Lateral interactions between CD276 and CD147 are essential for stemness in breast cancer: a novel insight from proximal proteome analysis.

Oncogenic cell-surface membrane proteins contribute to the phenotypic and functional characteristics of cancer stem cells (CSCs). We employed a proximity-labeling proteomic approach to quantitatively analyze the cell-surface membrane proteins in close proximity to CD147 in CSCs. Furthermore, we compared CSCs to non-CSCs to identify CSC-specific cell-surface membrane proteins that are closely interact with CD147 and revealed that lateral interaction between CD147 and CD276 concealed within the lipid raft microdomain in CSCs, confers resistance to docetaxel, a commonly used chemotherapy agent for various cancer types, including metastatic breast cancer. Moreover, we investigated the clinical relevance of CD147 and CD276 co-expression in HER2+ breast cancer (BC) and triple-negative breast cancer patients who underwent chemotherapy. We observed poor disease-free survival and Overall survival rates in patients of CD147 and CD276 (p = 0.04 and 0.08, respectively). Subsequent immunohistochemical analysis in independent cohorts of HER2+ BC support for the association between co-expression of CD147 and CD276 and a poor response to chemotherapy. Collectively, our study suggests that the lateral interaction between CD147 and its proximal partners, such as CD276, may serve as a poor prognostic factor in BC and a predictive marker for the critical phenotypic determinant of BC stemness.

Korean Red Ginseng suppresses bisphenol A-induced expression of cyclooxygenase-2 and cellular migration of A549 human lung cancer cell through inhibition of reactive oxygen species.

BACKGROUND: Korean Red Ginseng (KRG) is a natural product with antiinflammatory and anticarcinogenic effects. We have previously reported that the endocrine-disrupting compound bisphenol A (BPA)-induced cyclooxygenase-2 (COX-2) via nuclear translocation of nuclear factor-kappa B (NF-κB) and activation of mitogen-activated protein kinase and promoted the migration of A549. Here, in this study, we assessed the protective effect of KRG on the BPA-induced reactive oxygen species (ROS) and expression of COX-2 and matrix metalloproteinase-9 (MMP-9) in A549 cells. METHODS: The effects of KRG on the upregulation of ROS production and COX-2 and MMP-9 expression by BPA were evaluated by fluorescence-activated cell sorting (FACs) analysis, quantitative reverse transcription polymerase chain reaction, and western blotting. Antimigration ability by KRG was evaluated by migration assay in A549 cells. RESULTS: KRG significantly suppressed the BPA-induced COX-2, the activity of NF-κB, the production of ROS, and the migration of A549 cells. These effects led to the downregulation of the expression of MMP-9. CONCLUSIONS: Overall, our results suggest that KRG exerts an antiinflammatory effect on BPA-treated A549 cells via the suppression of ROS and downregulation of NF-κB activation and COX-2 expression which leads to a decrease in cellular migration and MMP-9 expression. These results provide a new possible therapeutic application of KRG to protect BPA-induced possible inflammatory disorders.

Biological Effects of Korean Red Ginseng Polysaccharides in Aged Rat Using Global Proteomic Approach.

Much has been written on the physiological benefits of Korean Red Ginseng (KRG). Among its various components, ginsenosides have been widely investigated for their various pharmacological effects. However, polysaccharides are a major KRG component that has not received scrutiny similar to that of ginsenosides. The present study aims to fill that gap in the existing literature and to investigate the possible functions of polysaccharide in KRG. The researchers evaluated proteomic changes in non-saponin fractions with rich polysaccharides (NFP) in KRG. Based on the serum analysis, proteomics analysis of the liver and the spleen was additionally conducted to identify related functions. We validated the suggested functions of NFP with the galactosamine-induced liver injury model and the cyclophosphamide-induced immunosuppression model. Then, we evaluated the antimetastatic potential of NFP in the lungs. Further proteomics analysis of the spleen and liver after ingestion confirmed functions related to immunity, cancer, hepatoprotection, and others. Then, we validated the suggested corresponding functions of the NFP in vivo model. NFP showed immune-enhancing effects, inhibited melanoma cell metastasis in the lung, and decreased liver damage. The results show that using the proteomic approach uncovers the potential effects of polysaccharides in KRG, which include enhancing the immune system and protecting the liver.

