Oligomannurarate sulfate inhibits CXCL12/SDF-1-mediated proliferation and invasion of human tumor cells in vitro.

AIM: JG6 is a novel marine-derived oligosaccharide that has shown to inhibit angiogenesis and tumor metastasis. In this study, we sought to identify the potential target responsible for the anti-cancer activity of JG6. METHODS: Human liver cancer cell line Bel-7402 and human cervical cancer cell line HeLa were examined. CXCL12-stimulated cell proliferation and migration were determined using a CCK-8 kit and a transwell assay, respectively. Western blotting was performed to examine the changes in CXCL12/CXCR4 axis. Molecular docking and surface plasmon resonance (SPR) were performed to characterize the possible interaction between JG6 and the CXCL12/CXCR4 axis. RESULTS: Treatment with CXCL12 potently stimulated the proliferation and migration in both Bel-7402 and HeLa cells. Co-treatment of the cells with JG6 (10, 50 and 100 µg/mL) dose-dependently impeded the CXCL12-stimulated cell proliferation and migration. Furthermore, CXCL12 rapidly induced phosphorylation of AKT, ERK, FAK and Paxillin in Bel-7402 and HeLa cells, whereas pretreatment with JG6 dose-dependently inhibited the CXCL12-induced phosphorylation of these proteins. The SPR assay showed that JG6 bound to CXCL12 with a high affinity. In molecular docking study, JG6 appeared to interact with CXCL12 via multiple polar interactions, including 6 ionic bonds and 7 hydrogen bonds. CONCLUSION: Inhibition of the CXCL12/CXCR4 axis by JG6 may account for its anticancer activity.

Establishment of platform for screening insulin-like growth factor-1 receptor inhibitors and evaluation of novel inhibitors.

AIM: The insulin-like growth factor-1 receptor (IGF1R) is over-expressed in a wide variety of tumors and contributes to tumor cell proliferation, metastasis and drug resistance. The aim of this study was to establish a sensitive screening platform to identify novel IGF1R inhibitors. METHODS: The catalytic domain of IGF1R was expressed using the Bac-to-Bac baculovirus expression system. The screening platform for IGF1R inhibitors was established based on ELISA. The binding profile of IGF1R with the inhibitors was predicted with molecular docking and then subjected to the surface plasmon resonance (SPR) approach. The growth inhibition of cancer cells by the inhibitors was assessed with MTT assay. Apoptosis was analyzed using flow cytometry and Western blotting. RESULTS: A naturally occurring small molecule compound hematoxylin was identified as the most potent inhibitor (IC50 value=1.8±0.1 µmol/L) within a library of more than 200 compounds tested. Molecular simulation predicted the possible binding mode of hematoxylin with IGF1R. An SPR assay further confirmed that hematoxylin bound directly to IGF1R with high binding affinity (Kd=4.2 × 10⁻6 mol/L). In HL-60 cancer cells, hematoxylin inactivated the phosphorylation of IGF1R and downstream signaling and therefore suppressed cell proliferation. Mechanistic studies revealed that hematoxylin induced apoptosis in HL-60 cells via both extrinsic and intrinsic pathways. CONCLUSION: A simple, sensitive ELISA-based screening platform for identifying IGF1R inhibitors was established. Hematoxylin was identified as a promising IGF1R inhibitor with effective antitumor activity that deserves further investigation.

Sulfated polymannuroguluronate inhibits Tat-induced SLK cell adhesion via a novel binding site, a KKR spatial triad.

