A novel c-Kit/phospho-prohibitin axis enhances ovarian cancer stemness and chemoresistance via Notch3-PBX1 and ß-catenin-ABCG2 signaling.

BACKGROUND: The underlying mechanism involved in ovarian cancer stemness and chemoresistance remains largely unknown. Here, we explored whether the regulation of c-Kit and plasma membrane prohibitin (PHB) affects ovarian cancer stemness and chemotherapy resistance. METHODS: Mass spectrum analysis and an in vitro kinase assay were conducted to examine the phosphorylation of PHB at tyrosine 259 by c-Kit. The in vitro effects of c-Kit on membrane raft-PHB in ovarian cancer were determined using tissue microarray (TMA)-based immunofluorescence, western blotting, immunoprecipitation, colony and spheroid formation, cell migration and cell viability assays. In vivo tumor initiation and carboplatin treatment were conducted in nude mice. RESULTS: We found that c-Kit and PHB colocalized in the raft domain and were positively correlated in human ovarian serous carcinoma. c-Kit interacted with PHB and facilitated the phosphorylation of PHB at tyrosine 259 (phospho-PHBY259) in the membrane raft to enhance ovarian cancer cell motility. The generation of SKOV3GL-G4, a metastatic phenotype of SKOV3 green fluorescent protein and luciferase (GL) ovarian cancer cells, in xenograft murine ascites showed a correlation between metastatic potential and stem cell characteristics, as indicated by the expression of c-Kit, Notch3, Oct4, Nanog and SOX2. Further study revealed that after activation by c-Kit, raft-phospho-PHBY259 interacted with Notch3 to stabilize Notch3 and increase the downstream target PBX1. Downregulation of raft-phospho-PHBY259 increased the protein degradation of Notch3 through a lysosomal pathway and inhibited the ß-catenin-ABCG2 signaling pathway. Moreover, raft-phospho-PHBY259 played an important role in ovarian cancer stemness and tumorigenicity as well as resistance to platinum drug treatment in vitro and in vivo. CONCLUSIONS: These findings thus reveal a hitherto unreported interrelationship between c-Kit and PHB as well as the effects of raft-phospho-PHBY259 on ovarian cancer stemness and tumorigenicity mediated by the Notch3 and ß-catenin signaling pathways. Targeting the c-Kit/raft-phospho-PHBY259 axis may provide a new therapeutic strategy for treating patients with ovarian cancer.

Autophagic degradation of SQSTM1 inhibits ovarian cancer motility by decreasing DICER1 and AGO2 to induce MIRLET7A-3P.

The relationship between macroautophagy/autophagy and miRNA in regulating cancer cell motility is not clearly delineated. Here, we found that induction of BECN1-dependent or -independent autophagy decreased ubiquitin-binding proteins SQSTM1/p62 and CALCOCO2/NDP52. Downregulation of SQSTM1 (but not CALCOCO2) led to a decrease of the miRNA-processing enzyme DICER1 and the miRNA effector AGO2. The autophagy-mediated reduction of levels of SQSTM1, DICER1 or AGO2 resulted in increased MIRLET7A-3P (but not MIRLET7A-5P or PRE-MIRLET7A miRNA) and suppressed ovarian cancer motility. The investigation of the MIRLET7A effects on cancer cell motility showed that synthetic MIRLET7A-3P (3 nM) inhibited, whereas MIRLET7A-5P (100 nM) increased cancer cell motility. Moreover, downregulation of MIRLET7A-3P with antisense of MIRLET7A-3P miRNA (MIRLET7A-3P inhibitor; 3 nM) reversed the nutrient depletion- and rVP1-mediated suppression of ovarian cancer cell motility. In addition, restoring SQSTM1, DICER1 and AGO2 with inhibition of autophagic degradation or overexpression of DICER1 and AGO2 reversed the autophagy-associated enhancement of MIRLET7A-3P and inhibition of motility. Examination of ovarian cancer tissue microarray further showed that the levels of SQSTM1, DICER1 and AGO2 in the tumor were higher than those in the non-tumor cells and negatively correlated with the levels of autophagy and MIRLET7A-3P. Our results demonstrated that induction of autophagy to decrease SQSTM1, DICER1 and AGO2 and increase MIRLET7A-3P is a potential therapeutic strategy for suppressing ovarian cancer cell motility. Abbreviations: ACTB: actin beta; AGO2: argonaute 2, RISC catalytic component; ATG: autophagy related; BCIP/NBT: 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium; BECN1: beclin 1, autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; DICER1: dicer 1, ribonuclease III; EBSS: Earle balanced salt solution; FBS: fetal bovine serum; HGF: hepatocyte growth factor; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MIRLET7A: microRNA LET-7A: MIR16: microRNA 16; MIR29C: microRNA 29C; miRNA: microRNA; MMP: matrix metallopeptidase; PRE-MIRNA: precursor microRNA; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RISC: RNA-induced silencing complex; rVP1: recombinant foot-and-mouth disease virus capsid protein VP1; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; WIPI: WD repeat domain, phosphoinositide interacting.

PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer.

Activation of IKK enhances NF-κB signaling to facilitate cancer cell migration, invasion and metastasis. Here, we uncover the existence of a negative feedback loop of IKK. The transcription factor PATZ1 induces protein phosphatase-4 (PP4) regulatory subunit 2 (PP4R2) in an IKK-dependent manner. PP4R2 enhances the binding of PP4 to phosphorylated IKK to inactivate IKK/NF-κB signaling during sustained stimulation by cellular stimuli such as growth factors and inflammatory mediators. Matched pair studies reveal that primary lung cancers express more PATZ1 and PP4R2 than lymph node metastases in patients. Ectopic PATZ1 decreases invasion/colonization of lung cancers and prolongs the survival of xenograft mice. These effects of PATZ1 are reversed by downregulating PP4R2. Our results suggest that PATZ1 and PP4R2 provide negative feedback on IKK/NF-κB signaling to prevent cancer cells from over-stimulation from cellular stimuli; a decline in PATZ1 and PP4R2 is functionally associated with cancer migration/invasion and agents enhancing PATZ1 and PP4R2 are worth exploring to prevent invasion/metastasis of lung cancers.

Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent.

Macrophage proliferation and migration are important for many facets of immune response. Here we showed that stimulation of macrophages with type B CpG oligodeoxynucleotides (CpG-B ODNs) such as CpG-ODN 1668 increased the production of anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1Ra) in a TLR9- and MyD88-dependent manner. The CpG-B ODNs-induced IL-1Ra increased macrophage migration and promoted macrophage proliferation by down-regulating the expression of a cell cycle negative regulator, p27 to increase cell population in the S phase. The induction of IL-1Ra by CpG-B ODNs was F-spondin dependent. Knockdown of F-spondin and IL-1Ra decreased CpG-B ODNs-induced macrophage migration whereas overexpression of IL-1Ra increased migration of those cells. These findings demonstrated novel roles for F-spondin and IL-1Ra in CpG-B ODNs-mediated cell proliferation and migration of macrophages.

MIG-7 and phosphorylated prohibitin coordinately regulate lung cancer invasion/metastasis.

