Heteronemin and Tetrac Induce Anti-Proliferation by Blocking EGFR-Mediated Signaling in Colorectal Cancer Cells.

Overexpressed EGFR and mutant K-Ras play vital roles in therapeutic resistance in colorectal cancer patients. To search for an effective therapeutic protocol is an urgent task. A secondary metabolite in the sponge Hippospongia sp., Heteronemin, has been shown to induce anti-proliferation in several types of cancers. A thyroxine-deaminated analogue, tetrac, binds to integrin αvß3 to induce anti-proliferation in different cancers. Heteronemin- and in combination with tetrac-induced antiproliferative effects were evaluated. Tetrac enhanced heteronemin-induced anti-proliferation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC). Heteronemin and tetrac arrested cell cycle in different phases. Combined treatment increased the cell accumulation in sub-G1 and S phases. The combined treatment also induced the inactivation of EGFR signaling and downregulated the phosphorylated ERK1/2 protein in both cell lines. Heteronemin and the combination showed the downregulation of the phosphorylated and total PI3K protein in HT-29 cells (KRAS WT CRC). Results by NanoString technology and RT-qPCR revealed that heteronemin and combined treatment suppressed the expression of EGFR and downstream genes in HCT-116 cells (KRAS MT CRC). Heteronemin or combined treatment downregulated genes associated with cancer progression and decreased cell motility. Heteronemin or the combined treatment suppressed PD-L1 expression in both cancer cell lines. However, only tetrac and the combined treatment inhibited PD-L1 protein accumulation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC), respectively. In summary, heteronemin induced anti-proliferation in colorectal cancer cells by blocking the EGFR-dependent signal transduction pathway. The combined treatment further enhanced the anti-proliferative effect via PD-L1 suppression. It can be an alternative strategy to suppress mutant KRAS resistance for anti-EGFR therapy.

Role of Integrin αvß3 in Doxycycline-Induced Anti-Proliferation in Breast Cancer Cells.

Doxycycline, an antibiotic, displays the inhibition of different signal transduction pathways, such as anti-inflammation and anti-proliferation, in different types of cancers. However, the anti-cancer mechanisms of doxycycline via integrin αvß3 are incompletely understood. Integrin αvß3 is a cell-surface anchor protein. It is the target for estrogen, androgen, and thyroid hormone and plays a pivotal role in the proliferation, migration, and angiogenic process in cancer cells. In our previous study, thyroxine hormones can interact with integrin αvß3 to activate the extracellular signal-regulated kinase 1/2 (ERK1/2), and upregulate programmed death-ligand 1 (PD-L1) expression. In the current study, we investigated the inhibitory effects of doxycycline on proliferation in two breast cancer cell lines, MCF-7 and MDA-MB-231 cells. Doxycycline induces concentration-dependent anti-proliferation in both breast cancer cell lines. It regulates gene expressions involved in proliferation, pro-apoptosis, and angiogenesis. Doxycycline suppresses cell cyclin D1 (CCND1) and c-Myc which play crucial roles in proliferation. It also inhibits PD-L1 gene expression. Our findings show that modulation on integrin αvß3 binding activities changed both thyroxine- and doxycycline-induced signal transductions by an integrin αvß3 inhibitor (HSDVHK-NH2). Doxycycline activates phosphorylation of focal adhesion kinase (FAK), a downstream of integrin, but inhibits the ERK1/2 phosphorylation. Regardless, doxycycline-induced FAK phosphorylation is blocked by HSDVHK-NH2. In addition, the specific mechanism of action associated with pERK1/2 inhibition via integrin αvß3 is unknown for doxycycline treatment. On the other hand, our findings indicated that inhibiting ERK1/2 activation leads to suppression of PD-L1 expression by doxycycline treatment. Furthermore, doxycycline-induced gene expressions are disturbed by a specific integrin αvß3 inhibitor (HSDVHK-NH2) or a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) kinase (MAPK/ERK, MEK) inhibitor (PD98059). The results imply that doxycycline may interact with integrin αvß3 and inhibits ERK1/2 activation, thereby regulating cell proliferation and downregulating PD-L1 gene expression in estrogen receptor (ER)-negative breast cancer MDA-MB-231 cells.

