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.

Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production.

BACKGROUND: The application of viral elements in tumor therapy is one facet of cancer research. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus has previously been demonstrated to induce apoptosis in cancer cell lines. Here, we aim to further investigate its apoptotic mechanism and possible anti-metastatic effect in murine models of hepatocellular carcinoma (HCC), one of the most common human cancers worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with rVP1 inhibited cell proliferation in two murine HCC cell lines, BNL and Hepa1-6, with IC50 values in the range of 0.1-0.2 µM. rVP1 also induced apoptosis in these cells, which was mediated by Akt deactivation and dissociation of Ku70-Bax, and resulted in conformational changes and mitochondrial translocation of Bax, leading to the activation of caspases-9, -3 and -7. Treatment with 0.025 µM rVP1, which did not affect the viability of normal hepatocytes, suppressed cell migration and invasion via attenuating CCL2 production. The production of CCL2 was modulated by Akt-dependent NF-κB activation that was decreased after rVP1 treatment. The in vivo antitumor effects of rVP1 were assessed in both subcutaneous and orthotopic mouse models of HCC in immune-competent BALB/c mice. Intratumoral delivery of rVP1 inhibited subcutaneous tumor growth as a result of increased apoptosis. Intravenous administration of rVP1 in an orthotopic HCC model suppressed tumor growth, inhibited intra-hepatic metastasis, and prolonged survival. Furthermore, a decrease in the serum level of CCL2 was observed in rVP1-treated mice. CONCLUSIONS/SIGNIFICANCE: The data presented herein suggest that, via inhibiting Akt phosphorylation, rVP1 suppresses the growth, migration, and invasion of murine HCC cells by inducing apoptosis and attenuating CCL2 production both in vitro and in vivo. Recombinant protein VP1 thus has the potential to be developed as a new therapeutic agent for HCC.

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.

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.

A putative polyketide-synthesis protein XC5357 from Xanthomonas campestris: heterologous expression, crystallization and preliminary X-ray analysis.

Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative yellow-pigmented bacterium and is the causative agent of black rot, one of the major worldwide diseases of cruciferous crops. It also synthesizes a variety of polyketide metabolites that lead to important antibiotics. XC5357 is a putative 12.2 kDa protein of unknown structure from Xcc that is likely to be essential for polyketide synthesis. It was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 43.7, b = 43.7, c = 46.5 A, alpha = 65.0, beta = 64.9, gamma = 73.4 degrees, and diffracted to a resolution of 1.85 A.