Growth factors and COX-2/PGE2 enhance lung cancer invasion/metastasis via PI3K/Akt and RAS/Raf. Here, we explored their mechanism of action further. We found first that higher levels of migration inducting gene-7 protein (MIG-7) and PHB phosphorylated at threonine 258 (phospho-PHBT258) are positively correlated with advanced stages of human lung cancer in tissue microarray. PGE2 or growth factors such as EGF, HGF and IGF-1 increased complex formation of phospho-PHBT258 with Ras, phospho-AktS473, phospho-Raf-1S338, MEKK1 and IKKα/ßS176/180 in the raft domain transiently within 1 hour and MIG-7 in the cytosol 12-24 hours later. Association of phospho-PHBT258 with MEKK1 but not MEKK3 activates IKK/IκB/NF-κB and MEK/ERK to increase cellular COX-2/PGE2 and an E-cadherin suppressor Snail leading to enhancement of epithelial-mesenchymal transition (EMT) and lung cancer migration/invasion. MIG-7, on the other hand, was induced by growth factors and PGE2 via Akt/GSK-3ß in a phospho-PHBT258 independent manner. MIG-7 increased two E-cadherin suppressors ZEB-1 and Twist to enhance EMT and cancer migration/invasion. Downregulating phospho-PHBT258 and MIG-7 had an additive effect on attenuating lung cancer invasion/metastasis and prolonging the survival of xenograft mice. Phospho-PHBT258 and MIG-7 may thus play complementary roles in the initiation and sustainment of the effects of growth factors and COX-2/PGE2 on cancer invasion/metastasis.

Recombinant viral capsid protein VP1 suppresses lung cancer metastasis by inhibiting COX-2/PGE2 and MIG-7.

Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus binds to integrins to modulate Akt/GSK3-ß signaling and suppress migration/invasion and metastasis of cancer cells, but the underlying molecular mechanism is unclear. Here, we showed that the rVP1-mediated inhibition of Akt/GSK3-ß signaling and cell migration/invasion was accompanied by downregulation in phosphatidylinositol (3,4,5)-triphosphate (PIP3), integrin-linked kinase (ILK) and IKK/NF-κB signaling as well as suppression of COX-2/PGE2 and MIG-7. Addition of PIP3 or overexpression of ILK reversed the rVP1-induced inhibition of IKK/NF-κB signaling, COX-2 and MIG-7. The rVP1-mediated downregulation of COX-2/PGE2 and MIG-7 led to not only attenuation of epithelial-mesenchymal transition, MMP2 activity and invasion of lung cancer cells in vitro but also decreased tumor growth and metastasis of lung cancer in xenograft mice. Moreover, downregulation of COX-2/PGE2 and MIG-7 significantly prolonged the overall and disease-free survival of lung cancer-bearing mice. These results suggest that rVP1 inhibits cancer invasion/metastasis, partly if not mainly, via downregulating integrin/PI3K/Akt, ILK and IKK/NF-κB signaling to suppress expression of COX-2/PGE2 and MIG-7.

MIG-7 controls COX-2/PGE2-mediated lung cancer metastasis.

More effective treatments for metastatic lung cancer remain a pressing clinical need. In this study, we identified migration inducting gene-7 (MIG-7) protein as critical for COX-2/prostaglandin E2 (PGE2)- and Akt/GSK-3ß-dependent tumor invasion/metastasis. COX-2/PGE2 activated EP4 to enhance Akt and GSK-3ß phosphorylation and ß-catenin/T-cell factor/lymphoid enhancer factor signaling leading to MIG-7 upregulation. RNAi-mediated attenuation of MIG-7 blocked COX-2/PGE2- and Akt/GSK-3ß-mediated migration/invasion effects. Furthermore, MIG-7 protein inhibited protein phosphatase 2A to sustain Akt/GSK-3ß phosphorylation and cancer-cell migration/invasion. Cancer cells overexpressing MIG-7 exhibited increased expression of ZEB-1 and Twist in parallel with epithelial-mesenchymal transition, metastasis and cancer lethality. MIG-7 protein level positively correlated with advanced stages of human lung cancers. MIG-7 thus offers a theranostic target for cancer metastases arising from aberrant activation of the cellular COX-2/PGE2 and Akt/GSK-3ß signaling pathways.

Recombinant protein rVP1 upregulates BECN1-independent autophagy, MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration.