AIM: Sulfated polymannuroguluronate (SPMG), a candidate anti-AIDS drug, inhibited HIV replication and interfered with HIV entry into host T lymphocytes. SPMG has high binding affinity for the transactivating factor of the HIV-1 virus (Tat) via its basic domain. However, deletion or substitution of the basic domain affected, but did not completely eliminated Tat-SPMG interactions. Here, we sought to identify other SPMG binding sites in addition to the basic domain. METHODS: The potential SPMG binding sites were determined using molecular simulation and a surface plasmon resonance (SPR) based competitive inhibition assay. The effect of SPMG on Tat induced adhesion was evaluated using a cell adhesion assay. RESULTS: The KKR domain, a novel high-affinity heparin binding site, was identified, which consisted of a triad of Lys12, Lys41, and Arg78. The KKR domain, spatially enclosed SPMG binding site on Tat, functions as another binding domain for SPMG. Further functional evaluation demonstrated that SPMG inhibits Tat-mediated SLK cell adhesion by directly binding to the KKR region. CONCLUSION: The KKR domain is a novel high-affinity binding domain for SPMG. Our findings provide important new insights into the molecular mechanisms of SPMG and a potential therapeutic intervention for Tat-induced cell adhesion.

PH006, a novel and selective Src kinase inhibitor, suppresses human breast cancer growth and metastasis in vitro and in vivo.

The central role of Src in tumor progression and metastasis has validated it as an attractive therapeutic target for the treatment of human breast cancer. The aim of this study was to identify potential Src kinase inhibitor, explore its activity, and mechanism of action in human breast cancer. A strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and high-throughput screening was adopted and a novel 6-hydrazinopurine-based inhibitor of c-Src kinase PH006 was obtained. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay. The binding mode between PH006 and Src was profiled by surface plasmon resonance approach and molecular simulation. The anti-proliferative activity was evaluated by Sulforhodamin B (SRB) and Colony formation. The anti-invasion and anti-migration activities were assessed by trans-well and wound healing assay. Results indicated that PH006 was an ATP-competitive Src inhibitor, which selectively inhibited c-Src with an IC50 of 0.38 µM among a panel of 14 diverse tyrosine kinases. PH006 potently inhibited c-Src phosphorylation and c-Src-dependent signal transduction, resulting in inhibition of cell proliferation, migration, and invasion in human breast cancer MDA-MB-231 cells. Further study demonstrated that the anti-proliferative activity of PH006 was ascribed to its capability to arrest cells in G1 phase, while its anti-motility activity was related to suppression of MMP2/9 and HGF secretion. Moreover, PH006 exhibited potent activity against tumor growth as well as metastasis of human breast cancer MDA-MB-435 xenograft beard in nude mice, which was accompanied with reduced Src/FAK signaling in tumor tissue. Taken together, PH006 is a novel selective inhibitor of c-Src and possesses potent activity against breast cancer growth and metastasis, which could be potentially developed as a lead candidate against breast cancers with elevated Src tyrosine kinase activity.

Oligomannurarate sulfate blocks tumor growth by inhibiting NF-kappaB activation.

AIM: 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-kappaB activation status was determined using immunofluorescence, Western blotting, DNA-binding and transcription-activity assays. The effect of JG3 on upstream components of the NF-kappaB pathway and on selected transcription factors were monitored by Western blotting. The antitumor effect of JG3 and its relation to NF-kappaB activation were evaluated using four different tumor xenograft models. RESULTS: We found that JG3 effectively inhibited NF-kappaB activation independent of heparanase expression. Our results indicate that JG3 inactivated NF-kappaB by interfering with the activation of upstream components of the NF-kappaB 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-kappaB was constitutively active (BEL-7402 liver carcinoma and MDA-MB-435s breast carcinoma), but did not affect the growth of tumors derived from NF-kappaB-negative cell lines (SGC-7901 gastric cancer and HO-8910 ovarian carcinoma). CONCLUSION: Our data indicate that NF-kappaB 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.

AIM: 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 beta1-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 beta1-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.Acta Pharmacologica Sinica (2009) 30: 1033-1038; doi: 10.1038/aps.2009.97; published online 22 June 2009.

Sulfated polymannuroguluronate, a novel anti-AIDS drug candidate, inhibits HIV-1 Tat-induced angiogenesis in Kaposi's sarcoma cells.

