Role In Cell Invasion Research Articles

Suppression of tumor invasion and migration in breast cancer cells following delivery of siRNA against Stat3 with the antimicrobial peptide PR39

PR39, a porcine cathelicidin rich in the amino acids proline and arginine can interact with the negatively charged component of the cell surface, and rapidly penetrate cell membranes. Therefore, we hypothesized that PR39, as a membrane penetrating peptide (MPP), could be exploited as a novel carrier to deliver siRNA into the cell cytoplasm in order to knockdown target gene expression. Firstly, a complex formation of PR39 with siRNA and its cellular colocalization were investigated in our studies. Further, we optimized the ratio of the PR39/siRNA complex, cell/complex incubation period and the concentration of siRNA. The results suggest that PR39 could form a complex with siRNA, and mediate translocation of the siRNA into 4T1 cells. The optimal ratio of siRNA with PR39 was 1:90 which was found to have a maximum Stat3 gene silencing effect after 48h treatment. Moreover, 4T1 cell proliferation, cell cycle, invasion and migration were investigated. The results suggested that Stat3 knockdown could not result in 4T1 cell proliferation inhibition and cell cycle arrest, while invasion and migration of 4T1 cells were strongly inhibited. Notably, the data also showed that in addition to inhibition of carcinogenesis, single PR39 may play a role in cell invasion and migration. PR39 and Stat3 siRNA displayed synergistic biological effects in inhibiting cell invasion and migration of 4T1 cells, which was more prominent compared with the popular Lipofectamine delivery system.

Overexpression of ADP‐ribosylation factor 1 in human gastric carcinoma and its clinicopathological significance

Gastric cancer is the sixth leading cause of cancer-related death in Taiwan, and the identification of related factors is essential to increase patient survival. ADP-ribosylation factor 1 (ARF1) was initially identified using 2-D electrophoresis combined with MALDI-time-of-flight mass spectrometry. ADP-ribosylation factor 1 belongs to the Ras superfamily or GTP-binding protein family and has been shown to enhance cell proliferation. In the current study, we evaluated the potential of ARF1 as a biomarker for gastric cancer detection. ADP-ribosylation factor 1 mRNA was upregulated in tumor tissues (compared with adjacent non-tumor tissues, n = 55) in approximately 67.2% of gastric cancer patients. Expression of ARF1 protein was additionally observed using Western blot and immunohistochemistry (IHC) analyses. The clinicopathological correlations of ARF1 were further evaluated. Elevated ARF1 expression was strongly correlated with lymph node metastasis (P = 0.008), serosal invasion (P = 0.046), lymphatic invasion (P = 0.035), and pathological staging (P = 0.010). Moreover, the 5-year survival rate for the lower ARF1 expression group (n = 50; IHC score < 90) was higher than that of the higher expression group (n = 60; IHC score ≥ 90) (P = 0.0228, log-rank test). To establish the specific function of ARF1 in human gastric cancer, isogenic ARF1-overexpressing cell lines were prepared. Our results showed that ARF1-overexpressing clones display enhanced cell proliferation, migration, and invasion. Furthermore, ARF1-overexpression might contribute to poor prognosis of patients. These findings collectively support the utility of ARF1 as a novel prognostic marker for gastric cancer and its role in cell invasion.

Abstract 1442: Expression of miRNA processing factor Dicer in cutaneous melanoma and its role in cell invasion