Physiological and pharmacological features of the non-saponin components in Korean Red Ginseng.

Panax ginseng, a medicinal plant, has been used as a blood-nourishing tonic for thousands of years in Asia, including Korea and China. P. ginseng exhibits adaptogen activity that maintains homeostasis by restoring general biological functions and non-specifically enhancing the body's resistance to external stress. Several P. ginseng effects have been reported. Korean Red Ginseng, in particular, has been reported in both basic and clinical studies to possess diverse effects such as enhanced immunity, fatigue relief, memory, blood circulation, and anti-oxidation. Moreover, it also protects against menopausal symptoms, cancer, cardiac diseases, and neurological disorders. The active components found in most Korean Red Ginseng varieties are known to include ginsenosides, polysaccharides, peptides, alkaloids, polyacetylene, and phenolic compounds. In this review, the identity and bioactivity of the non-saponin components of Korean Red Ginseng discovered to date are evaluated and the components are classified into polysaccharide and nitrogen compounds (protein, peptide, amino acid, nucleic acid, and alkaloid), as well as fat-soluble components such as polyacetylene, phenols, essential oils, and phytosterols. The distinct bioactivity of Korean Red Ginseng was found to originate from both saponin and non-saponin components rather than from only one or two specific components. Therefore, it is important to consider saponin and non-saponin elements together.

Korean Red ginseng extract inhibits glioblastoma propagation by blocking the Wnt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Korean Red ginseng extract (RG) is one of the most widely used traditional health functional food in Asia, which invigorates immunity and vital energy. RG have been suggested to inhibit proliferation, invasion, and inflammation in several cancer cell lines. Correspondingly, clinical studies have raised the possibility that RG could augment therapeutic efficacy in cancer patients. However, little is known about the anti-cancer effects of RG in glioblastoma (GBM), the most common and aggressive brain tumor for which effective therapeutic regimens need to be developed. AIM OF THIS STUDY: Here, we assessed the in vivo and in vitro anti-cancer properties of RG in a patient-derived xenograft mouse model and GBM stem cell (GSC) line. MATERIALS AND METHODS: We evaluated the anti-cancer effects of RG in patient-derived GBM xenograft mice with and without combined concurrent chemo- and radiation therapy (CCRT). Furthermore, we verified the in vitro effects of RG on the proliferation, cell death, and stem cell-like self-renewal capacity of cancer cells. Finally, we investigated the signaling pathway affected by RG, via which its anti-cancer effects were mediated. RESULTS: When combined with CCRT, RG impeded GBM progression by reducing cancer cell proliferation and ionized calcium-binding adapter molecule 1 (IBA1)-positive immune cell recruitment. The anti-cancer effects of RG were mediated by Rg3 and Rh2 ginsenosides. Rg3 promoted cell death while Rh2 did not. Furthermore, both Rg3 and Rh2 reduced cell viability and self-renewal capacity of GSCs by inhibiting Wnt/ß-catenin signaling. CONCLUSION: Therefore, our observations imply that RG could be applied to the GBM patients in parallel with CCRT to enhance therapeutic efficacy.

DATS sensitizes glioma cells to TRAIL-mediated apoptosis by up-regulation of death receptor 5 via ROS.

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising anticancer reagent for antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, due to repeat treat to cancer cells, highlighting the need for strategies to overcome TRAIL resistance. Here we present that in combination with diallyl trisulfide (DATS), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Surprisingly, we found that subtoxic concentrations of DATS significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. DATS dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. In addition, DATS enhances TRAIL-induced apoptosis through the downregulation of anti-apoptotic protein (Mcl-1) and the upregulation of DR5 receptors through actions on the ROS- induced-p53.

