Diabetes as a prognostic factor in HER-2 positive breast cancer patients treated with targeted therapy.

PURPOSE: Recent studies revealed that metabolic stress influences the outcomes of breast cancer treatment. We sought to evaluate the prognostic effect of type 2 diabetes and find the molecular mechanism of relapses in postoperative HER-2+ breast cancer patients treated with HER-2 targeted therapy. MATERIALS AND METHODS: We evaluated 190 HER-2+ breast cancer patients (pT1-4N0-2M0) who were treated with surgical resection and trastuzumab (HER-2 targeted therapy) between 2006 and 2015. Survival outcomes and failure patterns were compared between such patients with (n = 12) and without (n = 178) type 2 diabetes. RESULTS: The median follow-up period was 42.4 months (range 12.0-124.7 months). Twenty-one patients (11.1%) showed relapse (including nine patients with locoregional failure), and three patients (1.6%) died as a result of cancer relapse. One-third of the patients with diabetes experienced relapse (4/12, 33.3%). The 3-year disease-free survival (DFS) and overall survival (OS) rates were 90.7% and 98.6%, respectively. Diabetic patients showed shorter DFS compared with non-diabetic patients (p = 0.006, 74.1% vs. 91.9%). OS was also shorter in diabetic patients compared with non-diabetic patients (p = 0.017, 91.7% vs. 99.1%). Of our interest, the levels of HER-3 and its ligand neuregulin-1 were significantly increased in the tumor specimen in HER-2+ breast cancer patients suffering with type 2 diabetes than that in the euglycemic control group. CONCLUSIONS: Type 2 diabetes was associated with detrimental effects on survival in postoperative HER-2+ breast cancer patients who were treated with trastuzumab. The poor prognostic effect of diabetes in HER-2+ breast cancer patients could be associated with the high levels of HER-3 and neuregulin 1, thus it should be considered and evaluated more.

COL6A3-derived endotrophin links reciprocal interactions among hepatic cells in the pathology of chronic liver disease.

Extracellular matrix dysregulation is associated with chronic liver disease. CollagenVI-alpha3 chain (COL6A3) is a biomarker for hepatic fibrosis and poor prognosis of hepatocellular carcinoma (HCC), but its function in liver pathology remains unknown. High levels of COL6A3 and its cleaved product, endotrophin (ETP) in tumor-neighboring regions are strongly associated with poor prognosis in HCC patients. Here, we report that the high levels of ETP in injured hepatocytes induce JNK-dependent hepatocyte apoptosis and activate nonparenchymal cells to lead further activation of hepatic inflammation, fibrosis, and apoptosis. Nevertheless ETP per se showed limited phenotypic changes in normal liver tissues. Furthermore, inhibition of ETP activity by utilizing neutralizing antibodies efficiently suppressed the pathological consequences in chronic liver diseases. Our results implicate ETP mechanistically as a crucial mediator in reciprocal interactions among various hepatic cell populations in the pathogenesis of chronic liver disease, and it could be a promising therapeutic target particularly in individuals with high local levels of COL6A3. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lumazine synthase protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development.

PURPOSE: Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation. MATERIALS AND METHODS: Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo. RESULTS: We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8(+)T cells and OT-2-specific CD4(+)T cells. CONCLUSION: Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications.

Development of P22 viral capsid nanocomposites as anti-cancer drug, bortezomib (BTZ), delivery nanoplatforms.

Genetic and chemical engineering approaches are used to employ P22 viral capsids as nanoplatforms for developing an efficient delivery vehicle. Catechol ligands are chemically attached to the interior surface of P22 viral capsid for subsequent encapsulation of an anticancer drug, bortezomib (BTZ), through boronic acid-diol complexation. For targeted delivery, hepatocellular carcinoma (HCC)-targeting peptide (SP94, SFSIIHTPILPL) is synthesized and chemically conjugated to the exterior surface of the P22 viral capsid nanocomposites. Effective targeted delivery of synthesized P22 viral capsid nanocomposites is demonstrated by fluorescent cell imaging and the efficacy of delivered P22 viral capsid nanocomposites is evaluated using a cell viability assay.

Ferritin protein cage nanoparticles as versatile antigen delivery nanoplatforms for dendritic cell (DC)-based vaccine development.