The monocyte/macrophage is critical for regulating immune and antitumor responses. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus induces apoptosis and inhibits migration/metastasis of cancer cells. Here, we explored the effects of rVP1 on macrophages. Our results showed that rVP1 increased LC3-related autophagosome formation via WIPI1 and WIPI2 in a BECN1-independent manner. rVP1 treatment increased macrophage migration that was attenuated by knockdown of ATG5, ATG7, WIPI1 or WIPI2 and was abolished when both WIPI1 and WIPI2 were depleted. Treatment of macrophages with rVP1 increased matrix metalloproteinase-9 (MMP9) activity and phosphorylated mitogen-activated protein kinase 1/3 (MAPK1/3), two major mediators of cell migration. Knockdown of WIPI1, WIPI2, ATG5 and ATG7 but not BECN1 attenuated the rVP1-mediated increase in MAPK1/3 phosphorylation and MMP9 activity. These results indicated that rVP1 upregulated autophagy, MAPK1/3 phosphorylation and MMP9 activity to promote macrophage migration, which was dependent on WIPI1, WIPI2, ATG5 and ATG7 but not BECN1.

Recombinant viral protein VP1 suppresses HER-2 expression and migration/metastasis of breast cancer.

Breast cancer is one of the most common cancers in women worldwide and metastasis is the major cause of breast cancer death. Development of new therapeutic agents for inhibiting breast cancer metastasis is therefore an urgent need. We previously demonstrated that recombinant DNA-derived viral capsid protein VP1 (rVP1) of foot-and-mouth disease virus-induced apoptosis of MCF-7 breast cancer cells in vitro. Here, we investigated whether rVP1 exhibits any inhibitory effects on migration/metastasis and human epidermal growth factor receptor 2 (HER-2), a well-known biomarker for poor prognosis of breast cancer. The effects of rVP1 on cancer cell migration/invasion and metastasis were evaluated using Transwell migration assay and animal cancer models of metastasis. Western blotting, RT-PCR, flow cytometry, immunohistochemistry, and immunofluorescence staining techniques were used to investigate the effects of rVP1 on HER-2 and signal transduction mediators. Non-cytotoxic concentrations of rVP1-induced mesenchymal-epithelial transition and significantly suppressed AP-2α and HER-2 expression as well as the migration and invasion of a variety of breast cancer cell lines in a ß1-integrin-dependent manner in vitro. Gross and histopathologic examinations showed that rVP1 also suppressed metastasis of several breast cancer cell lines, including HER-2-overexpressing SK-BR-3 and BT-474 cells to lung, liver, or peripheral lymph node in orthotopic allograft/xenograft murine models. In addition, rVP1 significantly prolonged survival in breast cancer-bearing mice. Notably, no apparent side effects of rVP1 were detected, as shown by normal complete blood count levels and serum biochemistry profiles, including AST, ALT, BUN, and creatine. This study demonstrates that rVP1 suppresses the migration, invasion, and metastasis of breast cancer cells via binding to ß1 integrin receptor and down-regulation of AP-2α and HER-2 expression. The effectiveness of rVP1 on inhibiting migration/metastasis of breast cancer and HER-2 expression suggests that it may be suitable for serving as potential therapeutics for metastatic breast cancer particularly HER-2-overexpressing cancer.

3'poly-G-tailed ODNs inhibit F-spondin to induce cell death and neurite retraction in rat embryonic neurons.

The effects and mechanism of action of oligodeoxyribonucleotides containing CpG motif (CpG-ODNs) on neuron cells are largely unexamined. Here, we found that CpG-A ODNs but not other types of CpG-ODNs induced neurite retraction and cell apoptosis of rat embryonic neurons in a TLR9-independent manner. These effects of CpG-A ODNs were primarily due to the poly-guanosine at the 3' terminus (3'G-ODNs). Pull-down analysis showed that 3'G-ODNs associated with transcription factor Y-BOX1 (YB-1) to facilitate the translocation of YB-1 into the nucleus via the nuclear localizing sequence of YB-1. YB-1 then interacted with the promoter of F-spondin directly at -45 and -1,375 sites as demonstrated by chromatin immunoprecipitation (ChIP) analysis. Binding of YB-1 to F-spondin promoter resulted in downregulation of F-spondin expression. Overexpression of F-spondin rescued the cell death and neurite retraction induced by 3'G-ODNs in embryonic neuron cells. Taken together, these findings suggest that 3'G-ODNs enhance nucleus YB-1 to inhibit F-spondin leading to cell death and neurite retraction of embryonic neuron cells.