Protein-Induced Pluripotent Stem Cells Ameliorate Cognitive Dysfunction and Reduce Aß Deposition in a Mouse Model of Alzheimer's Disease.

Transplantation of stem cells into the brain attenuates functional deficits in the central nervous system via cell replacement, the release of specific neurotransmitters, and the production of neurotrophic factors. To identify patient-specific and safe stem cells for treating Alzheimer's disease (AD), we generated induced pluripotent stem cells (iPSCs) derived from mouse skin fibroblasts by treating protein extracts of embryonic stem cells. These reprogrammed cells were pluripotent but nontumorigenic. Here, we report that protein-iPSCs differentiated into glial cells and decreased plaque depositions in the 5XFAD transgenic AD mouse model. We also found that transplanted protein-iPSCs mitigated the cognitive dysfunction observed in these mice. Proteomic analysis revealed that oligodendrocyte-related genes were upregulated in brains injected with protein-iPSCs, providing new insights into the potential function of protein-iPSCs. Taken together, our data indicate that protein-iPSCs might be a promising therapeutic approach for AD. Stem Cells Translational Medicine 2017;6:293-305.

Metformin enhances TRAIL-induced apoptosis by Mcl-1 degradation via Mule in colorectal cancer cells.

Metformin is an anti-diabetic drug with a promising anti-cancer potential. In this study, we show that subtoxic doses of metformin effectively sensitize human colorectal cancer (CRC) cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which induces apoptosis. Metformin alone did not induce apoptosis, but significantly potentiated TRAIL-induced apoptosis in CRC cells. CRC cells treated with metformin and TRAIL showed activation of the intrinsic and extrinsic pathways of caspase activation. We attempted to elucidate the underlying mechanism, and found that metformin significantly reduced the protein levels of myeloid cell leukemia 1 (Mcl-1) in CRC cells and, the overexpression of Mcl-1 inhibited cell death induced by metformin and/or TRAIL. Further experiments revealed that metformin did not affect mRNA levels, but increased proteasomal degradation and protein stability of Mcl-1. Knockdown of Mule triggered a significant decrease of Mcl-1 polyubiquitination. Metformin caused the dissociation of Noxa from Mcl-1, which allowed the binding of the BH3-containing ubiquitin ligase Mule followed by Mcl-1ubiquitination and degradation. The metformin-induced degradation of Mcl-1 required E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Our study is the first report indicating that metformin enhances TRAIL-induced apoptosis through Noxa and favors the interaction between Mcl-1 and Mule, which consequently affects Mcl-1 ubiquitination.

Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line.

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO2) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/α-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.

Subcellular tissue proteomics of hepatocellular carcinoma for molecular signature discovery.

Hepatocellular carcinoma (HCC) is one of the leading causes of mortality from solid organ malignancy worldwide. Because of the complexity of proteins within liver cells and tissues, the discovery of therapeutic targets of HCC has been difficult. To investigate strategies for decreasing the complexity of tissue samples for detecting meaningful protein mediators of HCC, we employed subcellular fractionation combined with 1D-gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis. Moreover, we utilized a statistical method, namely, the Power Law Global Error Model (PLGEM), to distinguish differentially expressed proteins in a duplicate proteomic data set. Mass spectrometric analysis identified 3045 proteins in nontumor and HCC from cytosolic, membrane, nuclear, and cytoskeletal fractions. The final lists of highly differentiated proteins from the targeted fractions were searched for potentially translocated proteins in HCC from soluble compartments to the nuclear or cytoskeletal compartments. This analysis refined our targets of interest to include 21 potential targets of HCC from these fractions. Furthermore, we validated the potential molecular targets of HCC, MATR3, LETM1, ILF2, and IQGAP2 by Western blotting, immunohistochemisty, and immunofluorescent microscopy. Here we demonstrate an efficient strategy of subcellular tissue proteomics toward molecular target discovery of one of the most complicated human disease, HCC.