We utilized ferritin protein cage nanoparticles (FPCN) as antigen delivery nanoplatforms for DC-based vaccine development and investigated DC-mediated antigen-specific immune responses. Antigenic peptides, OT-1 (SIINFEKL) or OT-2 (ISQAVHAAHAEINEAGR) which are derived from ovalbumin, were genetically introduced either onto the exterior surface or into the interior cavity of FPCN. FPCN carrying antigenic peptides (OT-1-FPCN and OT-2-FPCN) were effectively delivered to DCs and processed within endosomes. Delivered antigenic peptides, OT-1 or OT-2, to DCs successfully induced antigen-specific CD8(+) or CD4(+) T cell proliferations both in vitro and in vivo. Naïve mice immunized with OT-1-FPCN efficiently differentiated OT-1 specific CD8(+) T cells into functional effector cytotoxic T cells resulting in selective killing of antigen-specific target cells. Effective differentiation of proliferated OT-2 specific CD4(+) T cells into functional CD4(+) Th1 and Th2 cells was confirmed with the productions of IFN-γ/IL-2 and IL-10/IL-13 cytokines, respectively. FROM THE CLINICAL EDITOR: In this study, the authors utilized ferritin protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development and investigated DC-mediated antigen-specific immune responses using strong model antigens derived from ovalbumin, suggesting potential future clinical applicability of this or similar techniques.

Implementation of P22 viral capsids as intravascular magnetic resonance T1 contrast conjugates via site-selective attachment of Gd(III)-chelating agents.

P22 viral capsids and ferritin protein cages are utilized as templating macromolecules to conjugate Gd(III)-chelating agent complexes, and we systematically investigates the effects of the macromolecules' size and the conjugation positions of Gd(III)-chelating agents on the magnetic resonance (MR) relaxivities and the resulting image contrasts. The relaxivity values of the Gd(III)-chelating agent-conjugated P22 viral capsids (outer diameter: 64 nm) are dramatically increased as compared to both free Gd(III)-chelating agents and Gd(III)-chelating agent-conjugated ferritins (outer diameter: 12 nm), suggesting that the large sized P22 viral capsids exhibit a much slower tumbling rate, which results in a faster T1 relaxation rate. Gd(III)-chelating agents are attached to either the interior or exterior surface of P22 viral capsids and the conjugation positions of Gd(III)-chelating agents, however, do not have a significant effect on the relaxivity values of the macromolecular conjugates. The contrast enhancement of Gd(III)-chelating agent-conjugated P22 viral capsids is confirmed by in vitro phantom imaging at a short repetition times (TR) and the potential usage of Gd(III)-chelating agent-conjugated P22 viral capsids for in vivo MR imaging is validated by visualizing a mouse's intravascular system, including the carotid, mammary arteries, the jugular vein, and the superficial vessels of the head at an isotropic resolution of 250 µm.

Fabrication of uniform layer-by-layer assemblies with complementary protein cage nanobuilding blocks via simple His-tag/metal recognition.

A capsid-forming enzyme, lumazine synthase isolated from hyperthermophile Aquifex aeolicus (AaLS), is prepared and utilized as a template for constructing nanobuilding blocks to fabricate uniform layer-by-layer (LbL) assemblies. Two functionally complementary AaLS protein cage nanoparticles (PCNs) are generated either by genetically introducing His-tags on the surface of wild-type AaLS PCNs or by chemically attaching metal chelates (Ni-NTA moiety) to the surface of cysteine-bearing AaLS PCNs individually. The multivalent displays of His-tags (AaLS-His6 PCN) and Ni-NTA ligands (AaLS-NTA-Ni PCN) on the surface of each complementary AaLS PCN are successfully demonstrated by mass spectrometric and surface plasmon resonance analyses. By using these two complementary AaLS PCNs, uniform LbL assemblies are constructed via simple recognition between His-tags and metal chelates without the aid of additional binding mediators. This approach illustrates the potential of fabricating uniform nanostructures using protein-based hybrid functional nanobuilding blocks.

Incorporation of thrombin cleavage peptide into a protein cage for constructing a protease-responsive multifunctional delivery nanoplatform.

