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In CTLs: High glucose-culture enhances thapsigargin-induced SOCE but decreases target recognition-induced Ca2+ influx. High glucose-culture regulates expression of ORAIs and STIMs without affecting glucose uptake. More high glucose-cultured CTLs are prone to necrosis after execution of killing.

Natural killer (NK) cells play a key role in eliminating pathogen-infected cells.Verbena officinalis(V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activities, but its roles in immune responses still remains largely elusive. In this work, investigated the regulation of inflammation and NK functionsby V. officinalisextract (VO-extract). In an influenza virus infection mouse model, oral administration of VO-extract alleviated lung injury, promoted maturation and activation of NK cells residing in the lung, and decreased the levels of inflammatory cytokines (IL-6, TNF-α and IL-1β) in the serum. We further analyzed the impact of five bioactive components of VO-extract on NK killing functions. Among them, Verbenalin enhanced NK killing efficiency significantly as determined by real-time killing assays based on plate-reader or high-throughput live-cell imaging in 3D using primary human NK cells. Further investigation showed that treatment of Verbenalin accelerated killing processes by reducing the contact time of NK cells with their target cells without affecting NK proliferation, expression of cytotoxic proteins, or lytic granule degranulation. Together, our findings reveal that low doses ofV. officinaliscan achieve a satisfactory anti-inflammation effect against viral infectionin vivo, andV. officinalisregulates activation, maturation and killing functions of NK cells. NK killing efficiency is enhanced by Verbenalin fromV. officinalis, suggesting a promising potential of verbenalin to fight viral infection.

After acute brain injuries various response cascades are evoked that direct the formation of the glial scar. Here, we report that acute lesions associated with a disruption of the blood-brain barrier trigger a re-programming within the oligodendrocyte lineage. In PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice with cortical injuries, we transiently found PLP transgene-labelled cells with activated GFAP promoter activity adjacent to the lesion site. We termed them AO cells, based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and NG2-CreERT2 mice we observed that major portions of AO cells surprisingly differentiated into astrocytes. Using repeated long-term in vivo two-photon laser-scanning microscopy (2P-LSM) we followed oligodendrocytes after injury. We observed their conversion into astrocytes via the AO cell stage with silencing of the PLP promoter and simultaneous activation of the GFAP promoter. In addition, we provide evidence that this oligodendrocyte-to-astrocyte conversion depends on local cues. At the lesion site higher expression levels of various glial differentiation factors were detected. And indeed, local injection of IL-6 promoted the formation of AO cells. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma. An altered environmental milieu affects gene expression programs of mature oligodendrocytes and induces a plastic differentiation stage with astrogliogenic potential via transitional AO cells.

Cytotoxic T lymphocytes (CTLs) are key players to eliminate tumorigenic or pathogen-infected cells using lytic granules (LG) and Fas ligand (FasL) pathways. Depletion of glucose leads to severely impaired cytotoxic function of CTLs. However, the impact of excessive glucose on CTL functions still remains largely unknown. Here we used primary human CD8+ T cells, which were stimulated by CD3/CD28 beads and cultured in medium either containing high glucose (HG, 25 mM) or normal glucose (NG, 5.6 mM). We found that in HG-CTLs, glucose uptake and glycolysis were enhanced, whereas proliferation remained unaltered. Furthermore, CTLs cultured in HG exhibited an enhanced CTL killing efficiency compared to their counterparts in NG. Unexpectedly, expression of cytotoxic proteins (perforin, granzyme A, granzyme B and FasL), LG release, cytokine/cytotoxic protein release and CTL migration remained unchanged in HG-cultured CTLs. Interestingly, additional extracellular Ca2+ diminished HG-enhanced CTL killing function. Our findings suggest that in an environment with excessive glucose, CTLs could eliminate target cells more efficiently, at least for a certain period of time, in a Ca2+-dependent manner.