Abstract Deregulated expression of miRNAs is a hallmark of various human cancers. One potential mechanism for the aberrant expression of miRNAs in cancers is their abnormal processing due to altered expression or function of their processing factors such as Dicer and Drosha. Although altered expression of several miRNAs have been reported in melanoma, the expression profile of miRNA processing factors in this cancer is unknown. In this study we examined the expression of Dicer protein in different stages of melanocytic lesions. Using tissue microarray and immunohistochemistry, we evaluated cytoplasmic Dicer expression in 32 dysplastic nevi, 77 primary melanomas, and 48 metastatic melanomas. Our data revealed that expression of Dicer has a significant but inverse correlation with progression of melanoma (P &amp;lt; 0.001). Accordingly, the number of samples with moderate-strong staining for Dicer was reduced from 81.2% in dysplastic nevi to 67.5% in primary melanoma and 40.8% in metastatic melanoma. The expression of Dicer was also negatively correlated with the American Joint Committee on Cancer (AJCC) staging of the melanoma samples (p=0.019). Moreover, the reduced Dicer expression was correlated with a poorer disease-specific 5-year survival of melanoma patients (P = 0.026). Multivariate Cox regression analysis revealed that reduced nuclear Dicer expression is an independent prognostic factor to predict patient outcome (P = 0.030). We also knocked down Dicer expression and used Boyden chamber migration assay to study its role in the invasion ability of MMRU human melanoma cells. Interestingly, knockdown of Dicer enhanced the invasion ability of MMRU cells by 2-fold. Our results demonstrate the critical role of miRNA machinery in the progression of melanoma, suggesting that Dicer may be a suitable prognostic marker for human melanoma patients as well as a potential therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1442. doi:10.1158/1538-7445.AM2011-1442

Involvement of Voltage-Gated Sodium Channels in Breast Cancer Cells Invasiveness and Regulation by Docosahexaenoic Acid

Voltage-gated sodium channels (NaV) are expressed in highly invasive metastatic cancer cells derived from epithelial tissues. In the highly invasive breast cancer cell line MDA-MB-231, we showed that the abnormal expression of NaV1.5 was responsible for a sustained inward sodium current at the membrane potential, which promotes the invasion of the extracellular matrix. We found that NaV1.5 activity leads to a perimembrane acidification, favourable for the pH-dependent proteolytic activity of cysteine cathepsins which play a predominant role in cell invasiveness. Moreover, NaV and the sodium-proton exchanger NHE1 (a pH regulator) seem to be colocalized in specialized lipid rafts domains, caveolae, where cathepsins-B are known to be secreted. Since docosahexaenoic acid (DHA, 22:6 n-3) has been reported to reduce the aggressiveness of some breast cancers, and to be a modulator of ion channels, we investigated its role on NaV activity and cell invasiveness. Supplementation of MDA-MB-231 cells culture medium with 1 µM DHA for five days affected neither cell migration nor proliferation but significantly reduced cell invasiveness, while oleic acid (OA, 18:1 n-9), used as a control, had no effect. Moreover, the combination of DHA with tetrodotoxin (specific NaV blocker) demonstrated no further effect on invasion, suggesting a possible regulation of NaV activity by DHA. Indeed, NaV currents were reduced with DHA but not with OA supplementation. These fatty acids were inefficient to reduce sodium currents when acutely applied, suggesting a middle- or long-term regulation of NaV channels. However, no effect on NaV mRNA or protein expression and no change in NaV raft localization was observed. Thus, DHA effects could occur through a modification in cholesterol/phospholipids composition of the plasma membrane and/or a modification in factors regulating NaV channels activity and/or addressing to the plasma membrane.

Toxoplasma gondii toxolysin 4 is an extensively processed putative metalloproteinase secreted from micronemes

Proteases play central roles in cell invasion by Toxoplasma gondii and other apicomplexan parasites. Herein we report the cloning and characterization of a novel secretory putative metalloproteinase, Toxolysin 4 (TLN4). T. gondii tachyzoites store TLN4 in the micronemes and secrete it in response to elevated calcium, suggesting a possible role in cell invasion. TLN4 is initially synthesized as a large (∼260kDa) precursor, which is extensively processed into multiple proteolytic fragments within the parasite secretory system. At least some of these proteolytic fragments remain associated in a large molecular complex. Whereas precomplementation with the TLN4 cDNA allowed disruption of the TLN4 gene, multiple attempts to directly knockout TLN4 without precomplementation failed. TLN4 knockout parasites were detected by PCR in transfected populations but were lost from the cultures during drug selection and growth suggesting that TLN4 contributes to parasite fitness.