Protein cages are spherical hollow supramolecules that are attractive nanoscale platforms for constructing cargo delivery vehicles. Using ferritin isolated from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Fn), we developed a multifunctional protein cage-based delivery nanoplatform that can hold cargo molecules securely, deliver them to the targeted sites, and release them to the targeted cells. The release is triggered by cleavage induced by the protease, thrombin. The thrombin cleavage peptide (GGLVPR/GSGAS) was inserted into the flexible loop region of Pf_Fn, which is located at a 4-fold axis of symmetry exposed on the surface of protein cages (Thr-Pf_Fn). Subsequently, the C-terminal glycine, which is situated in the interior cavity, was substituted with cysteine (G173C) to permit site-specific conjugation of cargo molecules. The introduced cysteine (G173C) was labeled with a fluorescent probe (F5M-Thr-Pf_Fn) for cell imaging and cargo release monitoring. The surface of F5M-Thr-Pf_Fn was further modified with biotins (F5M-Thr-Pf_Fn-NPB) as targeting ligands. The specific binding of dual functionalized F5M-Thr-Pf_Fn-NPB to the MDA MB 231 cell line, which overexpresses biotin-specific receptors on its surface, was confirmed by fluorescence microscopic analyses. The inserted thrombin cleavage peptides were effectively cleaved by thrombin, resulting in the release of the C-terminal helix in buffer and on the targeted cells without disruption of the cage architecture. Protein cage scaffolds that combine genetic and chemical modifications may serve as stimulus-responsive delivery nanoplatforms and provide opportunities for developing new types of theranostic nanoplatforms.

Developing an antibody-binding protein cage as a molecular recognition drug modular nanoplatform.

We genetically introduced the Fc-binding peptide (FcBP) into the loop of a self-assembled protein cage, ferritin, constituting four-fold symmetry at the surface to use it as a modular delivery nanoplatform. FcBP-presenting ferritin (FcBP-ferritin) formed very stable non-covalent complexes with both human and rabbit IgGs through the simple molecular recognition between the Fc region of the antibodies and the Fc-binding peptide clusters inserted onto the surface of FcBP-ferritin. This approach realized orientation-controlled display of antibodies on the surfaces of the protein cages simply by mixing without any complicated chemical conjugation. Using trastuzumab, a human anti-HER2 antibody used to treat patients with breast cancer, and a rabbit antibody to folate receptor, along with fluorescently labeled FcBP-ferritin, we demonstrated the specific binding of these complexes to breast cancer cells and folate receptor over-expressing cells, respectively, by fluorescent cell imaging. FcBP-ferritin may be potentially used as modular nanoplatforms for active targeted delivery vehicles or molecular imaging probes with a series of antibodies on demand.

Rutin inhibits osteoclast formation by decreasing reactive oxygen species and TNF-alpha by inhibiting activation of NF-kappaB.

Rutin, a glycoside of flavonol, inhibits osteoclast formation induced by receptor activator of NF-kappaB ligand (RANKL) in bone marrow-derived macrophages. It reduces reactive oxygen species produced by RANKL and its inhibitory effect results from reduced levels of TNF-alpha. Rutin also lowers NF-kappaB activation in response to RANKL.

Absence of 4-1BB increases cell influx into the peritoneal cavity in response to LPS stimulation by decreasing macrophage IL-10 levels.

Peritoneal injection of lipopolysaccharide (LPS) increased the influx of polymorphonuclear leukocytes and macrophages into the peritoneal cavity (PEC), with significantly higher cell numbers in the 4-1BB-deficient (KO) mice than in wild-type (WT) mice. The peritoneal macrophages of KO mice contained less IL-10 transcripts and protein than those of WT after LPS treatment, and immobilization of 4-1BB-Fc increased the level of IL-10. Injection of IL-10 resulted in lower cell numbers into the PEC of KO mice, suggesting that lower level of IL-10 is responsible for stimulated cell influx in KO mice due to lack of 4-1BB and 4-1BBL interaction.

Stimulation of osteoclastogenesis by enhanced levels of MIP-1alpha in BALB/c mice in vitro.

OBJECTIVES: We compared osteoclast (OC) formation in bone marrow-derived macrophages (BMM) from C57BL/6 (B/6) and BALB/c (B/c) mice. After stimulation of receptor activator of nuclear factor-kappaB ligand (RANKL), enhanced OC formation and higher level of macrophage inflammatory protein-1alpha (MIP-1alpha) were observed in the BMM from B/c mice. In this study, we determined whether MIP-1alpha is responsible for stimulated OC formation in the BMM. MATERIALS AND METHODS: OC formation was evaluated in BMM. Expression of MIP-1alpha during OC formation was analyzed at the mRNA and protein levels. Apoptosis of mature OCs was evaluated by observing the degradation of DNA. Activation of nuclear factor-kappaB (NF-kappaB) was measured by electrophoretic mobility shift assay. RESULTS: After stimulation by RANKL expression of MIP-1alpha at the mRNA and protein levels was much higher in BMM from B/c mice than in BMM from B/6 mice. Transcripts of the MIP-1alpha receptors, CCR1 and CCR5, were present at similar levels in unstimulated BMM of the two strains. Blockade of MIP-1alpha inhibited OC formation, and exogenously added MIP-1alpha stimulated it in RANKL-stimulated BMM. MIP-1alpha affected not only the early precursors but also mature OCs. It prevented apoptosis of mature OCs by activating NF-kappaB, and the effect of RANKL on survival was dependent on its ability to induce MIP-1alpha. CONCLUSIONS: MIP-1alpha, induced by RANKL during OC differentiation, increases OC formation by acting on OC progenitor cells, and prolongs survival of mature OC via signaling through NF-kappaB. The enhanced OC formation in BMM from B/c mice could be due to, at least in part, to their higher levels of MIP-1alpha.