Cortical neural circuits are complex but very precise networks of balanced excitation and inhibition (E/I). Yet, the molecular and cellular mechanisms that form the E/I balance are just beginning to emerge. Here, using conditional GABAB receptor-deficient mice we identified a GABA/TNF-related cytokine (TNFSF12)-mediated bidirectional communication pathway between Parvalbumin-positive (PV+) fast spiking interneurons and oligodendrocyte precursor cells (OPCs) that determines the density and function of interneurons in the developing medial prefrontal cortex (mPFC). Interruption of the GABAergic signaling to OPCs resulted in reduced myelination and hypoactivity of interneurons, strong changes of cortical network activities and impaired cognitive behavior. In conclusion, glial transmitter receptors are pivotal elements in finetuning distinct brain functions.

Progress in our understanding of mechanotransduction events requires noninvasive methods for the manipulation of forces at molecular scale in physiological environments. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the entangled polymer chains, which will in turn effectively “pull” on engaged cell membrane receptors (e.g., integrins, T cell receptors) within the illuminated area. Applied forces have physiologically-relevant magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate a functionality which at the moment cannot be achieved by other technologies for force application., Molecular scale force application in physiological environments is important for studying mechanotransduction. Here, the authors use a molecular machine to apply forces at cell-matrix and cell-cell junctions using light to trigger twisting actuation which then pulls on cell membrane receptors.

Mechanotransduction events in physiological environments are difficult to investigate, in part due to the lack of experimental tools to apply forces to mechanosensitive receptors remotely. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), here we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the polymer chains, which will in turn effectively “pulls” on engaged cell membrane receptors (integrins, cadherins…) within the illuminated area. Applied forces have the adequate magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in forcedependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate, for the first time, a functionality which at the moment cannot be achieved by any other means.

The actin-binding protein profilin1 (PFN1) plays a central role in actin dynamics, which is essential for cytotoxic T lymphocyte (CTL) functions. The functional role of PFN1 in CTLs, however still remains elusive. Here, we identify PFN1 as the only member of the profilin family expressed in primary human CD8+ T cells. Using in vitro assays, we find that PFN1 is a negative regulator of CTL-mediated elimination of target cells. Furthermore, PFN1 is involved in activation-induced lytic granule (LG) release, CTL migration and modulation of actin structures at the immunological synapse (IS). During CTL migration, PFN1 modulates the velocity, protrusion formation patterns and protrusion sustainability. In contrast, PFN1 does not significantly affect migration persistence and the rates of protrusion emergence and retraction. Under in vitro conditions mimicking a tumor microenvironment, we show that PFN1 downregulation promotes CTL invasion into a 3D matrix, without affecting the viability of CTLs in a hydrogen peroxide-enriched microenvironment. Highlighting its potential relevance in cancer, we find that in pancreatic cancer patients, PFN1 expression is substantially decreased in peripheral CD8+ T cells. Taken together, we conclude that PFN1 is a negative regulator for CTL-mediated cytotoxicity and may have an impact on CTL functionality in a tumor-related context.

In saliva and gingival crevicular fluid (GCF) soluble factors such as cytokines, chemokines and growth factors have shown a great potential serving as biomarkers for early detection and/or diagnosis of oral and systemic diseases. However, GCF and saliva, which one is a better source is still under debate. This study aimed to gain an overview of cytokines, chemokines and growth factors in saliva and GCF to pave the way for selecting suitable oral fluids for oral and systemic diseases. Multiplex cytokine assay was conducted to determine concentrations of cytokines, chemokines and growth factors in saliva and GCF samples from healthy subjects. The protocol for sample collection was carefully optimized. Stabilization, repeatability, and donor variation of the profiles were analyzed. We found that for different donors, cytokine and chemokine profiles showed unique patterns in saliva but similar patterns in GCF. In terms of growth factors, the profiles were individualized in saliva and GCF. All profiles stayed stable for the same healthy individual. In saliva, profiles of cytokines, chemokines and growth factors are individualized for different donors. In GCF, profiles of cytokines and chemokines are similar. Other factors, such as growth factors and T helper-related cytokines, are highly variable in donors. Profiles of soluble factors are not correlated in saliva and GCF. The comprehensive cytokine profiles in saliva and GCF reported in this work would serve as a good base for choosing promising cytokines for developing biomarkers in oral fluids.

