Malignant tumor cells engender second membrane-lined organelles for self-protection and tumor progression.

Cancer is a leading cause of mortality in humans, but the efficacy of current treatments for many cancers is limited, as they lack unique mechanistically defined targets. Here, we show that, upon malignant transformation, aggressive oncocells generate a second membrane exterior to their plasma membrane to form cytocapsulas (CCs) and cytocapsular tubes (CCTs), which all together constitute cytocapsular oncocells with pleotropic biological functions in cancer patient tissues in vivo. Proteomic and biochemical analyses revealed that the PMCA2 calcium pump is highly up-regulated in CCs and CCTs in malignant tumors but not in normal tissues, thus identifying a unique cancer biomarker and target for cancer therapy. Cytocapsular oncocells are universally present in solid cancers and appear in hematologic cancers in immune organs. Multi-cell malignant tumors are also enveloped by protective CC membranes. These cytocapsular tumors (CTs) generate numerous CCTs that form freeways for cancer cell metastasis to both neighboring and distant destinations. Entire cytocapsular tumor networks (CTNs) dominate physical cancer metastasis pathways in cancer patients in vivo. Later, CCTs invade micro blood vessels and release cytocapsular oncocells into the blood, providing a source of circulating tumor cells. CTNs interconnect cytocapsular tumors in primary and secondary cancer niches, creating larger cytocapsular tumor network systems (CTNSs). Primary and secondary CTNSs are in turn interconnected, forming dynamic and integrated CTNSs. Thus, interconnected cytocapsular oncocells, CTNs, and CTNSs coordinate cancer progression via the integrated cytocapsular membrane systems.

GPR54 deficiency reduces the Treg population and aggravates experimental autoimmune encephalomyelitis in mice.

GPR54 is highly expressed in the central nervous system and plays a crucial role in pubertal development. However, GRP54 is also expressed in the immune system, implying possible immunoregulatory functions. Here we investigated the role of GPR54 in T cell and immune tolerance. GPR54 deficiency led to an enlarged thymus, an increased number of thymocytes, and altered thymic micro-architecture starting around puberty, indicating GPR54 function in T-cell development through its regulatory effect on the gonadal system. However, flow cytometry revealed a significant reduction in the peripheral regulatory T cell population and a moderate decrease in CD4 single-positive thymocytes in prepubertal Gpr54-/- mice. These phenotypes were confirmed in chimeric mice with GPR54 deficient bone marrow-derived cells. In addition, we found elevated T cell activation in peripheral and thymic T cells in Gpr54-/- mice. When intact mice were immunized with myelin oligodendrocyte glycoprotein, a more severe experimental autoimmune encephalomyelitis (EAE) developed in the Gpr54-/- mice. Interestingly, aggravated EAE disease was also manifested in castrated and bone marrow chimeric Gpr54-/- mice compared to the respective wild-type control, suggesting a defect in self-tolerance resulting from GPR54 deletion through a mechanism that bypassed sex hormones. These findings demonstrate a novel role for GPR54 in regulating self-tolerant immunity in a sex hormone independent manner.

Cytocapsular tubes conduct cell translocation.

Cell locomotion is essential for multicellular organism embryo development, organ homeostasis, tissue regeneration, immune responses, and tumor metastasis. Here we report that single mammalian cells can generate two extracellular membranous compartments: cytocapsulae and cytocapsular tubes. Cells migrate in cytocapsulae and engender cytocapsular tubes, which exhibit pleiotropic biological functions and provide tubular routes for directed cell transportation. Ultrastructural analysis by electron microscope revealed that nanoprotrusions surround and anchor cytocapsular tubes in place. Multiple cytocapsular tubes interconnect and form networks supporting directed cell transportation in diverse directions. Enhanced translation initiation factor eIF4E up-regulates translation of transcripts encoding proteins important for organelle development. Thus, this study proposes a mechanism of directed cell translocation in cytocapsular tubes, which may facilitate the management of diseases, including tumor metastasis.

Identifying RISC Components Using Ago2 Immunoprecipitation and Mass Spectrometry.

Complex immunoprecipitation (Co-IP) is a powerful technique for precipitating an intact protein complex out of solution and cell lysates using an antibody that specifically binds to a particular protein in a large complex of proteins. Mass spectrometry (MS) is used to identify, sequence, and quantify proteins. RNA-induced silencing complexes (RISCs), Ago2 centered protein assemblies, are essential for miRNA mediated RNA decay and gene expression regulation; however, the complete list of RISCs is unknown. Here we describe methods used to combine IP and MS to identify new components of RISCs.

eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference.

MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centred RNA-induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis.

4EGI-1 targets breast cancer stem cells by selective inhibition of translation that persists in CSC maintenance, proliferation and metastasis.

Cancer death is a leading cause of global mortality. An estimated 14.1 million new cancer cases and 8.2 million cancer deaths occurred worldwide in 2012 alone. Cancer stem cells (CSCs) within tumors are essential for tumor metastasis and reoccurrence, the key factors of cancer lethality. Here we report that 4EGI-1, an inhibitor of the interaction between translation initiation factors eIF4E1 and eIF4G1 effectively inhibits breast CSCs through selectively reducing translation persistent in breast CSCs. Translation initiation factor eIF4E1 is significantly enhanced in breast CSCs in comparison to non-CSC breast cancer cells. 4EGI-1 presents increased cytotoxicity to breast CSCs compared to non-CSC breast cancer cells. 4EGI-1 promotes breast CSC differentiation and represses breast CSC induced tube-like structure formation of human umbilical vein endothelial cells (HUVECs). 4EGI-1 isomers suppress breast CSC tumorangiogenesis and tumor growth in vivo. In addition, 4EGI-1 decreases proliferation in and induces apoptosis into breast CSC tumor cells. Furthermore, 4EGI-1 selectively inhibits translation of mRNAs encoding NANOG, OCT4, CXCR4, c-MYC and VEGF in breast CSC tumors. Our study demonstrated that 4EGI-1 targets breast CSCs through selective inhibition of translation critical for breast CSCs, suggesting that selective translation initiation interference might be an avenue targeting CSCs within tumors.

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between ß-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells.

Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.

LGR4/GPR48 inactivation leads to aniridia-genitourinary anomalies-mental retardation syndrome defects.

AGR syndrome (the clinical triad of aniridia, genitourinary anomalies, and mental retardation, a subgroup of WAGR syndrome for Wilm's tumor, aniridia, genitourinary anomalies, and mental retardation) is a rare syndrome caused by a contiguous gene deletion in the 11p13-14 region. However, the mechanisms of WAGR syndrome pathogenesis are elusive. In this study we provide evidence that LGR4 (also named GPR48), the only G-protein-coupled receptor gene in the human chromosome 11p12-11p14.4 fragment, is the key gene responsible for the diseases of AGR syndrome. Deletion of Lgr4 in mouse led to aniridia, polycystic kidney disease, genitourinary anomalies, and mental retardation, similar to the pathological defects of AGR syndrome. Furthermore, Lgr4 inactivation significantly increased cell apoptosis and decreased the expression of multiple important genes involved in the development of WAGR syndrome related organs. Specifically, deletion of Lgr4 down-regulated the expression of histone demethylases Jmjd2a and Fbxl10 through cAMP-CREB signaling pathways both in mouse embryonic fibroblast cells and in urinary and reproductive system mouse tissues. Our data suggest that Lgr4, which regulates eye, kidney, testis, ovary, and uterine organ development as well as mental development through genetic and epigenetic surveillance, is a novel candidate gene for the pathogenesis of AGR syndrome.

Hypoxia-inducible factor-1α (HIF-1α) promotes cap-dependent translation of selective mRNAs through up-regulating initiation factor eIF4E1 in breast cancer cells under hypoxia conditions.

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF-1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels.

The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2ß.

Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2ß on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2ß. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2ß in switching PICs from an open to a closed state at start codons.

The roles of transmembrane domain helix-III during rhodopsin photoactivation.

BACKGROUND: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11-cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. PRINCIPAL FINDINGS: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 4'-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108-135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. SIGNIFICANCE: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.

Structure of the VP16 transactivator target in the Mediator.

The human Mediator coactivator complex interacts with many transcriptional activators and facilitates recruitment of RNA polymerase II to promote target gene transcription. The MED25 subunit is a critical target of the potent herpes simplex 1 viral transcriptional activator VP16. Here we determine the solution structure of the MED25 VP16-binding domain (VBD) and define its binding site for the N-terminal portion of the VP16 transactivation domain (TADn). A hydrophobic furrow, formed by a ß-barrel and two α-helices in MED25 VBD, interacts tightly with VP16 TADn. Mutations in this furrow prevent binding of VP16 TAD to MED25 VBD and interfere with the ability of overexpressed MED25 VBD to inhibit VP16-dependent transcriptional activation in vivo. This detailed molecular understanding of transactivation by the benchmark activator VP16 could provide important insights into viral and cellular gene activation mechanisms.