Recombinant viral capsid protein VP1 suppresses migration and invasion of human cervical cancer by modulating phosphorylated prohibitin in lipid rafts.

Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus inhibits invasion/metastasis of cancer cells. Here we studied its mechanism of action on human cervical cancer cells. The inhibition of cell invasion by rVP1 was accompanied with reduction in phosphatidylinositol (3,4,5)-triphosphate (PIP3), phospho-Akt S473, phosphorylated prohibitin (phospho-PHB) T258 in lipid rafts, dissociation of phospho-PHB T258 with Raf-1 and the inactivation of Raf-1/ERK. Addition of PIP3 or overexpression of constitutively active Akt and raft-anchored PHB T258 but not PHB T258I mutant protein reversed the inhibitory effects of rVP1. rVP1 inhibited cervical tumor growth and metastasis, and prolonged survival in xenograft mouse models. These results suggest that rVP1 inhibits cancer metastasis via de-phosphorylation of Akt and PHB T258 in lipid rafts to downregulate Raf/ERK signaling.

Recombinant viral protein promotes apoptosis and suppresses invasion of ovarian adenocarcinoma cells by targeting α5ß1 integrin to down-regulate Akt and MMP-2.

BACKGROUND AND PURPOSE: As prognosis for patients with metastatic ovarian cancer is generally poor, advances in treatment are needed. Here, we studied the mechanism of action of a recombinant viral capsid protein (rVP1) and explored its effect against ovarian tumour growth and metastasis in vivo. EXPERIMENTAL APPROACH: The human ovarian cancer cell line SKOV3 and BALB/cAnN-Foxn1 female nude mice were used. Effects of rVP1 on the viability, invasive ability, matrix metalloproteinase (MMP)-2 activity and cancer cell proliferation and metastasis were determined by cell proliferation assay, Matrigel invasion assay, gelatin zymographic analysis, as well as bioluminescence imaging and immunohistological analysis in xenograft mouse models respectively. Levels of total and phosphorylated focal adhesion kinase (FAK), PKB/Akt, phosphatase and tensin homologue (PTEN) and glycogen synthase kinase-3ß (GSK-3ß) were detected by Western blotting. KEY RESULTS: rVP1 promoted apoptosis and decreased invasion of human ovarian cancer cells. This effect of rVP1 was accompanied by activation of PTEN and GSK-3ß as well as down-regulation of FAK, Akt and MMP-2. Anti-integrin antibodies or overexpression of constitutively active Akt reversed the cellular effects of rVP1. Orthotopic and intraperitoneal xenograft mouse models demonstrated that rVP1 attenuated survival and metastasis of human ovarian cancer SKOV3 cell line in vivo through selective regulation of Akt and GSK-3ß activity as shown by bioluminescence imaging of mice and immunohistochemical analysis. CONCLUSION AND IMPLICATIONS: These results indicate that negative regulation of Akt signalling and MMP-2 by rVP1 may have the potential to suppress ovarian tumour growth and metastasis in vivo.

A fibrillar form of fibronectin induces apoptosis by activating SHP-2 and stress fiber formation.

Fibronectin (FN) is an endogenous ligand of integrins, which plays a critical role in cell adhesion and growth. Here, we converted globular FN (G-FN) into a fibrillar form (F-FN) and found that, even though both G-FN and F-FN interacted with integrin alpha5beta1, G-FN induced cellular proliferation, whereas F-FN resulted in apoptosis that was associated with deactivation of Akt/GSK-3beta and phosphorylation of SHP-2. SHP-2 inhibitor and anti-sense oligodeoxynucleotide decreased SHP-2 level and reversed the F-FN mediated apoptosis. F-FN also induced stress fiber formation associated with activation of RhoA, Rho kinase (ROCK), and filamin. Inhibition of ROCK by ROCK inhibitor or dominant negative plasmid treatment modulated F-FN mediated apoptosis. Pharmacological studies revealed that F-FN was effective in inhibiting the survival of SKOV-3 and MCF-7 cancer cells. These findings thus demonstrate that unlike G-FN, F-FN exhibits fibrillar structure to induce cell apoptosis that is associated with phosphorylation of SHP-2, activation of RhoA/ROCK and formation of stress fibers as well as deactivation of Akt/GSK-3beta.