Enhancement of T2* Weighted MRI Imaging Sensitivity of U87MG Glioblastoma Cells Using γ-Ray Irradiated Low Molecular Weight Hyaluronic Acid-Conjugated Iron Nanoparticles.

INTRODUCTION: It has been reported that low-molecular-weight hyaluronic acid (LMWHA) exhibits a potentially beneficial effect on cancer therapy through targeting of CD44 receptors on tumor cell surfaces. However, its applicability towards tumor detection is still unclear. In this regard, LMWHA-conjugated iron (Fe3O4) nanoparticles (LMWHA-IONPs) were prepared in order to evaluate its application for enhancing the T2* weighted MRI imaging sensitivity for tumor detection. METHODS: LMWHA and Fe3O4 NPs were produced using γ-ray irradiation and chemical co-precipitation methods, respectively. First, LMWHA-conjugated FITC was prepared to confirm the ability of LMWHA to target U87MG cells using fluorescence microscopy. The hydrodynamic size distribution and dispersion of the IONPs and prepared LMWHA-IONPs were analyzed using dynamic light scattering (DLS). In addition, cell viability assays were performed to examine the biocompatibility of LMWHA and LMWHA-IONPs toward U87MG human glioblastoma and NIH3T3 fibroblast cell lines. The ability of LMWHA-IONPs to target tumor cells was confirmed by detecting iron (Fe) ion content using the thiocyanate method. Finally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging and in vitro magnetic resonance imaging (MRI) were performed to confirm the contrast enhancement effect of LMWHA-IONPs. RESULTS: Florescence analysis results showed that LMWHA-FITC successfully targeted the surfaces of both tested cell types. The ability of LMWHA to target U87MG cells was higher than for NIH3T3 cells. Cell viability experiments showed that the fabricated LMWHA-IONPs possessed good biocompatibility for both cell lines. After co-culturing test cells with the LMWHA-IONPs, detected Fe ion content in the U87MG cells was much higher than that of the NIH3T3 cells in both thiocyanate assays and TOF-SIMs images. Finally, the addition of LMWHA-IONPs to the U87MG cells resulted in an obvious improvement in T2* weighted MR image contrast compared to control NIH3T3 cells. DISCUSSION: Overall, the present results suggest that LMWHA-IONPs fabricated in this study provide an effective MRI contrast agent for improving the diagnosis of early stage glioblastoma in MRI examinations.

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer.

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

Leptin-derived peptides block leptin-induced proliferation by reducing expression of pro-inflammatory genes in hepatocellular carcinoma cells.

The obesity-regulated gene, leptin, is essential for diet. Leptin resistance causes obesity and related diseases. Certain types of diet are able to decrease leptin resistance. However, leptin has been shown to be correlated with inflammation and stimulate proliferation of various cancers. Two synthetic leptin derivatives (mimetics), OB3 and [D-Leu-4]-OB3, show more effective than leptin in reducing obesity and diabetes in mouse models. OB3 inhibits leptin-induced proliferation in ovarian cancer cells. However, effects of these mimetics in hepatocellular carcinoma (HCC) have not been investigated. In the present study, we examined the effects of OB3 and [D-Leu-4]-OB3 on cell proliferation and gene expressions in human HCC cell cultures. In contrast to what was reported for leptin, OB3 and [D-Leu-4]-OB3 reduced cell proliferation in hepatomas. Both OB3 and [D-Leu-4]-OB3 stimulated expression of pro-apoptotic genes. Both compounds also inhibited expressions of pro-inflammatory, proliferative and metastatic genes and PD-L1 expression. In combination with leptin, OB3 inhibited leptin-induced cell proliferation and expressions of pro-inflammation-, and proliferation-related genes. Furthermore, the OB3 peptide inhibited phosphoinositide 3-kinase (PI3K) activation which is essential for leptin-induced proliferation in HCC. These results indicate that OB3 and [D-Leu-4]-OB3 may have the potential to reduce leptin-related inflammation and proliferation in HCC cells.