Cancer metastasis plays a major role in tumor deterioration. Metastatic processes are known to be regulated by hypoxic microenvironment and non-muscle myosin IIA (NMIIA). DT-13, a bioactive saponin monomer isolated from Ophiopogon japonicus, has been reported to inhibit various cancer metastasis, but whether NMIIA is involved in the anti-metastatic activity of DT-13 under hypoxia remains to be determined. Thus, this study aims to clarify the role of DT-13 in regulating 95D cell metastasis under hypoxic microenvironment and to further investigate whether NMIIA is involved in the anti-metastatic mechanism of DT-13. We found that DT-13 significantly inhibited 95D cells metastasis in vitro and in vivo. Furthermore, hypoxia significantly inhibited the expression of NMIIA and redistributed NMIIA to the cell periphery, whereas DT-13 reversed the hypoxic effects by upregulating the expression of NMIIA. Moreover, DT-13 treatment redistributed NMIIA to the nuclear periphery and reduced the formation of F-actin in 95D cells. In addition, we found that the Raf-ERK1/2 signaling pathway is involved in regulation of NMIIA by DT-13. Collectively, these findings support NMIIA as a target of DT-13 to prevent lung cancer metastasis.

Chemokine (C-X-C motif) ligand 16 (CXCL16) is a new angiogenic factor inducing angiogenesis via extracellular signal-regulated kinases pathway. To further understand the molecular mechanism underlying CXCL16‑induced angiogenesis, we explored involvement of other relevant pathways in CXCL16-induced angiogenesis. In the present study, we investigated the mechanisms underlying CXCL16-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). CXCL16 promoted HUVEC proliferation, tube formation and migration. Enzyme-linked immunosorbent assay revealed that CXCL16 induced vascular endothelial growth factor secretion from HUVECs. Western blot analysis showed that CXCL16 increased the level of hypoxia‑inducible factor 1α, p-extracellular signal-regulated kinases (ERK), p-p38 and p-Akt dose- and time-dependently. ERK-, p38- and Akt-selective inhibitors significantly suppressed HUVEC proliferation, migration, tube formation and hypoxia-inducible factor 1α (HIF-1α) expression induced by CXCL16. Furthermore, CXCL16 peptides induced CXCL16 secretion via ERK, p38 and Akt pathways, which was suppressed by HIF-1α-selective inhibitor PX12. Our data suggest that CXCL16 induces angiogenesis in autocrine manner via ERK, Akt, p38 pathways and HIF-1α modulation.

Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency. With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived H2O2 was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells. In addition, we found that integrin β chains (β1, β2 and β7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of H2O2-producing bystander cells reduces target cell search time and enhances NK killing efficiency.

The saponin monomer of dwarf lilyturf tuber, DT-13, exhibits anticancer activity by reducing human breast cancer cell adhesion and migration under hypoxia. To further investigate the anticancer activity of DT-13, we investigated whether DT-13 exhibits anti-angiogenic activity. DT-13 showed no effect on human umbilical vein endothelial cell proliferation but inhibited tube formation and migration under normoxia and hypoxia. Moreover, DT-13 significantly reduced density of vessels in vivo observed from a chicken chorioallantoic membrane model. Western blotting results showed that DT-13 suppressed the increased level of hypoxia-inducible factor 1α, p-extracellular signal-regulated kinase 1/2 and p-Akt induced by hypoxia. Enzyme-linked immunosorbent assay revealed that vascular endothelial growth factor excretion was suppressed by DT-13. DT-13 inhibited migration and tube formation induced by vascular endothelial growth factor under normoxia and hypoxia. In addition, DT-13 reduced the level of p-vascular endothelial growth factor receptor 2 and p-Akt induced by vascular endothelial growth factor. Our data suggest that DT-13 inhibits angiogenesis under normoxia and hypoxia and also inhibits angiogenesis induced by vascular endothelial growth factor via targeting at multi elements.