IQGAP1 Plays an Important Role in the Invasiveness of Thyroid Cancer

This study was designed to explore the role of IQGAP1 in the invasiveness of thyroid cancer and its potential as a novel prognostic marker and therapeutic target in this cancer. We examined IQGAP1 copy gain and its relationship with clinicopathologic outcomes of thyroid cancer and investigated its role in cell invasion and molecules involved in the process. We found IQGAP1 copy number (CN) gain ≥ 3 in 1 of 30 (3%), 24 of 74 (32%), 44 of 107 (41%), 8 of 16 (50%), and 27 of 41 (66%) of benign thyroid tumor, follicular variant papillary thyroid cancer (FVPTC), follicular thyroid cancer (FTC), tall cell papillary thyroid cancer (PTC), and anaplastic thyroid cancer, respectively, in the increasing order of invasiveness of these tumors. A similar tumor distribution trend of CN ≥ 4 was also seen. IQGAP1 copy gain was positively correlated with IQGAP1 protein expression. It was significantly associated with extrathyroidal and vascular invasion of FVPTC and FTC and, remarkably, a 50%-60% rate of multifocality and recurrence of BRAF mutation-positive PTC (P = 0.01 and 0.02, respectively). The siRNA knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion and colony formation. Coimmunoprecipitation assay showed direct interaction of IQGAP1 with E-cadherin, a known invasion-suppressing molecule, which was upregulated when IQGAP1 was knocked down. This provided a mechanism for the invasive role of IQGAP1 in thyroid cancer. In contrast, IQGAP3 lacked all these functions. IQGAP1, through genetic copy gain, plays an important role in the invasiveness of thyroid cancer and may represent a novel prognostic marker and therapeutic target for this cancer.

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11. Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

Doubles Game: Src-Stat3 versus p53-PTEN in Cellular Migration and Invasion

We have recently shown that Src induces the formation of podosomes and cell invasion by suppressing endogenous p53, while enhanced p53 strongly represses the Src-induced invasive phenotype. However, the mechanism by which Src and p53 play antagonistic roles in cell invasion is unknown. Here we show that the Stat3 oncogene is a required downstream effector of Src in inducing podosome structures and related invasive phenotypes. Stat3 promotes Src phenotypes through the suppression of p53 and the p53-inducible protein caldesmon, a known podosome antagonist. In contrast, enhanced p53 attenuates Stat3 function and Src-induced podosome formation by upregulating the tumor suppressor PTEN. PTEN, through the inactivation of Src/Stat3 function, also stabilizes the podosome-antagonizing p53/caldesmon axis, thereby further enhancing the anti-invasive potential of the cell. Furthermore, the protein phosphatase activity of PTEN plays a major role in the negative regulation of the Src/Stat3 pathway and represses podosome formation. Our data suggest that cellular invasiveness is dependent on the balance between two opposing forces: the proinvasive oncogenes Src-Stat3 and the anti-invasive tumor suppressors p53-PTEN.

CDH3/P-cadherin overexpression in breast carcinomas: its regulatory mechanisms, the role in cell invasion, and the association with cancer stem cell properties