Thyroid hormone-induced expression of inflammatory cytokines interfere with resveratrol-induced anti-proliferation of oral cancer cells.

Thyroid hormone, L-thyroxine (T4), induces inflammatory genes expressions and promotes cancer growth. It also induces expression of the checkpoint programmed death-ligand 1 (PD-L1), which plays a vital role in cancer progression. On the other hand, resveratrol inhibits inflammatory genes expressions. Moreover, resveratrol increases nuclear inducible cyclooxygenase (COX)-2 accumulation, complexes with p53, and induces p53-dependent anti-proliferation. In this study, we investigated the effect of T4 on resveratrol-induced anti-proliferation in oral cancer. T4 increased the expression and cytoplasmic accumulation of PD-L1. Increased expressions of pro-inflammatory genes, interleukin (IL)-1ß and transforming growth factor (TGF)-ß1, were shown to stimulate PD-L1 expression. T4 stimulated pro-inflammatory and proliferative genes expressions, and oral cancer cells proliferation. In contrast, resveratrol inhibited those genes and activated anti-proliferative genes. T4 retained resveratrol-induced COX-2 in cytoplasm and prevented COX-2 nuclear accumulation when resveratrol treated cancer cells. A specific signal transducer and activator of transcription 3 (STAT3) inhibitor, S31-201, blocked T4-induced inhibition and restored resveratrol-induced nuclear COX-2 accumulation. By inhibiting the T4-activated STAT3 signal transduction axis with S31-201, resveratrol was able to sequentially reestablish COX-2/p53-dependent gene expressions and anti-proliferation. These findings provide a novel understanding of the inhibitory effects of T4 on resveratrol-induced anticancer properties via the sequential expression of PD-L1 and inflammatory genes.

Enhancement by Nano-Diamino-Tetrac of Antiproliferative Action of Gefitinib on Colorectal Cancer Cells: Mediation by EGFR Sialylation and PI3K Activation.

Drug resistance complicates the clinical use of gefitinib. Tetraiodothyroacetic acid (tetrac) and nano-diamino-tetrac (NDAT) have been shown in vitro and in xenografts to have antiproliferative/angiogenic properties and to potentiate antiproliferative activity of other anticancer agents. In the current study, we investigated the effects of NDAT on the anticancer activities of gefitinib in human colorectal cancer cells. ß-Galactoside α-2,6-sialyltransferase 1 (ST6Gal1) catalyzes EGFR sialylation that is associated with gefitinib resistance in colorectal cancers, and this was also investigated. Gefitinib inhibited cell proliferation of HT-29 cells (K-ras wild-type), and NDAT significantly enhanced the antiproliferative action of gefitinib. Gefitinib inhibited cell proliferation of HCT116 cells (K-ras mutant) only in high concentration, and this was further enhanced by NDAT. NDAT enhancedd gefitinib-induced antiproliferation in gefitinib-resistant colorectal cancer cells by inhibiting ST6Gal1 activity and PI3K activation. Furthermore, NDAT enhanced gefitinib-induced anticancer activity additively in colorectal cancer HCT116 cell xenograft-bearing nude mice. Results suggest that NDAT may have an application with gefitinib as combination colorectal cancer therapy.

Inhibitory Effect of Anoectochilus formosanus Extract on Hyperglycemia-Related PD-L1 Expression and Cancer Proliferation.