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method.

BACKGROUND: The measurement of serum hepcidin, a peptide hormone that regulates iron metabolism, is clinically important to the understanding of iron homeostasis in health and disease. To date, the quantification of serum hepcidin levels by conventional immunological detection methods has proven problematic due to challenges in obtaining high quality antibodies which demonstrate good reproducibility. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been employed recently for more sensitive quantification of hepcidin; however, this method has high background levels and therefore less than optimal specificity. METHODS: In order to increase the specificity of the mass spectrometry based assay, we developed a robust, ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS) protocol using multiple selected reaction monitoring (mSRM) for quantification of hepcidin levels in urine and serum of human subjects. With this assay, we assessed levels of hepcidin before and for up to 8 h after oral ingestion of ferrous sulfate in ten adult human subjects without known disease. RESULTS: The linear response of hepcidin quantitation on each instrument was measured, and the correlation coefficients of these calibrations were r(2)=0.9512±0.0202 (n=5) for urine and r(2)=0.9709±0.0291 (n=5) for serum [r(2)=mean±SD]. Compared to baseline, the levels of urinary hepcidin between 2-4 h and 4-8 h of both women and men showed significant increases with p<0.05 and p<0.001, respectively. The levels of serum hepcidin between 4 h and 8 h in both women and men showed significant increases, compared with baseline values, with both p<0.01. Interestingly, we also observed some degree of oscillation of levels, occurring at later time points. CONCLUSIONS: We have developed and validated a new method for measuring hepcidin concentrations in human serum and urine and used it to demonstrate early increases with iron supplement in both urinary and serum levels of hepcidin, which return to baseline levels, except in urine samples from men.

Discovery of putative pancreatic cancer biomarkers using subcellular proteomics.

Pancreatic cancer (PC) is a highly aggressive disease that frequently remains undetected until it has progressed to an advanced, systemic stage. Successful treatment of PC is hindered by the lack of early detection. The application of proteomic analysis to PC combined with subcellular fractionation has introduced new possibilities in the field of biomarker discovery. We utilized matched pairs of pancreas tumor and non-tumor pancreas from patients undergoing tumor resection. The tissues were treated to obtain cellular protein fractions corresponding to cytosol, membrane, nucleus and cytoskeleton. The fractions were then separated by molecular weight and digested with trypsin, followed by liquid chromatography and tandem mass spectrometry. The spectra obtained were searched using Sequest engine and combined into a single analysis file to obtain a semi-quantitative number, spectral count, using Scaffold software. We identified 2393 unique proteins in non-tumor and cancer pancreas. Utilizing PLGEM statistical analysis we determined 104 proteins were significantly changed in cancer. From these, we further validated four secreted proteins that are up-regulated in cancer and have potential for development as minimally-invasive diagnostic markers. We conclude that subcellular fractionation followed by gel electrophoresis and tandem mass spectrometry is a powerful strategy for identification of differentially expressed proteins in pancreatic cancer.

Inhibitory activity of ginsenosides isolated from processed ginseng on platelet aggregation.

Seven ginsenosides, namely Rg6 (1), F4 (2), Rk3 (3), Rh4 (4), Rs3 (5), Rs4 (6) and Rs5 (7) isolated from processed ginseng were evaluated for their effects on platelet aggregation induced by adenosine diphosphate (ADP), collagen, arachidonic acid (AA) and U46619 (thromboxane A2 mimetic drug). Ginsenosides Rg6, F4 and Rk3 showed inhibitory activity (IC50 = 76 microM, 114 microM and 128 microM, respectively) on AA-induced platelet aggregation. The corresponding IC50 values were comparable to that of acetylsalicylic acid (ASA) (63 microM). Compared to ASA (IC50 = 468 microM) ginsenosides Rg6, F4, Rk3 and Rh4 were found to be more inhibitive (IC50 = 286 microM, 87 microM, 187 microM and 119 microM, respectively) against U46619-induced aggregation. On the other hand, most of the ginsenosides (Rg6, F4, Rh4, Rs3, Rs5) showed negligible effects on ADP and collagen-induced platelet aggregation. The acetylated ginsenosides (Rs3, Rs4 and Rs5) had only mild effects on aggregation induced by four stimulators.