One of the most basic characteristics of cancer cells is the loss of cell-cell adhesion and acquirement of invasive properties. During cancer progression, the regulation and expression of cell-cell adhesion molecules (like cadherins) play a pivotal role. In most invasive tumors, E-cadherin is downregulated and N-cadherin is de novo expressed (known as epithelial-to-mesenchymal transition). However, in some invasive carcinomas, E-cadherin expression is maintained, with the concomitant de novo expression of another cadherin – P-cadherin. Their co-expression occurs frequently in breast carcinomas, which show a worst patient prognosis when compared with patients harboring tumors with loss of E-cadherin as single event. In the last ten years, our group has been mainly interested in understanding the role of P-cadherin overexpression in cancer cells, as well as the gene regulatory mechanisms behind its aberrant expression. P-cadherin has been extensively studied concerning its function and prognostic value in breast cancer. Its overexpression has been identified in 30% of invasive carcinomas, being highly associated with proliferative lesions of high histological grade and decreased patient survival. Recently, we showed that P-cadherin overexpression confers an invasive capacity to breast cancer cells, inducing the secretion of MMPs, which are responsible by the cleavage of its extracellular domain, giving rise to a P-cadherin soluble form. We proved that this fragment is a pro-invasive factor, which needs to be inhibited to render cancer cells non-invasive. Still, we identified the intracellular signaling pathway that regulates and activates the P-cadherin (CDH3) gene promoter, inducing P-cadherin overexpression in breast cancer cells. We found that an antiestrogen is able to increase CDH3 promoter activity, as well as to induce activating histone epigenetic modifications at putative C/EBPβ binding sites in the CDH3 gene promoter. We showed, for the first time, that C/EBPβ is able to regulate P-cadherin overexpression. Furthermore, our recent data supports the idea that P-cadherin has a role in cancer stem cell biology. We found that breast cancer cell lines, presenting the highest levels of P-cadherin, show the highest expression of the stem cell markers CD49f, CD44 and CD24, as well as ALDH1 activity. Importantly, we reproduced these results in primary tumors, where we found that P-cadherin overexpression occurs in breast carcinomas with a cancer stem cell phenotype (CD44+CD24-/low) and ALDH1 expression. These results are now being experimentally addressed by mammosphere, 2D and 3D culture in vitro assays, to be then confirmed in vivo. In conclusion, our results open new avenues in breast cancer treatment, since P-cadherin is likely to be a good therapeutic target for invasive carcinomas overexpressing this protein. Scientifically, our studies will help to understand better the pathogenesis of breast cancer and other cancer models involving cadherin’s alterations.

Production of Interferon-Inducible Protein-10 and Its Role as an Autocrine Invasion Factor in Nasal Natural Killer/T-Cell Lymphoma Cells

Nasal natural killer (NK)/T-cell lymphoma is associated with Epstein-Barr virus and has poor prognosis because of local invasion and/or multiple dissemination. Recently, the role of chemokines/chemokine receptors in tumor proliferation and invasion has been shown. In this study, we examined whether the specific chemokines were related to the tumor behaviors in nasal NK/T-cell lymphoma. A chemokine protein array was used to examine specific chemokines produced by SNK-6 and SNT-8 (Epstein-Barr virus-positive nasal NK/T-cell lymphoma lines). The expression of interferon gamma-inducible protein 10 (IP-10) and the IP-10 receptor CXCR3 was investigated by ELISA and flow cytometry. Cell growth and invasion were assessed by the MTT and Matrigel invasion assays, respectively. Immunohistologic staining and ELISA were used to examine IP-10 expression in biopsies and sera from patients, respectively. IP-10 was specifically produced by SNK-6 and SNT-8. Moreover, CXCR3 was expressed on the NK cell lines. Functionally, IP-10 did not affect cell proliferation but enhanced cell invasion. In biopsy samples, IP-10 and CXCR3 expressions were detected in the lymphoma cells. Serum IP-10 levels in the patients were much higher than those of healthy controls and the levels were decreased during the complete remission phase after treatments. These results suggest that IP-10 may play an important role in cell invasion in nasal NK/T-cell lymphoma through an autocrine mechanism.

Positive regulation of spondin 2 by thyroid hormone is associated with cell migration and invasion

The thyroid hormone 3,3',5-triiodo-L-thyronine (T(3)) regulates growth, development, and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). Microarray analyses were performed previously to study the mechanism of regulation triggered by T(3) treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T(3). However, the underlying mechanism and the physiological role of T(3) in the regulation of spondin 2 are not clear. To verify the microarray results, spondin 2 was further investigated using semi-quantitative reverse transcription-PCR and western blotting. After 48 h of T(3) treatment in the HepG2-TR alpha 1#1 cell line, spondin 2 mRNA and protein levels increased by 3.9- to 5.7-fold. Similar results were observed in thyroidectomized rats. To localize the regulatory region in spondin 2, we performed serial deletions of the promoter and chromatin immunoprecipitation assays. The T(3) response element on the spondin 2 promoter was localized in the -1104/-1034 or -984/-925 regions. To explore the effect of spondin 2 on cellular function, spondin 2 knockdown cell lines were established from Huh7 cells. Knockdown cells had higher migration ability and invasiveness compared with control cells. Conversely, spondin 2 overexpression in J7 cells led to lower migration ability and invasiveness compared with control cells. Furthermore, this study demonstrated that spondin 2 overexpression in some types of hepatocellular carcinomas is TR dependent. Together, these experimental findings suggest that spondin 2, which is regulated by T(3), has an important role in cell invasion, cell migration, and tumor progression.

