of Prenylated Xanthones and Polycyclic Acylphloroglucinols from the Leaves of Garcinia nujiangensis

Bioassay-guided fractionation of the acetone extract of the leaves of Garcinia nujiangensis resulted in the isolation of two new prenylated xanthones, nujiangexanthones A (1) and B (2), three new polycyclic polyprenylated acylphloroglucinols, nujiangefolins A-C (3-5), and 10 known related analogues. The structures of compounds 1-5 were elucidated by interpretation of their spectroscopic data. Compounds 3 and 4 are unusual polycyclic polyprenylated acylphloroglucinols in which the enol hydroxy group forms a six-membered ring with a benzene ring carbon. The compounds isolated were evaluated for their cytotoxic effects against 11 cancer cell lines and immortalized MIHA normal liver cells, and the test substances demonstrated selectivity toward the cancer cells. Isojacareubin (6) was found to be the most potent cytotoxic compound of those tested.

Guttiferone K induces autophagy and sensitizes cancer cells to nutrient stress-induced cell death.

BACKGROUND Medicinal plants have long been an excellent source of pharmaceutical agents. Autophagy, a catabolic degradation process through lysosomes, plays an important role in tumorigenesis and cancer therapy. PURPOSE Through a screen designed to identify autophagic regulators from a library of natural compounds, we found that Guttiferone K (GUTK) can activate autophagy in several cancer cell lines. The objective of this study is to investigate the mechanism by which GUTK sensitizes cancer cells to cell death in nutrient starvation condition. METHODS Cell death analysis was performed by propidium iodide staining with flow cytometry or Annexin V-FITC/PI staining assay. DCFH-DA staining was used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Cell viability was measured by MTT assay. RESULTS Exposure to GUTK was observed to markedly induce GFP-LC3 puncta formation and activate the accumulation of LC3-II and the degradation of p62 in HeLa cells, suggesting that GUTK is an autophagy inducer. Importantly, hydroxychloroquine, an autophagy inhibitor, was found to significantly prevent GUTK-induced cell death in nutrient starvation conditions, suggesting that the cell death observed is largely dependent on autophagy. We further provide evidence that GUTK inhibits Akt phosphorylation, thereby inhibiting the mTOR pathway in cancer cells during nutrient starvation. In addition, GUTK causes the accumulation of reactive oxygen species (ROS) and the phosphorylation of JNK in EBSS, which may mediate both autophagy and apoptosis. CONCLUSION These data indicate that GUTK sensitizes cancer cells to nutrient stress-induced cell death though Akt/mTOR dependent autophagy pathway.

Simultaneous determination of ten compounds in rat plasma by UPLC-MS/MS: Application in the pharmacokinetic study of Ma-Zi-Ren-Wan

Ma-Zi-Ren-Wan (MZRW) is a classic Chinese formula which has been used to treat human constipation in China for over 2000 years. In order to make good and rational use of this formula in the future, this paper presents the first attempt to track the pharmacokinetic features of MZRW in rat using rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Ten chemical components of MZRW, namely, rhein, emodin, aloe emodin, hesperidin, naringin, amygdalin, albiflorin, paeoniflorin, magnolol and honokiol, were simultaneously determined in rat plasma after a single oral administration (10g/kg body weight) of MZRW to rats. Geniposide and liquiritin were used as internal standards. The separation was performed on a Waters ACQUITY BEH C18 column (100mm×2.1mm, 1.7μm). The detection was conducted by multiple-reaction monitoring (MRM) in negative ionization mode. Two highest abundant MRM transitions without interference were optimized for each analyte. This method was well validated in terms of linearity, precision, accuracy, recovery, matrix effect and stability. All calibration curves had good linearity (r(2)>0.995) over the concentration range from 3.9 to 125.0ng/mL for emodin, 3.9-500.0ng/mL for amygdalin, 2.0-4000.0ng/mL for naringin and hesperidin, 3.9-2000.0ng/mL for magnolol, 7.8-2000.0ng/mL for rhein and 3.9-4000.0ng/mL for albiflorin, paeoniflorin, aloe emodin and honokiol. The intra-day and inter-day precision (relative standard deviation) was within 15%, the accuracy (relative error) ranged from -13.6% to 15.1%, and the lower limit of quantification in plasma ranged between 2.0ng/mL and 7.8ng/mL. Extraction recovery, matrix effect and stability were satisfactory. The validated method was successfully applied to a pharmacokinetic study of these ten compounds after oral administration of MZRW to rats. The pharmacokinetic parameters of each compound can facilitate clinical studies in the future.