Enhanced osteoclastogenesis in 4-1BB-deficient mice caused by reduced interleukin-10.

UNLABELLED: Enhanced osteoclastogenesis was observed in bone marrow-derived macrophage cells from 4-1BB-deficient mice than in those from wildtype mice. 4-1BB and 4-1BB ligand interaction may play a role at a certain stage of osteoclast formation through increased level of IL-10, a negative regulator of osteoclastogenesis. INTRODUCTION: 4-1BB is an inducible T-cell costimulatory molecule and a member of the TNF receptor family. The expression pattern of 4-1BB and 4-1BB ligand (4-1BBL) has suggested that 4-1BB plays a role not only in various responses related to innate immunity but also in bone metabolism. MATERIALS AND METHODS: Osteoclast formation was evaluated in bone marrow-derived macrophage cells (BMMs) from wildtype and 4-1BB-deficient (4-1BB-/-) mice. Expression of interleukin-10 (IL-10) during osteoclast formation was analyzed at the mRNA and protein levels. RESULTS: Expression of IL-10 was higher in RANKL-stimulated wildtype BMMs than 4-1BB-/- BMMs. When 4-1BBL was stimulated with 4-1BB-Fc fusion protein, the expression of IL-10 in BMMs increased. Neutralization of IL-10 was not as effective in preventing inhibition by IL-10 of osteoclast differentiation in 4-1BB-/- BMMs as in wildtype BMMs. When IL-10 was added to the culture medium, osteoclast formation was inhibited more efficiently in the 4-1BB-/- BMMs than in the wildtype BMMs. CONCLUSIONS: Interaction of 4-1BB and 4-1BBL stimulates IL-10 production through 4-1BBL signaling. 4-1BBL plays a role at a certain stage of osteoclast formation, and IL-10 may mediate this effect. The elevated level of osteoclastogenesis in 4-1BB-/- BMMs may thus be caused, in part, by a lower level of IL-10.

Soluble glucocorticoid-induced tumor necrosis factor receptor stimulates osteoclastogenesis by down-regulation of osteoprotegerin in bone marrow stromal cells.

Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) is a potent stimulator of osteoclastogenesis. The mechanism by which it induces osteoclastogenesis was studied by culturing bone-marrow-derived macrophages (BMM) with conditioned medium from mouse bone marrow stromal cells. GITR and GITR ligand (GITRL) were expressed on the surface of bone marrow stromal cells, and sGITR-induced osteoclastogenesis was inhibited by anti-GITRL Ab, indicating that stimulatory effect of osteoclastogenesis by sGITR involved signaling via GITRL. Bone marrow stromal cells up-regulated cyclooxygenase-2 (COX-2) and produced prostaglandin E(2) (PGE(2)) early in their response to sGITR, and the stimulation of osteoclastogenesis was markedly inhibited by NS398, a COX-2 inhibitor. Later, sGITR markedly reduced the steady-state level of osteoprotegerin (OPG) mRNA and increased receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA. NS398 blocked the sGITR-induced reduction of OPG mRNA but did not significantly affect the sGITR-induced rise in RANKL mRNA. This suggests that down-regulation of OPG by PGE(2) is involved in sGITR-induced osteoclast (OC) formation in the presence of conditioned medium from mouse bone marrow stromal cells.

A signal through 4-1BB ligand inhibits receptor for activation of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis by increasing interferon (IFN)-beta production.