Regulation of embryonic kidney branching morphogenesis and glomerular development by KISS1 receptor (Gpr54) through NFAT2- and Sp1-mediated Bmp7 expression.

G-protein-coupled receptor 54 (Gpr54, KISS1 receptor) plays critical roles in puberty regulation, tumor metastasis suppression, and vasoconstriction. Bone morphogenetic protein-7 (Bmp7) is required for kidney organogenesis. However, whether Gpr54 is involved in embryonic kidney development and how Bmp7 expression is regulated in the kidney are largely unknown. Here we report that Gpr54 deletion leads to kidney branching morphogenesis and glomerular development retardation in embryonic kidneys in vivo and in explanted kidneys in vitro. Gpr54 inactivation results in a high risk of low glomerular number in adult kidneys. Gpr54 is expressed in condensed mesenchyme at E12.5 and epithelial cells of proximal and distal tubules and collecting ducts at E17.5 and P0 mouse kidney. Deletion of Gpr54 decreases Bmp7 expression and Smad1 phosphorylation in the developing kidney. Using chromatin immunoprecipitation and luciferase assays, we demonstrate that Gpr54 regulates NFAT2- and Sp1-mediated Bmp7 transcription. Furthermore, we show that NFAT2 cooperates with Sp1 to promote Bmp7 transcription activation. Together, these data suggest that Gpr54 regulates Bmp7 expression through NFAT2 and Sp1 and plays an important role in embryonic kidney branching morphogenesis and glomerular development.

Kisspeptin-10, a KISS1-derived decapeptide, inhibits tumor angiogenesis by suppressing Sp1-mediated VEGF expression and FAK/Rho GTPase activation.

Kisspeptin-10 (Kp-10), a decapeptide derived from the primary translation product of KISS1 gene, has been reported previously to be a key hormone for puberty and an inhibitor for tumor metastasis via the activation of G protein-coupled receptor 54. However, whether Kp-10 inhibits angiogenesis, which is critical for tumor growth and metastasis and other human diseases, is still unknown. Here we show that Kp-10 significantly inhibits human umbilical vein endothelial cell (HUVEC) migration, invasion, and tube formation, key processes in angiogenesis. Using chicken chorioallantoic membrane assay and vascular endothelial growth factor (VEGF)-induced mouse corneal micropocket assay, we show that Kp-10 inhibits angiogenesis in vivo. Furthermore, Kp-10 inhibits tumor growth in severe combined immunodeficient mice xenografted with human prostate cancer cells (PC-3) through inhibiting tumor angiogenesis, whereas Kp-10 has little effect on the proliferation of HUVECs and human prostate cancer cells. In deciphering the underlying molecular mechanisms, we show that Kp-10 suppresses VEGF expression by inhibiting the binding of specificity protein 1 to VEGF promoter and by blocking the activation of c-Src/focal adhesion kinase and Rac/Cdc42 signaling pathways in HUVECs, leading to the inhibition of tumor angiogenesis.

Regulation of bone formation and remodeling by G-protein-coupled receptor 48.

G-protein-coupled receptor (GPCR) 48 (Gpr48; Lgr4), a newly discovered member of the glycoprotein hormone receptor subfamily of GPCRs, is an orphan GPCR of unknown function. Using a knockout mouse model, we have characterized the essential roles of Gpr48 in bone formation and remodeling. Deletion of Gpr48 in mice results in a dramatic delay in osteoblast differentiation and mineralization, but not in chondrocyte proliferation and maturation, during embryonic bone formation. Postnatal bone remodeling is also significantly affected in Gpr48(-/-) mice, including the kinetic indices of bone formation rate, bone mineral density and osteoid formation, whereas the activity and number of osteoclasts are increased as assessed by tartrate-resistant acid phosphatase staining. Examination of the molecular mechanism of Gpr48 action in bone formation revealed that Gpr48 can activate the cAMP-PKA-CREB signaling pathway to regulate the expression level of Atf4 in osteoblasts. Furthermore, we show that Gpr48 significantly downregulates the expression levels of Atf4 target genes/proteins, such as osteocalcin (Ocn; Bglap2), bone sialoprotein (Bsp; Ibsp) and collagen. Together, our data demonstrate that Gpr48 regulates bone formation and remodeling through the cAMP-PKA-Atf4 signaling pathway.

Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signaling pathways.

Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action.

A novel peptide from human apolipoprotein(a) inhibits angiogenesis and tumor growth by targeting c-Src phosphorylation in VEGF-induced human umbilical endothelial cells.

Many angiogenesis inhibitors are derived from large plasma proteins. Previous studies showed that the Kringle5-like domain (termed KV) in human apolipoprotein (a) is a potential antiangiogenic factor. However, its active region and the underling molecular mechanism remain elusive. Here, we identified an 11-amino acid peptide (named KV11) as the key region for the antiangiogenic function of the KV domain of apolipoprotein (a). We demonstrate that KV11 inhibits angiogenesis in vitro by suppressing human umbilical vein endothelial cell migration and microtubule formation. KV11 inhibits angiogenesis in chicken chorioallantoic membrane assays and mouse corneal micropocket angiogenesis assays in vivo. KV11 peptide shows no effect on tumor cell growth or proliferation, but significantly inhibits tumor growth in SCID mouse xenograft tumor model (p < 0.01) by preventing tumor angiogenesis. We elucidate that KV11 peptide suppresses angiogenesis and tumor progression by targeting the c-Src/ERK signaling pathways. Together, these studies provide the first evidence that KV11 from apolipoprotein KV domain has anti-angiogenesis functions and may be an anti-tumor drug candidate.

Zerumbone down-regulates chemokine receptor CXCR4 expression leading to inhibition of CXCL12-induced invasion of breast and pancreatic tumor cells.

CXC chemokine receptor 4 (CXCR4), initially linked with leukocyte trafficking, is now known to be expressed in various tumors including breast, ovary, prostate, gastrointestinal, head and neck, bladder, brain, and melanoma. This receptor mediates homing of tumor cells to specific organs that express the ligand CXCL12 for this receptor. Thus, agents that can down-regulate CXCR4 expression have potential against cancer metastasis. In this study, we report the identification of zerumbone, a component of subtropical ginger (Zingiber zerumbet), as a regulator of CXCR4 expression. This sesquiterpene down-regulated the expression of CXCR4 on HER2-overexpressing breast cancer cells in a dose- and time-dependent manner. The decrease in CXCR4 by zerumbone was found to be not cell type specific as its expression was abrogated in leukemic, skin, kidney, lung, and pancreatic cancer cell lines. The down-regulation of CXCR4 was not due to proteolytic degradation but rather to transcriptional regulation, as indicated by down-regulation of mRNA expression, inhibition of nuclear factor-kappaB activity, and suppression of chromatin immunoprecipitation activity. Suppression of CXCR4 expression by zerumbone correlated with the inhibition of CXCL12-induced invasion of both breast and pancreatic cancer cells. An analogue of zerumbone, alpha-humulene, which lacks the carbonyl group, was found to be inactive in inducing CXCR4 down-regulation. Overall, our results show that zerumbone is a novel inhibitor of CXCR4 expression and thus has a potential in the suppression of cancer metastasis.

Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways.

Thymoquinone, a component derived from the medial plant Nigella sativa, has been used for medical purposes for more than 2,000 years. Recent studies reported that thymoquinone exhibited inhibitory effects on cell proliferation of many cancer cell lines and hormone-refractory prostate cancer by suppressing androgen receptor and E2F-1. Whether thymoquinone inhibits tumor angiogenesis, the critical step of tumor growth and metastasis, is still unknown. In this study, we found that thymoquinone effectively inhibited human umbilical vein endothelial cell migration, invasion, and tube formation. Thymoquinone inhibited cell proliferation and suppressed the activation of AKT and extracellular signal-regulated kinase. Thymoquinone blocked angiogenesis in vitro and in vivo, prevented tumor angiogenesis in a xenograft human prostate cancer (PC3) model in mouse, and inhibited human prostate tumor growth at low dosage with almost no chemotoxic side effects. Furthermore, we observed that endothelial cells were more sensitive to thymoquinone-induced cell apoptosis, cell proliferation, and migration inhibition compared with PC3 cancer cells. Thymoquinone inhibited vascular endothelial growth factor-induced extracellular signal-regulated kinase activation but showed no inhibitory effects on vascular endothelial growth factor receptor 2 activation. Overall, our results indicate that thymoquinone inhibits tumor angiogenesis and tumor growth and could be used as a potential drug candidate for cancer therapy.