Moesin/ezrin: a specific role in cell metastasis?

Barsh, G.S. (2006). Regulation of pigment type switching by agouti, melanocortin signaling, attractin and mahoganoid. In The Pigmentary System, 2nd edn. J.J. Nordlund, R.E. Boissy, V.J. Hearing, R.A. King, W.S. Oetting, and J.P. Ortonne, eds (Massachusetts: Blackwell Publishing), pp. 395–409. Candille, S.I., Kaelin, C.B., Cattanach, B.M., Yu, B., Thompson, D.A., Nix, M.A., Kerns, J.A., Schmutz, S.M., Millhauser, G.L., and Barsh, G.S. (2007). A b-defensin mutation causes black coat color in domestic dogs. Science 318, 1418–1423. Chakrabarti, O., and Hegde, R.S. (2009). Functional depletion of mahogunin by cytosolically exposed prion protein contributes to neurodegeneration. Cell 137, 1136–1147. Hida, T., Wakamatsu, K., Sviderskaya, E.V. et al. (2009). Agouti protein, mahogunin, and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes: a cAMP-independent pathway. Pigment Cell Melanoma Res. 22, 623–634. Jiao, J., Kim, H.Y., Liu, R.R., Hogan, C.A., Sun, K., Tam, L.M., and Gunn, T.M. (2009). Transgenic analysis of the physiological functions of Mahogunin Ring Finger-1 isoforms. Genesis 47, 524–534.

Novel putative glycosylphosphatidylinositol-anchored micronemal antigen of Plasmodium falciparum that binds to erythrocytes.

We have identified a new Plasmodium falciparum erythrocyte binding protein that appears to be located in the micronemes of the merozoite stage of the parasite and membrane linked through a glycosylphosphatidylinositol (GPI) anchor. The protein is designated GPI-anchored micronemal antigen (GAMA) and was identified by applying a set of selection criteria to identify previously uncharacterized merozoite proteins that may have a role in cell invasion. The protein is also present in the proteomes of the sporozoite and ookinete micronemes and is conserved throughout the genus. GAMA contains a novel domain that may be constrained by disulfide bonds and a predicted C-terminal hydrophobic sequence that is presumably replaced by the GPI. The protein is synthesized late during schizogony, processed into two fragments that are linked by a disulfide bond, and translocated to an apical location, which is probably the micronemes. In a proportion of free merozoites GAMA can also be detected on the parasite surface. Following erythrocyte invasion the bulk of the protein is shed in a soluble form, although a short C-terminal fragment may be carried into the newly invaded red blood cell. The protein was shown to bind reversibly to erythrocytes and therefore represents a new example of a host cell binding protein.

A Mediator of Rho-dependent Invasion Moonlights as a Methionine Salvage Enzyme

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named “mediator of RhoA-dependent invasion (MRDI),” that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

The expression of β-adrenoceptors in human pancreatic cancer cell lines and their roles in cell invasiveness