Traditional herb medicine, golden thread (Anoectochilus formosanus Hayata) has been used to treat various diseases. Hyperglycemia induces generation of reactive oxygen species (ROS) and enhancement of oxidative stress which are risk factors for cancer progression and metastasis. In this study, we evaluated hypoglycemic effect of A. formosanus extracts (AFEs) in an inducible hyperglycemia animal model and its capacity of free-radical scavenging to establish hyperglycemia-related carcinogenesis. AFE reduced blood glucose in hyperglycemic mice while there was no change in control group. The incremental area under blood glucose response curve was decreased significantly in hyperglycemic mice treated with AFE in a dose-dependent manner. AFE and metformin at the same administrated dose of 50 mg/kg showed similar effect on intraperitoneal glucose tolerance test in hyperglycemic mice. Free-radical scavenger capacity of AFE was concentration dependent and 200 µg/ml of AFE was able to reduce more than 41% of the free radical. Treatment of cancer cells with AFE inhibited constitutive PD-L1 expression and its protein accumulation. It also induced expression of pro-apoptotic genes but inhibited proliferative and metastatic genes. In addition, it induced anti-proliferation in cancer cells. The results suggested that AFE not only reduced blood glucose concentration as metformin but also showed its potential use in cancer immune chemoprevention/therapy via hypoglycemic effect, ROS scavenging and PD-L1 suppression.

CME-1, a novel polysaccharide, suppresses iNOS expression in lipopolysaccharide-stimulated macrophages through ceramide-initiated protein phosphatase 2A activation.

CME-1, a novel water-soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti-oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME-1, namely anti-inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)-stimulated RAW 264.7 cells with CME-1 concentration-dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME-1-treated RAW 264.7 cells, LPS-induced IκBα degradation and the phosphorylation of p65, Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)-specific inhibitor, significantly reversed the CME-1-suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up-regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME-1-induced PP2A activation and its subsequent suppression of LPS-activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS-induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME-1. Furthermore, the role of ceramide signalling pathway and anti-oxidative property were also demonstrated in CME-1-mediated inhibition of LPS-activated primary peritoneal macrophages. In conclusion, CME-1 suppressed iNOS expression by up-regulating ceramide-induced PP2A activation and reducing ROS production in LPS-stimulated macrophages. CME-1 is a potential therapeutic agent for treating inflammatory diseases.

Inhibitory mechanisms of CME-1, a novel polysaccharide from the mycelia of Cordyceps sinensis, in platelet activation.

OBJECTIVE: CME-1 is a polysaccharide purified from the mycelia of medicinal mushroom Cordyceps sinensis, its molecular weight was determined to be 27.6 kDa by using nuclear magnetic resonance and gas chromatography-mass spectrometry. The initiation of arterial thromboses is relevant to various cardiovascular diseases (CVDs) and is believed to involve platelet activation. Our recent study exhibited that CME-1 has potent antiplatelet activity via the activation of adenylate cyclase/cyclic AMP ex vivo and in vivo. METHODS: The aggregometry, and immunoblotting were used in this study. RESULTS: In this study, the mechanisms of CME-1 in platelet activation is further investigated and found that CME-1 inhibited platelet aggregation as well as the ATP-release reaction, relative intracellular [Ca(+2)] mobilization, and the phosphorylation of phospholipase C (PLC)γ2 and protein kinase C (PKC) stimulated by collagen. CME-1 has no effects on inhibiting either convulxin, an agonist of glycoprotein VI, or aggretin, an agonist of integrin α2ß1 stimulated platelet aggregation. Moreover, this compound markedly diminished thrombin and arachidonic acid (AA) induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 2, c-Jun N-terminal kinase 1, and Akt. Treatment with SQ22536, an inhibitor of adenylate cyclase, markedly diminished the CME-1-mediated increasing of cyclic AMP level and reversed prostaglandin E1- or CME-1-mediated inhibition of platelet aggregation and p38 MAPK and Akt phosphorylation stimulated by thrombin or AA. Furthermore, phosphodiesterase activity of human platelets was not altered by CME-1. CONCLUSION: The crucial finding of this study is that the antiplatelet activity of CME-1 may initially inhibit the PLCγ2-PKC-p47 cascade, and inhibit PI3-kinase/Akt and MAPK phosphorylation through adenylate cyclase/ cyclic AMP activation, then inhibit intracellular [Ca(+2)] mobilization, and, ultimately, inhibit platelet activation. The novel role of CME-1 in antiplatelet activity indicates that this compound exhibits high therapeutic potential for treating or preventing CVDs.

