Combination of lithium chloride and pEGFP-N1-BmK CT effectively decreases proliferation and migration of C6 glioma cells

Deleterious invasiveness of glioma cells into the normal brain tissue is endorsed by its inherent ability to regulate the receptor-mediated adhesive properties, extracellular matrix degradation and remodeling and elevated secretory ability of metalloproteinase (MMPs) such as MMP-2. By doing so, it will create an intercellular space for the invasion of glioma cells. Here, we reported that combination of gene therapy Buthus martensii Karsch (BmK) CT, a type of scorpion toxin peptide, with lithium chloride (LiCl), clinically used as mood stabilizer, could inhibit the migration and invasion of C6 glioma cells. The results showed that concomitant administration of LiCl and pEGFP-N1-BmK CT on glioma cells would hamper pro-MMP2 secretion and in the meantime, inhibited its proliferation in a synergistic manner. These results try to extrapolate the potential interplay between the combined treatment of LiCl and BmK CT with signaling pathways β-catenin, MMP, GSK-3 in C6 glioma cells. This strategy can stand for a novel approach designated for the development of a new method for glioma therapy.     

Adenovirus-mediated expression of BmK CT suppresses growth and invasion of rat C6 glioma cells

BmK CT, one of the key toxins in the venom of the scorpion, Buthus martensii Karsch, can interact specifically with glioma cells as a chloride channel blocker and inhibit the invasion and migration of those cells via MMP-2. A recombinant adenovirus, Ad-BmK CT, was constructed and characterized by in vitro and in vivo studies, using MTT cytotoxicity assay and the glioma C6/RFP (red fluorescence protein)/BALB/c allogeneic athymic nude mice model, respectively. The adenovirus-mediated expression of BmK CT displayed a high activity in suppressing rat C6 glioma cells growth and invasion thereby suggesting that this recombinant adenovirus may be a powerful method for treating glioblastoma.     

A potential strategy for high-grade gliomas: combination treatment with lithium chloride and BmK CT

Therapies for high-grade gliomas (HHG) that have strong tendency of infiltration and resistance to chemotherapies are currently unavailable. Here, we report that lower-dose combination therapy of Buthus martensii Karsch (BmK) CT, a type of scorpion toxin peptide, and LiCl, clinically used as mood stabilizer, could synergistically inhibit the migration, invasion and proliferation of C6 glioma cells. The decreased invasiveness of C6 glioma cells was accompanied by inhibited activation, catalytic activity and/or expression of matrix metalloproteinase-2. Moreover, TOPfalsh luciferase reporter and immunofluorescence staining showed altered localization pattern of β-catenin at the leading edge of 2D scratch. Our results suggested that the combination treatment of lithium and BmK CT may constitute a novel and potential strategy for HHG therapy.     

Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. Despite the advances in surgical and medical management, the mechanisms underlying human osteosarcoma progression and metastasis remain to be elucidated. Gene expression profiles were compared by the cDNA microarray technique between two different human osteosarcoma sublines, MNNG/HOS and 143B, which differ greatly in spontaneous pulmonary metastatic potential. Here we report an enhanced expression of matrix metalloproteinase (MMP)-1 in the highly metastatic human osteosarcoma cell line 143B. Moreover, the in vitro invasion activity of 143B cells was MMP-1-dependent. The activator protein (AP)-1 binding site in the MMP-1 gene promoter was required for the constitutive expression of MMP-1 in 143B cells. Two AP-1 components, c-Jun and Fra-1, were phosphorylated, and bound to the AP-1 binding site of the MMP-1 promoter in 143B cells. Activated c-Jun and Fra-1 were essential for MMP-1 gene expression in 143B cells. Mitogen-activated protein kinase pathways including the c-Jun NH₂-terminal kinase and the extracellular signal-regulated kinase activate c-Jun and Fra-1 and thereby regulate c-Jun/Fra-1 mediated events, establishing the mitogen-activated protein kinase/AP-1/MMP-1 axis as important in 143B cells. These data suggest that MMP-1 plays a central role in osteosarcoma invasion. Accordingly, MMP-1 might be a biomarker and therapeutic target for invasive osteosarcomas and pulmonary metastases.     

