Xianliang Xin

Effect of acidic oligosaccharide sugar chain on scopolamine-induced memory impairment in rats and its related mechanisms

In this study we evaluated the effect of a novel, marine-derived, acidic oligosaccharide on scopolamine-induced amnesia in rats using the Morris water maze test. The results show that 30-day administration of this oligosaccharide, referred to as acidic oligosaccharide sugar chain (AOSC), to rats attenuates memory impairment by scopolamine, as evaluated by shortened escape latency, swimming distance, and increased swimming time of rats with memory impairment induced by scopolamine in the quadrant where the platform is placed. The data additionally suggest that an appropriate dose of scopolamine, a traditional muscarinic receptor antagonist, elevates oxidative damage in brain, characterized by inactivation of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and consequently, inhibition of ATPase in the hippocampus and cerebral cortex. AOSC ameliorates oxidative injuries caused by scopolamine by increasing the activities of SOD, GSH-Px, and ATPase. Further investigation by flow cytometry revealed that AOSC significantly reduces the overloading of intracellular free calcium ion ([Ca2+]i), thus suppressing apoptosis induced by H2O2 in human neuroblastoma SH-SY5Y cells. These findings suggest that AOSC can induce cognitive improvement via its antioxidant activity.

Oligomannurarate Sulfate, a Novel Heparanase Inhibitor Simultaneously Targeting Basic Fibroblast Growth Factor, Combats Tumor Angiogenesis and Metastasis

Inhibitors of tumor angiogenesis and metastasis are increasingly emerging as promising agents for cancer therapy. Recently, heparanase inhibitors have offered a new avenue for such work because heparanase is thought to be critically involved in the metastatic and angiogenic potentials of tumor cells. Here, we report that oligomannurarate sulfate (JG3), a novel marine-derived oligosaccharide, acts as a heparanase inhibitor. Our results revealed that JG3 significantly inhibited tumor angiogenesis and metastasis, both in vitro and in vivo, by combating heparanase activity via binding to the KKDC and QPLK domains of the heparanase molecule. The JG3-heparanase interaction was competitively inhibited by low molecular weight heparin (4,000 Da) but not by other glycosaminoglycans. In addition, JG3 abolished heparanase-driven invasion, inhibited the release of heparan sulfate-sequestered basic fibroblast growth factor (bFGF) from the extracellular matrix, and repressed subsequent angiogenesis. Moreover, JG3 inactivated bFGF-induced bFGF receptor and extracellular signal-regulated kinase 1/2 phosphorylation and blocked bFGF-triggered angiogenic events by directly binding to bFGF. Thus, JG3 seems to inhibit both major heparanase activities by simultaneously acting as a substrate mimetic and as a competitive inhibitor of heparan sulfate. These findings suggest that JG3 should be considered as a promising candidate agent for cancer therapy.

Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates Akt signaling.

O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling.

The marine-derived oligosaccharide sulfate (MdOS), a novel multiple tyrosine kinase inhibitor, combats tumor angiogenesis both in vitro and in vivo.

Despite the emerging success of multi-targeted protein tyrosine kinase (PTK) inhibitors in cancer therapy, significant side effects and resistance concerns seems to be avoided unlikely. The aim of the present study was to identify novel multi-targeting PTK inhibitors. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay (ELISA). The antiproliferative activities in human microvascular endothelial cells (HMECs) were evaluated by sulforhodamine (SRB) assay. The phosphorylation of kinases and their downstream molecules was probed by western blot analysis. The binding mode between MdOS and PTKs was profiled by surface plasmon resonance (SPR) approach and molecular simulation. Tube formation assay, rat aortic ring method and chicken chorioallantoic membrane assay were combined to illustrate the in vitro and in vivo anti-angiogenic effects. Results indicated that MdOS, a novel marine-derived oligosaccharide sulfate, exhibited a broad-spectrum PTK inhibitory action. At an enzymatic level, MdOS inhibited HER2, EGFR, VEGFR, PDGFR, c-Kit, FGFR1 and c-Src, with little impact on FGFR2. In cellular settings, MdOS inhibited phosphorylation of PTKs, exemplified by HER2, EGFR and VEGFR2, and downstream molecules of Erk1/2 and AKT. Further studies demonstrated that MdOS acted as an ATP-competitive inhibitor via directly binding to the residues of entrance rather than those of the ATP-binding pocket. Furthermore, MdOS inhibited proliferation and tube formation of HMECs, arrested microvessel outgrowth of rat aortic rings and hindered the neovascularization of chick allantoic membrane. Taken together, results presented here indicated that MdOS exhibited anti-angiogenic activity in a PTK-dependent manner and make it a promising agent for further evaluation in PTK-associated cancer therapy.