Ca2+ microdomains and spatially resolved Ca2+ signals are highly relevant for cell function. In T cells, local Ca2+ signaling at the immunological synapse (IS) is required for downstream effector functions. We present experimental evidence that the relocation of the MTOC towards the IS during polarization drags mitochondria along with the microtubule network. From time-lapse fluorescence microscopy we conclude that mitochondria rotate together with the cytoskeleton towards the IS. We hypothesize that this movement of mitochondria towards the IS together with their functionality of absorption and spatial redistribution of Ca2+ is sufficient to significantly increase the cytosolic Ca2+ concentration. To test this hypothesis we developed a whole cell model for Ca2+ homoeostasis involving specific geometries for mitochondria and use the model to calculate the spatial distribution of Ca2+ concentrations within the cell body as a function of the rotation angle and the distance from the IS. We find that an inhomogeneous distribution of PMCA pumps on the cell membrane, in particular an accumulation of PMCA at the IS, increases the global Ca2+ concentration and decreases the local Ca2+ concentration at the IS with decreasing distance of the MTOC from the IS. Unexpectedly, a change of CRAC/Orai activity is not required to explain the observed Ca2+ changes. We conclude that rotation-driven relocation of the MTOC towards the IS together with an accumulation of PMCA pumps at the IS are sufficient to control the observed Ca2+ dynamics in T-cells during polarization.

Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including extracellular regulated kinase (ERK)1/2 and protein kinase B (AKT) as well as the G-protein-coupled receptor (GPCR) membrane-type bile acid receptor (TGR5/M-BAR). Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be sensitive to pertussis toxin (PTX) and dominant-negative Gα(i) in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid-activated phylogenetic family (expressed in HEK293 cells) identified sphingosine-1-phosphate receptor 2 (S1P(2) ) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P-induced activation of ERK1/2 and AKT were significantly inhibited by JTE-013, a S1P(2) antagonist, in primary rat hepatocytes. JTE-013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knockdown of the expression of S1P(2) by a recombinant lentivirus encoding S1P(2) shRNA markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P(2) knock out (S1P(2) (-/-) ) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P(2) can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily through S1P(2) in primary rodent hepatocytes.

Tumor stroma plays an important role in cancer development. Stromal fibroblasts often represent the majority of stromal cells within various types of human carcinomas, and they are competent to promote the growth of cancer cells and to enhance tumor angiogenesis. However, the effect of known anti-cancer drugs on stromal cells has not been thoroughly investigated. Topotecan (TPT) is a semi-synthetic analogue of camptothecin, and several studies have shown that TPT inhibited angiogenesis via its direct effect on vascular endothelial cells. Here, we studied the effect of TPT on pro-angiogenesis action of hypoxic fibroblasts (MRC-5 cells). Growth inhibition was analyzed by MTT assay, while transwell assay and tube formation assay were used to evaluate the inhibition by TPT of hypoxic MRC-5 cell angiogenic activity in vitro. ELISA and Western blot analysis were used to investigate the related mechanism. Pretreatment of MRC-5 with TPT remarkably attenuated the induction of migration and tube formation of HUVECs by conditioned medium from hypoxic MRC-5 cells. In addition, topotecan decreased hypoxia-induced VEGF production by MRC-5 cells. Moreover, topotecan inhibits HIF-1α and α-SMA protein expression in hypoxic MRC-5 cells. Our data suggest that TPT inhibits hypoxic fibroblast angiogenic activity via downregulation of HIF-1α and prevention of fibroblast differentiation to myofibroblast.