Platelet antiaggregating activity of ginsenosides isolated from processed ginseng.

Four dammarane glycosides, namely ginsenosides Rk1 (1), Rg5 (2), 20(S)-Rg3 (3), and 20(R)-Rg3 (4), isolated from a new processed ginseng, were evaluated for their inhibitory activity against platelet aggregation induced by adenosine diphosphate (ADP), collagen, arachidonic acid (AA) and U46619 (thromboxane A2 mimetic agent). Ginsenoside Rk1 and Rg5 inhibited AA-induced platelet aggregation in a dose dependent manner. Their activity against AA-induced platelet aggregation were found to be 8-22 fold higher than that of a known antiplatelet drug acetylsalicylic acid (ASA). They also inhibited U46619-induced platelet aggregation. Ginsenoside 20(S)-Rg3 and 20(R)-Rg3 showed mild inhibitory activity against AA and U46619-induced aggregation.

Augmentation of U46619 induced human platelet aggregation by aspirin.

Aspirin is known to suppress platelet function markedly. However, aspirin at concentrations higher than 1 mM was observed to augment 1.3 microM U46619 (a stable thromboxane receptor (TP receptor) agonist) induced human platelet aggregation in this study. Moreover, at a concentration as low as 250 microM aspirin increased the aggregation induced by U46619 in 13% of normal and healthy individuals. The degree of platelet aggregation and the amount of ATP release were enhanced in U46619 stimulated platelet rich plasma by the addition of aspirin (>250 microM). U46619 was previously reported to inhibit forskolin-stimulated adenyl cyclase and to reduce the cAMP formation. Both of the augmentation effects of aspirin on U46619-induced aggregation and ATP release were blocked by MeSAMP, a P2Y(12) receptor antagonist. U46619 induced aggregation was suppressed by the addition of ADP scavenger (CP/CPK) with no significant change on ATP measured and the effect of CP/CPK could not be reversed by aspirin. In addition, aspirin augmented the inhibitory effect of U46619 on the cAMP production. Our present results suggested that the potentiation effect of aspirin on U46619 induced aggregation was related with the secreted ADP and the subsequent P2Y(12)/Gi related signaling.

Anti-cancer effect of Betulin on a human lung cancer cell line: a pharmacoproteomic approach using 2 D SDS PAGE coupled with nano-HPLC tandem Mass Spectrometry.

Betulin is a representative compound of Betula platyphylla, a tree species belonging to the Betulaceae family. In this investigation, we revealed that betulin showed anticancer activity on human lung cancer A549 cells by inducing apoptosis and changes in protein expression profiles were observed. Upon flow cytometry analysis, the surface of betulin-treated cells was found to be annexin-V positive and propidium iodide (PI) negative, which indicated that the cells were apoptotic. In order to identify the molecular players involved in betulin-induced apoptosis, cellular proteins were applied to two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2 D SDS PAGE) for differential proteomic analysis. As a result, four downregulated proteins and three upregulated proteins were identified by nano-HPLC MS/MS. The four downregulated proteins were poly(rC)-binding protein 1, isoform 1 of 3-hydroxyacyl-CoA dehydrogenase type 2, heat shock protein 90-alpha 2, and enoyl-CoA hydratase; the three upregulated proteins were aconitate hydratase, malate dehydrogenase, and splicing factor arginine/serine-rich 1. These differentially expressed proteins explained the cytotoxicity of betulin against human lung cancer A549 cells, and the proteomic approach was thus shown to be a potential tool for understanding the pharmacological activities of pharmacophores.