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Background

Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.

Methodology/Principal Findings

MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of αVβ3, α6β1 and α9β1 integrins in xenograft cells. Treatment with bicistronic constructs reduced αVβ3, α6β1 and α9β1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.

Conclusions/Significance

Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.     

Display of <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus GP64 on the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Spore Coat

To investigate whether Bombyx mori immunized with Bacillus subtilis spore displaying GP64 escape from the B. mori nucleopolyhedrovirus (BmNPV) attack, a recombinant integrative plasmid named pJS700-GP64 was constructed, which carries a recombinant cotC-Gp64 gene under the control of the cotC promoter. In this study, pJS700-GP64 was transformed into B. subtilis 168 (trp⁻) competent cells, an amylase (amyE) inactivated mutant was selected, and was confirmed to be a double cross-over integrant, cotC-Gp64 fragment of which was integrated into B. subtilis chromosome. Gp64 was expressed on the spore surface and recognized by Gp64-specific antibody. Results of B. mori when challenged with BmNPV indicated that B. mori vaccinated with the recombinant spores possessed resistance to the invasion of BmNPV at some degree.     

uPAR and cathepsin B shRNA impedes TGF-β1-driven proliferation and invasion of meningioma cells in a XIAP-dependent pathway

Overexpression of transforming growth factor β1 (TGF-β1) has been linked to immune suppression, tumor angiogenesis, tumor cell migration, tumor cell survival, and tumor cell invasion in many cancers. In the present study, we found abundant expression of TGF-β1 in the microenvironment of four different pathological types of meningioma tumors. TGF-β1 induced invasion in malignant meningioma cells with an associated upregulation of urokinase-type plasminogen activator (uPA), uPAR, cathepsin B, and MMP-9, and this increase in proliferation was coupled with the expression of anti-apoptotic and pro-survival signaling molecules. In addition to the intense immunoreactivity of meningioma tumors to X-linked inhibitor to apoptosis (XIAP), its knockdown abolished the TGF-β1-induced proliferation of these cells. The stimulation of XIAP expression and the activation of pSMAD-2 is mediated by phosphatidylinositol 3-kinase (PI3K)- and MEK-dependent pathways, and the addition of anti-TGF-β1 antibodies prevented their expression with a consequent decrease in invasion. Bicistronic shRNA constructs targeting uPAR and cathepsin B (pUC) quenched TGF-β1-driven invasion and survival of meningioma cells by downregulation of XIAP and pSMAD-2 expression. Animal models with intracranial tumors showed elevated levels of TGF-β1, XIAP and pSMAD-2, and pUC treatment prevented this increased expression. Thus, targeted silencing of TGF-β1-induced signaling by pUC in meningioma would provide new treatment approaches for management of meningioma.     

MMP-2 siRNA inhibits radiation-enhanced invasiveness in glioma cells

Background

Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.

Methodology/Principal Findings

We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.

Conclusion/Significance

MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients.     

Protease Nexin-1 affects the migration and invasion of C6 glioma cells through the regulation of urokinase Plasminogen Activator and Matrix Metalloproteinase-9/2

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6 + 27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion.     

SfP53 and filamentous actin (F-actin) are the targets of viral pesticide AcMNPV-<Emphasis Type="Italic">Bm</Emphasis>K IT (P10/PH) in host <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> 9 cells

Objectives

To analyze the anti-insect mechanism of viral pesticide AcMNPV-BmK IT(P10/PH) in the host Spodoptera frugiperda 9 (Sf9) cells.