We tested whether any intracellular signals are transmitted through 4-1BB/CD137 ligand (4-1BBL), using a 4-1BB-Fc fusion protein and 4-1BB-deficient mice. Immobilized 4-1BB-Fc fusion protein strongly inhibited osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) derived from bone marrow macrophages (BMM). Incubation of BMM with M-CSF increased 4-1BBL mRNA and surface expression of 4-1BBL protein. Cross-linking 4-1BBL with immobilized 4-1BB-Fc also dramatically reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) derived from the BMM from 4-1BB-deficient mice, suggesting that the inhibitory effect of immobilized 4-1BB on osteoclastogenesis is due to a signal through 4-1BBL. Reverse signaling by 4-1BB-Fc increased the level of interferon (IFN)-beta in BMM and neutralization of IFN-beta reversed the inhibitory effect of immobilized 4-1BB-Fc. Inhibition of osteoclastogenesis by immobilized 4-1BB-Fc is, therefore, at least in part, due to elevation of the level of the negative regulator, IFN-beta in BMM.

Corn silk induced cyclooxygenase-2 in murine macrophages.

Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE2. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-kappaB), indicating that COX-2 induction proceeds also via the NF-kappaB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE2 elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.

Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) stimulates osteoclast differentiation in response to receptor activator of NF-kappaB ligand (RANKL) in osteoclast cells.

We found that treatment of osteoclast (OC) precursors with soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) promoted osteoclastogenesis in the presence of macrophage colony-stimulating factor (M-CSF) and receptor for activation of nuclear factor-kappaB ligand (RANKL). Low levels of GITR and its ligand were expressed on the surface of OC precursor cells after incubation with RANKL. Stimulation of osteoclastogenesis by sGITR was blocked by neutralization with anti-GITR ligand antibody (Ab), whereas endogenous GITR did not affect osteoclastogenesis, indicating that enhancement of osteoclastogenesis by sGITR involves signaling via GITR ligand. The addition of sGITR decreased the level of interferon (IFN)-beta, and blockade of endogenous IFN-beta did not affect osteoclastogenesis stimulated by sGITR. We conclude that sGITR enhances osteoclastogenesis by acting on OC precursor cells to lower the level of IFN-beta.

Sulforaphane inhibits osteoclastogenesis by inhibiting nuclear factor-kappaB.

We show that sulforaphane inhibits osteoclastogenesis in the presence of macrophage colony-stimulating factor (M-CSF) and receptor for activation of nuclear factor-kB ligand (RANKL) in osteoclast (OC) precursors. Sulforaphane, an aliphatic isothiocyanate, is a known cancer chemo-preventative agent with anti-oxidative properties. Nuclear factor-kB (NF-kB) is a critical transcription factor in RANKL-induced osteoclastogenesis, and electrophoretic mobility shift assays (EMSAs) and assay of NF-kB-mediated secreted alkaline phosphatase (SEAP) revealed that sulforaphane selectively inhibited NF-kappaB activation induced by RANKL. Inhibition may involve interaction of sulforaphane with thiol groups, since it was prevented by reducing agents.

The soluble glucocorticoid-induced tumor necrosis factor receptor causes cell cycle arrest and apoptosis in murine macrophages.

In order to clarify the mechanism by which soluble GITR (sGITR) inhibits the survival of murine macrophages we examined its effect on the macrophage cell cycle. Soluble GITR induced G1 phase arrest followed by apoptosis. It also reduced the expression of cyclins D2 and A, and of cdk4, resulting in reduced cdk2 and cdk4 activities. These findings suggest that sGITR arrests division of the macrophages in G1 by lowering the activities of cdk2 and cdk4, and that this leads to apoptosis.

Soluble glucocorticoid-induced TNF receptor (sGITR) induces inflammation in mice.

Glucocorticoid-induced TNF receptor (GITR) was a new member of the TNF/nerve growth factor receptor (TNFR/ NGFR) family and induced in murine T cells by dexamathasone. Recombinant soluble GITR (sGITR) induced an inflammation in peritoneal membrane and changes in spleen after i.p. injection of 3 mg/kg in C57BL/6 mice. Spleen was enlarged and percentage of neutrophils and monocytes were increased. The area of red pulp in spleen was increased, while that of white pulp was decreased after GITR injection. The thickening of membrane and neutrophil infiltration was observed in peritoneal membrane with increased myeloperoxidase activity. At later time, neutrophil infiltration moved to inside the tissue with tissue damage. GITR ligand and GITR were expressed constitutively on the surface of spleen cells and cells from peritoneal fluid. In contrast, no significant change in the spleen and in peritoneal membrane was observed in mice treated with LPS. GITR may play a role in body's inflammatory processes.