Distinguishing Radix Angelica sinensis from different regions by HS-SFME/GC–MS

An automated headspace solvent free microextraction (HS-SFME) based gas chromatography/mass spectrometry (GC/MS) was developed for discrimination of Radix Angelica sinensis (RAS) from different cultivation regions. The MS data were subjected to principal component analysis (PCA) and hierarchical clustering analysis (HCA) to rapidly find the potential characteristic components of RAS from top-geoherb region and non top-geoherb region. Totally, fifty-one volatile organic compounds (VOCs) were identified, in which β-ocimene, α-pinene, 3-methylbutanal, heptanes, butanal were identified as potential markers for distinguishing RAS from top-geoherb region and non top-geoherb region. Sulphur dioxide was detected in some commercial RAS samples, which implied that sulphur-fumigation might be the main reason for the quality inconsistencies of commercial RAS samples. These results suggested that RAS from top-geoherb region and non-top geoherb region could be discriminated by the method. And characteristic chemical markers found in current study can be used for ensuring consistent quality of top-geoherb of RAS.

Cambogin Induces Caspase-Independent Apoptosis through the ROS/JNK Pathway and Epigenetic Regulation in Breast Cancer Cells.

Cambogin is a polycyclic polyprenylated acylphoroglucinol (PPAP) from the Garcinia genus, which has been used traditionally for cancer treatment across Southeastern Asia. In this study, we found that cambogin inhibited breast cancer cell proliferation and induced cell apoptosis in vitro. Cambogin induced the activation of the caspase-independent mitochondrial apoptotic pathway, as indicated by an increase in the ratio of Bax/Bcl-2 and the nuclear translocation of apoptosis inducing factor (AIF). Two-dimensional gel electrophoresis and mass spectrometry revealed that the expression of proteins involving in the radical oxygen species (ROS) pathway was among the most affected upon cambogin treatment. Cambogin enhanced cellular ROS production, and induced the activation of the ASK1–MKK4/MKK7–JNK/SAPK signaling pathway. Pretreatment with ROS scavenger N-acetylcysteine (NAC), an antioxidant, or the JNK inhibitor SP600125 was able to restore cell viability in the presence of cambogin. Importantly, cambogin treatment led to the activation of activating transcription factor-2 (ATF-2) and the trimethylation of histone H3K9 in the activator protein 1 (AP-1) binding region of the Bcl-2 gene promoter. Finally, cambogin exhibited a potential antitumor effect in MCF-7 breast cancer xenografts without apparent toxicity. Taken in conjunction, the present study indicates that cambogin can induce breast adenocarcinoma cell apoptosis and therefore represents therapeutic potential for cancer treatment. Mol Cancer Ther; 14(7); 1738–49. ©2015 AACR.

The natural compound Guttiferone F sensitizes prostate cancer to starvation induced apoptosis via calcium and JNK elevation.

BackgroundIn a cytotoxicity screen in serum-free medium, Guttiferone F showed strong growth inhibitory effect against prostate cancer cells.MethodsProstate cancer cells LNCaP and PC3 were treated with Guttiferone F in serum depleted medium. Sub-G1 phase distributions were estimated with flow cytometry. Mitochondrial disruption was observed under confocal microscope using Mitotracker Red staining. Gene and protein expression changes were detected by real-time PCR and Western blotting. Ca2+ elevation was examined by Fluo-4 staining under fluorescence microscope. PC3 xenografts in mice were examined by immunohistochemical analysis.ResultsGuttiferone F had strong growth inhibitory effect against prostate cancer cell lines under serum starvation. It induced a significant increase in sub-G1 fraction and DNA fragmentation. In serum-free medium, Guttiferone F triggered mitochondria dependent apoptosis by regulating Bcl-2 family proteins. In addition, Guttiferone F attenuated the androgen receptor expression and phosphorylation of ERK1/2, while activating the phosphorylation of JNK and Ca2+ flux. Combination of caloric restriction with Guttiferone F in vivo could increase the antitumor effect without causing toxicity.ConclusionsGuttiferone F induced prostate cancer cell apoptosis under serum starvation via Ca2+ elevation and JNK activation. Combined with caloric restriction, Guttiferone F exerted significant growth inhibition of PC3 cells xenograft in vivo. Guttiferone F is therefore a potential anti-cancer compound.