In the following study, we prepared a double-chain miniprotein with each chain containing three linear repeats of the self-peptide gonadotropin-releasing hormone (GnRH3), the hinge region of human IgG1 (hinge), and a T-helper epitope from the measles virus protein (MVP). The di-GnRH3-hinge-MVP miniprotein was conjugated to purified recombinant heat shock protein 65 (Hsp65) of Mycobacterium bovis and used to immunize BALB/c mice primed with subcutaneous injection of Bacillus Calmette-Gurerin (BCG) in the absence of adjuvants. After anti-GnRH antibodies were successfully produced, mice were inoculated with H22 cells as a solid tumor. The results showed that after GnRH was inhibited by anti-GnRH antibodies the testosterone levels in sera markedly decreased (P < 0.01) and the testicle weights reduced as well (P < 0.05) in GnRH3-hinge-MVP-Hsp65-immunized mice. The average weight of tumors in mice treated with GnRH3-hinge-MVP-Hsp65 was significantly lower than in mice treated with saline only (neutral control, P < 0.001), or less than in mice treated with Hsp65 (negative control, P < 0.005). The data reported here demonstrated that GnRH3-hinge-MVP-Hsp65 could significantly attenuate the progression of liver tumor in mice transplanted with H22 cells, and might develop to be palliative treatment of hepatocellular carcinoma (HCC) patients in the future.

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I–treated animals. Taken together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer–induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer. Cancer Res; 72(3); 726–35. ©2012 AACR.

Background The role of B Lymphocyte Stimulator (BLyS) in the survival of malignant B cells and the maintenance of normal B cell development and homeostasis has been intensively studied in the literature. However, the influence of BLyS on breast cancer progression remains unclear. The study aimed to investigate the effect of hypoxia on BLyS regulation, cell migratory response to BLyS and the possible molecular mechanisms. Methods In this study, we examined the role of BLyS in the migration of human breast cancer cells by transwell assay. We also explored whether BLyS and its receptors expressed in human breast cancer cell lines by immunofluorescence and Western Blotting. Then we detected the expression level of BLyS in both normoxic and hypoxic conditions by real time-PCR and Western Blotting. Pathways involved were confirmed by Western Blotting, immunofluorescence, transwell assay and luciferase assay. Results According to our study, the expression level of BlyS was increased in human breast cancer cell lines in hypoxic conditions. Up-regulation of this protein led to activation and nuclear translocation of NF-kappa B p65. We also found that the number of migrated cells was increased in the presence of BLyS and inhibition of phosphorylation of Akt attenuated the enhanced migratory response. Conclusions It suggested that better understanding of BLyS, an immunopotentiator, may offer a potential therapeutic target for the treatment of human breast cancers. In addition, BLyS promoted breast cancer cells migration, underscoring the necessity of appropriate applications of immunopotentiators to cancer treatment.

The chemokine CC motif receptor 5 (CCR5) and its ligands have been reported to be associated with cancer progression and metastasis. Although recent researches have demonstrated a fundamental role of hypoxia in cancer, the effect of hypoxia on the expression and function of CCR5 and its ligands in cancer cells is unknown. Here, we investigated the status of CCR5 and its ligands in cancer cells under hypoxic conditions. Quantitative polymerase chain reaction, western blotting and immunofluorescence staining showed that hypoxia induced a strong increase of CCR5 expression. Dual luciferase assay and mRNA stability analysis indicated that hypoxia‐induced CCR5 mRNA expression relied on both transcriptional and posttranscriptional mechanisms. We detected the expression of CCR5 ligands and found that chemokine CC motif ligand 5 (CCL5) was induced under hypoxia. Recombinant human CCL5 stimulated cell migration rather than cell proliferation under hypoxia, and neutralization of CCL5 inhibited hypoxia‐induced migration of cancer cells. Similarly, overexpression of CCR5 increased cell migration, and knockdown of CCR5 attenuated hypoxia‐mediated cell migration. We further showed that hypoxia‐inducible factor‐1α (HIF‐1α) was involved in CCR5 and CCL5 regulation under hypoxia. HIF‐1α mRNA levels were highly correlated with CCR5 mRNA and CCL5 mRNA levels in clinical samples. CCR5 and CCL5 were highly expressed in breast cancer lymph nodes metastases. Taken together, our data suggest that CCR5‐CCL5 interaction promotes cancer cell migration under hypoxia. (Cancer Sci 2012; 103: 904–912)