F-spondin plays a critical role in murine neuroblastoma survival by maintaining IL-6 expression.

F-spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro-2a cells expressed a significant level of IL-6, but only trace amounts of IL-12, tumor necrosis factor alpha and nitric oxide. Knock-down of F-spondin mRNA in murine neuroblastoma NB41A3 and Neuro-2a cells using small interfering RNAs led to decreased IL-6 levels along with lower resistance to serum starvation and cytotoxic amyloid beta(1-42) (Abeta(1-42)) peptide. Restoring decline of F-spondin or IL-6 induced by F-spondin knock-down through adding exogenous F-spondin, IL-6 or over-expressing F-spondin reversed the cell death induced by Abeta(1-42) peptide or serum starvation. The decrease of IL-6 level was positively correlated with decrease of NF-kappaB and inhibition of p38 mitogen-activated protein kinase (MAPK). Over-expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL-6 production, restored the decrease of p38 induced by F-spondin knock-down, and rescued the cells from death caused by Abeta(1-42) peptide. Taken together, these results suggest that F-spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL-6 level via a MEKK/p38 MAPK/NF-kappaB-dependent pathway.

Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway.

BACKGROUND: Numerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. As well, the detailed mechanism of the cytotoxic effects is not well characterized. In this study, we have developed a novel process to convert native proteins into the fibrillar form. We used globular bovine serum albumin (BSA) as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein. RESULTS: We induced BSA, a non-cytotoxic globular protein, to become fibril by a novel process involving Superdex-200 column chromatography in the presence of anionic or zwittergenic detergent(s). The column pore size was more important than column matrix composite in fibril formation. The fibrillar BSA induced apoptosis in BHK-21 cell as well as breast cancer cell line T47D. Pre-treating cells with anti-integrin antibodies blocked the apoptotic effect. Fibrillar BSA, but not globular BSA, bound to integrin, dephosphorylated focal adhesion kinase (FAK), Akt and glycogen synthase kinase-3beta (GSK-3beta). CONCLUSION: We report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3beta/caspase-3 signaling pathway. Our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism(s) of the etiologic agents.

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes.

BACKGROUND: Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. METHODS: We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164) of FMDV VP1. RESULTS: The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-gamma. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. CONCLUSION: Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

Effects of CpG-B ODN on the protein expression profile of swine PBMC.

The CpG motif within bacterial DNA is a potent immuno-stimulatory moiety. Here, using a 2-D electrophoretic approach, we investigated the effect of synthetic oligodeoxynucleotide containing a B type CpG motif (CpG-B ODN) on the protein expression profile of swine peripheral blood mononuclear cells (PBMC). We found that several proteins including spondin 1, N-acetolactate alpha linked acidic dipeptidase; V kappa light chain, T cell receptor variable alpha chain, heat shock protein (Hsp) 60, Hsp70, KIAA0857 protein, and PNAS-146 were up-regulated in PBMC by CpG-B ODN stimulation. Further studies showed that CpG-B ODN-mediated Hsp60, Hsp70 and Hsp90 expressions were closely associated with the TLR9 signalling pathway. Pretreatment with inhibitors of Hsp70, Hsp90 and TLR9 all blocked the CpG-B ODN-mediated anti-apoptotic effect in swine PBMC. These results suggest that CpG-B ODN treatment of swine PBMC may enhance the expression of biologically active proteins, notably spondin 1, V kappa light chain, T cell receptor variable alpha chain and Hsps, which may play an important role in CpG-B ODN-mediated activation of immune responses and enhancement of swine PBMC survival.