Anti-proliferative and gene expression actions of resveratrol in breast cancer cells in vitro.

We have used a perfusion bellows cell culture system to investigate resveratrolinduced anti-proliferation/apoptosis in a human estrogen receptor (ER)-negative breast cancer cell line (MDA-MB-231). Using an injection system to perfuse media with stilbene, we showed resveratrol (0.5 - 100 µM) to decrease cell proliferation in a concentration-dependent manner. Comparison of influx and medium efflux resveratrol concentrations revealed rapid disappearance of the stilbene, consistent with cell uptake and metabolism of the agent reported by others. Exposure of cells to 10 µM resveratrol for 4 h daily × 6 d inhibited cell proliferation by more than 60%. Variable extracellular acid-alkaline conditions (pH 6.8 - 8.6) affected basal cell proliferation rate, but did not alter anti-proliferation induced by resveratrol. Resveratrol-induced gene expression, including transcription of the most up-regulated genes and pro-apoptotic p53-dependent genes, was not affected by culture pH changes. The microarray findings in the context of induction of anti-proliferation with brief daily exposure of cells to resveratrol-and rapid disappearance of the compound in the perfusion system-are consistent with existence of an accessible initiation site for resveratrol actions on tumor cells, e.g., the cell surface receptor for resveratrol described on integrin αvß3.

Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP.

INTRODUCTION: CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation. METHODS: The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study. RESULTS: CME-1 (2.3-7.6 µM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B2 (TxB2) and hydroxyl radical (OH(●)) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB2 formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation. CONCLUSION: This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and MAPKs), ultimately inhibiting platelet activation. This novel role of CME-1 indicates that CME-1 exhibits high potential for application in treating and preventing CVDs.

Anti-cancer effects of CME-1, a novel polysaccharide, purified from the mycelia of Cordyceps sinensis against B16-F10 melanoma cells.

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in the invasion and migration of cancer cells. In melanoma, several signaling pathways are constitutively activated. Among these, the mitogen-activated protein kinase (MAPKs) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Therefore, the inhibition of MAPK signaling might be a crucial role for the treatment of melanoma cancer. AIMS: We examined the anticancer effect of CME-1, a novel water-soluble polysaccharide fraction, isolated from Cordyceps sinensis mycelia on B16-F10 melanoma cells. MATERIALS AND METHODS: B16-F10 cells were exposed to different concentrations of CME-1 (250, 500 and 800 µg/ml) for 24 h in 5% CO² incubator at 37°C. Western blot analysis was performed to detect the expression of MMP-1, p-p38 MAPK, p-ERK1/2, and IkB-α in B16-F10 cells. Cell migration test was performed by wound healing migration assay. RESULTS: CME-1 suppresses cell migration in a concentration-dependent manner. Western blotting analysis revealed that CME-1 led to the reduction on the expression levels of MMP-1 and down regulated the expression of phosphorylated extracellular signal-regulated kinase (ERK1/2 and p38 mitogen-activated protein kinase (p38 MAPK). CME-1 restored the IkB-degradation in B16F10 cells. CONCLUSIONS: These results indicate that CME-1 inhibited MMP-1 expressions in B16F10 melanoma cells through either NF-kB or ERK/p38 MAPK down regulation thereby inhibiting B16F10 cell migration. Therefore, we proposed that CME-1 might be developed as a therapeutic potential candidate for the treatment of cancer metastasis.

MALDI-TOF MS analysis of native and permethylated or benzimidazole-derivatized polysaccharides.