Objective To investigate the effect and mechanism of β-adrenoceptors on norepi-nephrine-induced invasiveness of pancreatic cancer cell lines. Methods The expression of β-adrenocep-tors mRNA in human pancreatic cancer cell lines MiaPaCa-2 and BxPC3 was detected by using RT-PCR. The cells were randomly divided into control group.10 mol/L NE intervention group, 1 mol/L propranolol intervention group and NE + propranolol intervention group. After 48 h , transwell invasiveness test was used to examine the changes in invasive ability of MiaPaCa-2. The expression of MMP-2, MMP-9 and VEGF mRNA was measured by semi-quantitative RT-PCR. The levels of MMP-2 , MMP-9 and VECF proteins were assayed by immunocytochemistry. Results Both MiaPaCa-2 and BxPC3 expressed β1-and β2-adrenocep-tors. The absorbance ( A) values of invasive cells in NE, NE + propranolol, propranolol and control groups were 0.78±0.02 ,0.32±0.03 ,0.26±0.01 and 0.28±0.02 , respectively, and those in NE intervention group were significantly higher than in control and NE + propranolol groups ( P 0. 05) . In NE group , the expression index of MMP-2 , MMP-9 and VEGF mRNA was 0. 87±0.02 , 1.04±0.02 and 0. 92±0. 01 , and the gray value of the protein expression was 131.20±2.34,105.32±7.21 and 115.60 ±5. 03 , respectively, which were higher than those in control and NE + propranolol groups ( P 0.05 ) . Conclusion β-adrenoceptors play an important role in the process of norepinephrine-induced invasiveness of pancreatic cancer cells. NE can promote the invasiveness of MiaPaCa-2 through up-regulating the expression of MMP-2 , MMP-9 and VEGF via β-adre-noceptors. Key words: Pancreatic carcinoma; Invasiveness; β-adrenoceptors; Norepinephrine

The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation

Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.

The Urokinase Plasminogen Activator (uPA) System in Uterine Natural Killer Cells in the Placental Bed During Early Pregnancy

The urokinase plasminogen activator (uPA) system plays pivotal roles in cell invasion, adhesion and migration. Roles for uterine natural killer (uNK) cells in regulating extravillous trophoblast (EVT) invasion and spiral artery remodeling have been proposed. Placental bed biopsies from early pregnancy were obtained from three gestational age groups (8–10, 12–14 and 15–20 weeks). Total caseinase activity in the placental bed was studied using casein in situ zymography. Localisation of uPA, uPA receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and -2 in the placental bed was investigated by immunohistochemistry. CD56 + uNK cells were separated from collagenase-digested decidual cells using an immunomagnetic technique, and uPA activity was measured in isolated cell culture supernatants by casein/plasminogen gel zymography (8–10 and 12–14 weeks' gestation, n = 10 each group). uPAR in cell lysates and PAI-1 and -2 secretion in supernatants were measured by Western blotting. Caseinase activity was stronger in decidua than myometrium as shown by in situ zymography. uPA localised strongly to uNK cells, especially at 8–10 weeks. Moderate uPAR localisation on uNK cells also observed. There was very weak immunostaining of uNK cells for PAI-1 and PAI-2. In casein gel zymography, uPA activity was similar in uNK cell culture supernatant compared with total unseparated decidual cells. uPAR in uNK cell lysates was significantly stronger than in total decidual cell lysates. PAI-1 and PAI-2 were not detected in uNK cell culture supernatants by Western blot analysis. These results suggest that uNK cells may regulate EVT invasion and spiral artery remodeling via the uPA system.

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Deltaku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.

Toll-like receptor 2 mediates invasion via activating NF-κB in MDA-MB-231 breast cancer cells

MDA-MB-231 breast cancer cells have a high invasive potential, yet the mechanisms involved are not known. This study showed that Toll-like receptor 2 (TLR2) was highly expressed in MDA-MB-231 cells and played a critical role in cell invasion. Compared with the poorly invasive MCF-7 cells, MDA-MB-231 cells expressed 10.5-fold more TLR2. Using TLR2 agonist pg-LPS and TLR2 neutralizing antibody, we found that TLR2 activation significantly promoted MDA-MB-231 invasion, whereas TLR2 blockade diminished this capacity. TLR2 activation enhanced the activity of NF-κB and induced phosphorylation of TAK1 and IκBα in the TLR2/NF-κB signaling pathway in MDA-MB-231, but not in MCF-7 cells. TLR2 activation increased IL-6, TGF-β, VEGF and MMP9 secretion, which are associated with TLR2-NF-κB signaling. We demonstrated that TLR2 is a critical receptor responsible for NF-κB signaling activity and highly invasive capacity of MDA-MB-231 cells.