Results

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)- mediated expression of BmK IT, regulated by P10 protein promoter (P10) and polyhedrosis promoter (PH), promoted the replication of progeny virus in host Sf9 cells. AcMNPV-BmK IT(P10) could accelerate the budding process (or speed) of budded virus (BV) in Sf9 cells. The impact of AcMNPV-BmK IT(P10) on the nuclear polymerization of filamentous actin (F-actin) participated in regulating the accelerated budding process. Unexpectedly, both AcMNPV-BmK IT(P10) and AcMNPV-BmK IT(PH) delayed the nuclear polymerization of F-actin and promoted the clearance of F-actin in the nucleus. SfP53, an important apoptosis factor, was involved in the regulation of AcMNPV-BmK IT(P10/PH) in Sf9 cells. AcMNPV-BmK IT(P10/PH) could also delay and promote the nuclear recruitment of SfP53 after 27 h post infection (h p.i.).

Conclusion

SfP53 and F-actin are the targets of viral pesticide AcMNPV-BmK IT (P10/PH) in host Sf9 cells, which provides the experimental basis for the development of recombinant baculovirus biopesticides.

     

Functional study of active residues scorpion insect toxin BmK IT from Buthus martensii Karsch

Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating. An excitatory insect toxin, BmK IT, is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is a toxin with a non-glutamate residue at the relevant position in the excitatory scorpion β-toxin subfamily. In this study, the mutants of recombinant BmK IT (BmK IT (I25E), BmK IT (E15G), BmK IT C-terminal (TKSYCDVQIN) truncated) were achieved by site-directed mutagenesis. Biological activity of BmK IT and its mutants confirmed these residues or peptides played key roles in BmK IT. BmK IT (I25E) could increase the sensitivity of BmK IT, but BmK IT(E15G) could decrease the sensitivity of BmK IT on Sf9 cells. BmK IT truncated C-terminal hydrophobic amino acids could cross the species boundaries and was effective on mammalian C6 cells. To date, several excitatory insect toxins have been isolated and identified from the venom of Buthus martensii Karsch. However, no functional data are available and therefore its classification in the family of excitatory insect toxins remains putative and is just based on its high similarity with the other toxins of this family. These results verified I25, E15 and C-terminal (TKSYCDVQIN) in BmK IT played key roles in the interaction of the BmK IT and its receptor- sodium channels on the surface of insect cells and laid a foundation for further structural and functional analysis of BmK IT.     

Integrin inhibition promotes atypical anoikis in glioma cells

Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation and motility. They are expressed by glioma cells and may contribute to their malignant phenotype. Integrin inhibition may therefore represent a promising therapeutic strategy. GL-261 and SMA-560 glioma cells grown under standard conditions uniformly detached and formed large cell clusters after integrin gene silencing or pharmacological inhibition using EMD-121974, a synthetic Arg-Gly-Asp-motif peptide, or GLPG0187, a nonpeptidic integrin inhibitor. After 120 h, the clusters induced by integrin inhibition decayed and cells died. In contrast, when cells were cultured under stem cell (sphere) conditions, no disaggregation became apparent upon integrin inhibition, and cell death was not observed. As poly-HEMA-mediated detachment had similar effects on cell viability as integrin inhibition, we postulated that cell death may result from detachment alone, which was confirmed using various permissive and nonpermissive substrates. No surrogate markers of apoptosis were detected and electron microscopy confirmed that necrosis represents the dominant morphology of detachment-induced cell death. In addition, integrin inhibition resulted in the induction of autophagy that represents a survival signal. When integrins were inhibited in nonsphere glioma cells, the TGF-β pathway was strongly impaired, whereas no such effect was observed in glioma cells cultured under sphere conditions. Cell death induced by integrin inhibition was rescued by the addition of recombinant transforming growth factor-β (TGF-β) and accelerated by exposure to the TGF-β receptor inhibitor, SD-208. In summary, cell death following integrin inhibition is detachment mediated, represents an atypical form of anoikis involving necrosis as well as autophagy, and is modulated by TGF-β pathway activity.     

Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9

The expression of matrix metalloproteinases (MMPs) produced by cancer cells has been associated with the high potential of metastasis in several human carcinomas, including breast cancer. Several pieces of evidence demonstrate that protein tyrosine phosphatases (PTP) have functions that promote cell migration and metastasis in breast cancer. We analyzed whether PTP inhibitor might control breast cancer invasion through MMP expression. Herein, we investigate the effect of 4-hydroxy-3,3-dimethyl-2H benzo[g]indole-2,5(3H)-dione (BVT948), a novel PTP inhibitor, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. The expression of MMP-9 and cell invasion increased after TPA treatment, whereas TPA-induced MMP-9 expression and cell invasion were decreased by BVT948 pretreatment. Also, BVT948 suppressed NF-κB activation in TPA-treated MCF-7 cells. However, BVT948 didn’t block TPA-induced AP-1 activation in MCF-7 cells. Our results suggest that the PTP inhibitor blocks breast cancer invasion via suppression of the expression of MMP-9. [BMB Reports 2013; 46(11): 533-538]     

Eukaryotic expression and immunogenicity of Ancylostoma ceylanicum calreticulin

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes human and animal intestines, causing malnutrition and iron-deficiency anemia. Calreticulin is a multifunctional protein involved in all stages of parasitic infection. Studies have found that parasites can secret calreticulin to regulate the host's immune response. To explore the immunogenicity of the eukaryotic expression plasmid of Ancylostoma ceylanicum calreticulin (Ace-CRT), we constructed a recombinant Ace-CRT eukaryotic expression plasmid (pEGFP-N3-Ace-CRT). Successful expression of the target protein in Human Embryonic Kidney (HEK) 293 T cells was confirmed by indirect immunofluorescence and Western blot analysis. BALB/c mice were immunized with pEGFP-N3-Ace-CRT plasmid. Measuring IgG antibody levels in immunized mice sera by ELISA showed that the recombinant plasmid stimulated IgG antibody production in mice. Spleen lymphocytes were collected from vaccinated mice to determine the proportion of T cell subsets and the expression levels of cytokines. Flow cytometry revealed that the percentage of CD3 + CD4+ and CD3 + CD8+ T cells in mice spleen in the immunization group was significantly higher than that in the control group. Recombinant plasmid immunization increased IL-4, IL-10, IL-12, and IL-13 expression while decreasing IL-5, IL-6, and INF-γ in mice spleens. These results indicate that the eukaryotic plasmid constructed in this study had good immunogenicity and mainly induced a T helper 2 response in the host, laying a foundation for screening candidate molecules for anti-hookworm vaccines.     

Role of fucosyltransferase IV in epithelial–mesenchymal transition in breast cancer cells

Epithelial–mesenchymal transition (EMT) is a crucial step in tumor progression and has an important role during cancer invasion and metastasis. Although fucosyltransferase IV (FUT4) has been implicated in the modulation of cell migration, invasion and cancer metastasis, its role during EMT is unclear. This study explores the molecular mechanisms of the involvement of FUT4 in EMT in breast cancer cells. Breast cancer cell lines display increased expression of FUT4, which is accompanied by enhanced appearance of the mesenchymal phenotype and which can be reversed by knockdown of endogenous FUT4. Moreover, FUT4 induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt, and inactivation of GSK3β and nuclear translocation of NF-κB, resulting in increased Snail and MMP-9 expression and greater cell motility. Taken together, these findings indicate that FUT4 has a role in EMT through activation of the PI3K/Akt and NF-κB signaling systems, which induce the key mediators Snail and MMP-9 and facilitate the acquisition of a mesenchymal phenotype. Our findings support the possibility that FUT4 is a novel regulator of EMT in breast cancer cells and a promising target for cancer therapy.