Philinopside E, a New Sulfated Saponin from Sea Cucumber, Blocks the Interaction between Kinase Insert Domain-Containing Receptor (KDR) and αvβ3 Integrin via Binding to the Extracellular Domain of KDR

Vascular endothelial growth factor (VEGF) signaling pathway is essential for tumor angiogenesis and has long been recognized as a promising target for cancer therapy. Current view holds that physical interaction between αvβ3 integrin and kinase insert domain-containing receptor (KDR) is important in regulating angiogenesis and tumor development. We have reported previously that a new marine-derived compound, philinopside E (PE), exhibited the antiangiogenic activity via inhibition on KDR phosphorylation and downstream signaling. Herein, we have further demonstrated that PE specifically interacts with KDR extracellular domain, which is distinct from conventional small-molecule inhibitors targeting cytoplasmic kinase domain, to block its interaction with VEGF and the downstream signaling. We also noted that PE markedly suppresses αvβ3 integrin-driven downstream signaling as a result of disturbance of the physical interaction between KDR and αvβ3 integrin in HMECs, followed by disruption of the actin cytoskeleton organization and decreased cell adhesion to vitronectin. All of these findings substantiate PE to be an unrecognized therapeutic class in tumor angiogenesis and, more importantly, help appeal the interest of the therapeutic potential in angiogenesis and cancer development via targeting integrin-KDR interaction in the future.

A new marine-derived sulfated polysaccharide from brown alga suppresses tumor metastasis both in vitro and in vivo.

Herein, we report that marine-derived sulfated polysaccharide (MSP), a new kind of polysaccharide extracted from brown alga, exhibits the anti-migration effect in vitro and potently suppress metastasis of Lewis lung carcinoma in vivo. Adhesion assays demonstrated that MSP inhibits the heterogenous adhesion on fibronectin. Further studies revealed that MSP decreased FN-induced MDA-MB-435 migration, accompanied by its potent regulatory effect on actin filament reassembling. In addition, MSP significantly inactivated the phosphorylation of FAK and subsequent ERK1/2 in MDA-MB-435 cells. All these actions may be the results of MSP binding to FN, promising the therapeutic potential of MSP in tumor metastasis.

Oligomannurarate sulfate blocks tumor growth by inhibiting NF-κB activation

AbstractAim:JG3, a novel marine-derived oligosaccharide, significantly inhibits angiogenesis and tumor metastasis by blocking heparanase activity. It also arrests tumor growth, an effect that is not fully explained by its anti-heparanase activity. Here we sought to identify the mechanisms underlying JG3-mediated inhibition of tumor growth.Methods:Heparanase expression was assessed by RT-PCR and Western blotting. NF-κB activation status was determined using immunofluorescence, Western blotting, DNA-binding and transcription-activity assays. The effect of JG3 on upstream components of the NF-κB pathway and on selected transcription factors were monitored by Western blotting. The antitumor effect of JG3 and its relation to NF-κB activation were evaluated using four different tumor xenograft models.Results:We found that JG3 effectively inhibited NF-κB activation independent of heparanase expression. Our results indicate that JG3 inactivated NF-κB by interfering with the activation of upstream components of the NF-κB pathway without generally affecting the nuclear translocation of transcription factors. Further, in vivo studies demonstrated that JG3 effectively arrested the growth of tumors derived from cell lines in which NF-κB was constitutively active (BEL-7402 liver carcinoma and MDA-MB-435s breast carcinoma), but did not affect the growth of tumors derived from NF-κB-negative cell lines (SGC-7901 gastric cancer and HO-8910 ovarian carcinoma).Conclusion:Our data indicate that NF-κB mediates the JG3-induced arrest of tumor growth. These results define a new mechanism of action of JG3 and highlight the potential for JG3 as a promising lead molecule in cancer therapy.