Adhesion and migration of tumor cells are crucial steps in tumor invasion and metastasis. In the present study, we investigated the effects of saponin monomer 13 of dwarf lilyturf tuber (DT-13) on metastasis of human breast cancer cells (MDA-MB-435) during hypoxia. The effects and molecular mechanisms of DT-13 on MDA-MB-435 cells metastatic phenotype in vitro and in vivo were evaluated by RNA interference; quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. DT-13 had no significant effects on cell adhesion and migration under normoxia conditions. Under hypoxic conditions, MDA-MB-435 adhesion to vitronectin was inhibited by about 43.5% or 60.8% after exposure of the cells to DT-13 at 1 microM or 10 microM, respectively. DT-13 decreased the migratory response by hypoxia at 1 or 10 microM, and inhibition ratios were 20% and 30%, respectively. DT-13 inhibited hypoxia-induced expression of alphavbeta3 integrin, tissue factor (TF) and early growth response gene-1 (Egr-1) and decreased excretion of matrix metalloproteinase 9 (MMP-9) of MDA-MB-435 cells under hypoxic conditions. After Egr-1 short interference RNA (siRNA) treatment, DT-13 could still inhibit the up-regulation of TF mRNA and protein levels and its pro-coagulant activity (PCA) under hypoxia. In nude mice, DT-13 decreased extravasation of MDA-MB-435 cells in the lung after tail vein injection. Our data suggest that DT-13 inhibits MDA-MB-435 cells metastasis during hypoxia via regulation of TF, and the effect of DT-13 on TF is partly mediated by Egr-1.

The aim of this study was to investigate the effect of topotecan (TPT) on cancer cell migration. Growth inhibition of TPT was analyzed by MTT assay, and cancer cell migration was measured by transwell double chamber assay. To verify the effect of TPT on the chemokine receptors CXCR4 and CCR7, quantitative PCR, semi-quantitative PCR and Western blot analysis were performed. The secretion of MMP-2 and MMP-9 was detected by enzyme-linked immunosorbent assay (ELISA) and gelatin zymography. To evaluate possible contributions of CCR7 to MMP secretion, the overexpression vectors pcDNA3.1+-CCR7 and CCR7 siRNA were transiently transfected into MDA-MB-435 cells. TPT inhibited cancer cell migration in a dose-dependent manner. Additionally, TPT significantly decreased the expression of CCR7 in both MDA-MB-435 and MDA-MB-231 cells and moderately reduced the expression of CXCR4 in MDA-MB-435 cells. The secretion of MMPs (MMP-2, MMP-9) was also inhibited by TPT. Overexpression of CCR7 increased the secretion of MMP-2/9 and cancer cell migration, whereas knockdown of CCR7 reduced active MMP-2/9 production and migration of MDA-MB-435 cells. TPT inhibited cancer cell migration by down-regulation of CCR7 and MMPs (MMP-2 and MMP-9).

The chemokine and chemokine receptor families have important roles in tumorigenesis. Although CXCR4 and CCR7 have been reported to be associated with cancer metastasis, the role of other chemokine receptors in cancer is poorly understood. We explored the status of CXCR6 in hypoxia-induced cell migration. Breast cancer cells and human umbilical vein endothelial cells (HUVEC) expressed CXCR6, and showed appreciable chemotactic migration to CXCL16. Significant accumulation of CXCR6 mRNA and protein during hypoxia was observed. Overexpression of CXCR6 increased cell migration, and knockdown of CXCR6 attenuated hypoxia-mediated cell migration and MMP-2 secretion. To investigate possible mechanisms regulating CXCR6 expression during hypoxia, we detected the expression of HIFs and found that HIF-1alpha was involved in CXCR6 regulation. CXCR6 and HIF-1alpha were highly expressed in breast cancer lymph nodes metastases. Our data suggest CXCR6 contributes significantly to cell migration during hypoxia.