MALDI-TOF MS provides rapid and sensitive analyses of larger biomolecules. However, MS analyses of polysaccharide have been reported to have lower sensitivity compared to peptides and proteins. Here, we investigated some polysaccharides chemically derivatized by permethylation and ortho-phenylene diamine (OPD) tagging. Methylated glycan is obviously able to improve the sensitivity for mass spectrometry detection. Oxidative condensation by UV-activation tagging to saccharides by OPD and peptide-OPD also improve the sensitivity of MALDI-TOF MS analyses. Polysaccharides including dextran, glucomannan, arabinoxylan, arabinogalactan and beta-1,3-glucan, isolated from nutritional supplements of Ganoderma lucidum and Saccharomyces pastorianus were measured using MALDI-TOF MS with 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix. These glycans were also derivatized to methylated and benzimidazole-tagged glycans by chemical transformation for molecular weight analysis. The derivatized polysaccharides showed excellent MALDI-TOF MS signal enhancement in the molecular weight range from 1 to 5 kDa. Here, we demonstrate an efficient method to give glycan-benzimidazole (glycan-BIM) derivatives for polysaccharide determination in MALDI-TOF MS. Therefore, permethylated or benzimidazole-derivatized polysaccharides provide a new option for polysaccharide analysis using MALDI-TOF MS.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis.

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance.

A potent sphingomyelinase inhibitor from Cordyceps mycelia contributes its cytoprotective effect against oxidative stress in macrophages.

A novel water-soluble polysaccharide fraction, CME-1, with a molecular mass of 27.6 kDa and containing mannose and galactose in a respective ratio of 4:6, was prepared from Cordyceps sinensis mycelia and identified by NMR and GC-MS. In the current study, we examined whether CME-1 has anti-inflammatory effects in RAW264.7 cells. The ability of CME-1 to inhibit H(2)O(2)-induced cell death in RAW264.7 cells was assessed by using an MTT assay and annexin V/propidium iodide double staining; we found that CME-1 protected cells against H(2)O(2)-induced injury. H(2)O(2)-induced intracellular oxidative stress and mitochondrial membrane depolarization were also diminished with CME-1 treatment. We evaluated the hydroxyl radical scavenging ability of CME-1 by using the DMPO-electron spin resonance technique, which indicated that CME-1 acts as an intracellular antioxidant in a concentration-dependent manner through a mechanism other than its scavenging activity. Activities of both neutral and acid sphingomyelinases (SMases) were assessed in vitro, and results showed that the CME-1 inhibited activities of both neutral and acid SMases in a concentration-dependent manner. CME-1 reduced H(2)O(2) treatment-elevated C16- and C18-ceramide levels measured by LC/MS/MS in RAW264.7 cells. Results suggest that CME-1 protects RAW264.7 cells against oxidative stress through inhibition of SMase activity and reduction of C16- and C18-ceramide levels.

Nuclear betaII-tubulin associates with the activated notch receptor to modulate notch signaling.

The Notch signal pathway plays important roles in proliferation, apoptosis, and differentiation. Abnormalities in Notch signaling are linked to many human diseases. After ligand binding, Notch signaling is activated through the cleavage of Notch receptors to release and translocate the Notch intracellular domain into the nucleus. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, can modulate downstream target genes via C promoter-binding factor 1-dependent and -independent pathways. To further dissect the Notch1 signaling pathway, we screened the N1IC-associated proteins using a yeast two-hybrid system and identified nuclear beta(II)-tubulin as a candidate for the N1IC-associated proteins. It was suggested that the presence of beta(II)-tubulin in nuclei might be correlated with the cancerous state of cells. However, the function of beta(II)-tubulin locating in the nucleus still is unknown. Herein, we show that the complex of alpha- and beta(II)-tubulin is associated with N1IC in cancer cells by a coimmunoprecipitation analysis. The ankyrin domain of the Notch1 receptor alone was sufficient to associate with beta(II)-tubulin. Furthermore, alpha- and beta(II)-tubulin were localized in the nucleus and formed a complex with N1IC. Treatment with Taxol increased the amounts of nuclear alpha- and beta(II)-tubulin in K562 and HeLa cells and promoted the C promoter-binding factor 1-dependent transactivation activity of N1IC. We also show that nuclear beta(II)-tubulin was bound on the C promoter-binding factor 1 response elements via the association with N1IC. These results suggest that nuclear beta(II)-tubulin can modulate Notch signaling through interaction with N1IC in cancer cells.