Marine-derived oligosaccharide sulfate (JG3) suppresses heparanase-driven cell adhesion events in heparanase over-expressing CHO-K1 cells

AbstractAim:To elucidate the detailed mechanisms underlying the appreciable effects of JG3, a novel marine-derived oligosaccharide, on cell migration using a Chinese hamster ovary (CHO) cell line stably over-expressing heparanase.Methods:A retrovirus infection system was used to establish a CHO-K1 cell line stably transfected with heparanase. Immunocytochemistry was used to assess cell morphology. Flow cytometry was selected to analyze the activation of β1-integrin, and Western blotting was used to analyze the downstream effects on the cell adhesion pathway. An affinity precipitation assay was used to determine activation of the small GTPases, Rac1, and RhoA.Results:JG3 abolished heparanase-driven formation of focal adhesions and cell spreading. Although JG3 failed to block the heparanase-triggered activation of β1-integrin or the phosphorylation of Src, the oligosaccharide caused a significant dephosphorylation of FAK and subsequent inactivation of Erk. Furthermore, JG3 was found to arrest the activation of Rac1.Conclusion:All these findings help form an alternative view to understand the mechanisms underlying the inhibitory effects of JG3 on cell motility.

N-acetylglucosaminyltransferase V mediates cell migration and invasion of mouse mammary tumor cells 4TO7 via RhoA and Rac1 signaling pathway.

In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behavior. Using small interfering RNA against GnT-V, we found that the expression of GnT-V and β1,6GlcNAc branching were significantly reduced which was particularly accompanied by the arrest in both cell migration and invasion as compared to the negative control. Moreover, the suppressed GnT-V expression by siRNA technique inactivated the signaling molecules including Rac1, cofilin, Erk and Akt, and activated RhoA levels in cells lacking GnT-V, but revealed no impact on Cdc42 activity. All these notions disclose for the first time that GnT-V and β1, 6GlcNAc branching mediate the cell migration and invasion in Rac1-positive and RhoA-negative regulatory manners.

Oligomannurarate sulfate sensitizes cancer cells to doxorubicin by inhibiting atypical activation of NF‐κB via targeting of Mre11

Aberrant regulation of nuclear factor kappa B (NF‐κB) transcription factor is involved in cancer development, progression and resistance to chemotherapy. JG3, a marine‐derived oligomannurarate sulfate, was reported as a heparanase and NF‐κB inhibitor to significantly block tumor growth and metastasis in various animal models. However, the detailed functional mechanism remains unclear. Here, we report that JG3 inhibits NF‐κB activation by specifically antagonizing the doxorubicin‐triggered Ataxia‐telangiectasia‐mutated kinase (ATM) and the sequential MEK/ERK/p90Rsk/IKK signaling pathway but does not interfere with TNF‐α‐mediated NF‐κB activation. This selective inactivation of the specific NF‐κB cascade is attributed to the binding capacity of JG3 for Mre11, a major sensor of DNA double‐strand breaks (DSB). Based on this selective mechanism, JG3 showed synergistic effect with doxorubicin in a panel of tumor cells and did not affect immune system function as shown in the in vivo delayed‐type hypersensitivity (DTH) and hemolysis assays. All these highlight the clinical potential of JG3 as a favorable sensitizer in cancer therapy. In addition, identification of Mre11 as a potential target in the development of NF‐κB inhibitors provides a platform for the further development of effective anticancer agents.