Kaposi's sarcoma (KS), a neoplasm often associated with iatrogenic and acquired immunosuppression, is characterized by prominent angiogenesis. Angiogenic factors released from KS and host cells and HIV viral products-the protein Tat are reported to be involved in angiogenesis. Mounting evidence further suggests that multiple angiogenic activities of Tat contribute to AIDS-associated Kaposi's sarcoma (AIDS-KS). Herein, we report that sulfated polymannuroguluronate (SPMG), a novel anti-AIDS drug candidate now undergoing phase II clinical trial, significantly eliminated Tat-induced angiogenesis in SLK cells both in vitro and in vivo. SPMG significantly and dose-dependently inhibits proliferation, migration, and tube formation by SLK cells. SPMG also dramatically arrested Tat-driven KDR phosphorylation and blocked the interaction between Tat and integrin beta1, thus inhibiting the phosphorylation of the downstream kinases of FAK, paxillin and MAPKs. In addition, SPMG was noted to block the release of bFGF and VEGF from ECM. All these collectively favor an issue that SPMG functions as a promising therapeutic against Tat-induced angiogenesis and pathologic events relevant to AIDS-KS, which adds novel mechanistic profiling to the anti-AIDS action of SPMG.

Philinopside E, a new sulfated saponin from sea cucumber, blocks the interaction between kinase insert domain-containing receptor (KDR) and alphavbeta3 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 alpha(v)beta(3) 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 alpha(v)beta(3) integrin-driven downstream signaling as a result of disturbance of the physical interaction between KDR and alpha(v)beta(3) 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.

Pseudolarix acid B, a new tubulin-binding agent, inhibits angiogenesis by interacting with a novel binding site on tubulin.

Tubulin-binding agents have received considerable interest as potential tumor-selective angiogenesis-targeting drugs. Herein, we report that pseudolarix acid B (PAB), isolated from the traditional Chinese medicinal plant Pseudolarix kaempferi Gordon, is a tubulin-binding agent. We further demonstrate that PAB significantly and dose-dependently inhibits proliferation, migration, and tube formation by human microvessel enthothelial cells. It is noteworthy that PAB eliminated newly formed endothelial tubes and microvessels both in vitro and in vivo. In addition, PAB dramatically arrested the cell cycle at G2/M phase. PAB also induced endothelial cell retraction, intercellular gap formation, and promoted actin stress fiber formation in conjunction with disruption of the tubulin and actin cytoskeletons. All of these effects occurred at noncytotoxic concentrations of PAB. We found that these effects of PAB are attributable to depolymerization of tubulin by direct interaction with a distinct binding site on tubulin compared with those of colchicine and vinblastine. Taken together, these findings show that PAB is a candidate antiangiogenic agent for use in cancer therapy, and they provide proof of principle for targeting this novel binding site on tubulin as a new strategy for treating cancer.

[Effects of acidic oligose on differentially expressed genes in the mice model of Alzheimer's disease by microarray].

AIM: To investigate the molecular mechanism of protective effect of acidic oligose 971 on Alzheimer's disease mouse model by using microarray. METHODS: Balb/c mice were randomly divided into control group, beta-AP(25-35) i.c.v. injected group and 971-treated group. The learning-memory ability of mice was tested by Morris water maze experiment. Total RNA of the cerebral cortex was extracted from the mice of each group. cDNA microarrays containing 1176 genes were used to investigate the gene expression pattern of each group. Expressions of 5 genes were randomly selected for further confirmation by RT-PCR. RESULTS: Icv injection of beta-AP(25-35) caused significant impairments in spatial and working memory performances of mice in Morris water maze and which were relieved by the treatment of 971. Up- and down- regulated genes were 19 and 12 in beta-AP(25-35)-injected group vs control group, respectively. Up- and down- regulated genes were 13 and 4, respectively, in 971-treated group vs beta-AP(25-35)-injected group. RT-PCR results indicated that 5 genes showed identical results to that of the microarray. CONCLUSION: The protective effect of 971 on learning and memory ability of beta-AP(25-35)-treated mouse may be related to the expression changes of genes involved in cell cycle, DNA repair, nerve growth, synaptic plasticity and immune response, etc.