Identification and characterization of anticancer compounds targeting apoptosis and autophagy from Chinese native Garcinia species.

Natural compounds from medicinal plants are important resources for drug development. Active compounds targeting apoptosis and autophagy are candidates for anti-cancer drugs. In this study, we collected Garcinia species from China and extracted them into water or ethanol fractions. Then, we performed a functional screen in search of novel apoptosis and autophagy regulators. We first characterized the anti-proliferation activity of the crude extracts on multiple cell lines. HeLa cells expressing GFP-LC3 were used to examine the effects of the crude extracts on autophagy. Their activities were confirmed by Western blots of A549 and HeLa cells. By using bioassay guided fractionation, we found that two caged prenylxanthones from Garcinia bracteata, neobractatin and isobractatin, can significantly induce apoptosis and inhibit autophagy. Our results suggest that different Garcinia species displayed various degrees of toxicity on different cancer cell lines. Furthermore, the use of a high content screening assay to screen natural products was an essential method to identify novel autophagy regulators.

Oblongifolin C inhibits metastasis by up-regulating keratin 18 and tubulins

Tumor metastasis is the main cause of cancer-related patient death. In this study, we performed a wound healing migration screen to search for a metastatic inhibitor within our library of natural compounds. We found that oblongifolin C (OC), a natural compound extracted from Garcinia yunnanensis Hu, is an effective inhibitor of metastasis in human esophageal squamous carcinoma Eca109 cells. The transwell migration and matrigel invasion assay results also showed that OC inhibits the migration of Eca109 cells and HepG2 cells. OC can increase the expression of tubulin, indicating that OC inhibits metastasis via tubulin aggregation. In addition, the Western blotting, real-time PCR, and immunostaining results indicated that OC increases the expression of keratin18. Furthermore, the knockdown of keratin 18 by small interfering RNAs inhibited the expression of tubulin and increased the metastasis of cancer cells, suggesting that keratin 18 is the upstream signal of tubulin and plays a vital role in metastasis. A subsequent study in a tail vein injection metastasis model showed that OC can significantly inhibit pulmonary metastasis, as revealed by immunohistochemistry staining. Taken together, our results suggest that OC inhibits metastasis through the induction of the expression of keratin 18 and may be useful in cancer therapy.

Chemical comparison of two dosage forms of Hemp Seed Pills by UHPLC-Q-ToF–MS/MS and multivariate statistical techniques

Hemp seed soft gel capsule (HSSGC) is a modernised dosage form that is derived from a traditional Chinese patent medicine, Hemp Seed Pills (HSP). Two dosage forms claim the same therapeutic effects; however, their chemical components and chemical equivalency are unclear. In the present study, an ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-ToF-MS)-based chemical profiling approach was proposed to rapidly evaluate the chemical differences between HSP and HSSGC as model dosage forms. Samples of the two dosage forms were subjected to UHPLC-ToF-MS analysis. The datasets of retention time (TR) and mass-to-charge ratio (m/z) pairs, ion intensities and sample codes were processed with principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) to holistically compare the difference between these two dosage form samples. A clear classification trend was observed in the score plot, and a loading bi-plot was generated in which the variables are correlated with the group and the samples that were observed. The important chemical components that caused differences among the samples were explored with a Variables Importance Projection (VIP) index. Using the proposed approach, global chemical differences were found between the two dosage forms and among samples of the same dosage form. The most important components that are related to the differences were identified and most of them were attributed to Fructus Aurantii Immaturus. It is suggested that this newly established approach could be used for pre-clinical trial chemical equivalence study or the quality evaluation of the traditional medicinal products with large variations in quality.