The Promoting Effect of the Extracellular Matrix Peptide TNIIIA2 Derived from Tenascin-C in Colon Cancer Cell Infiltration.

The extracellular matrix (ECM) molecule tenascin C (TNC) is known to be highly expressed under various pathological conditions such as inflammation and cancer. It has been reported that the expression of TNC is correlated with the malignant potential of cancer. In our laboratory, it was found that the peptide derived from the alternative splicing domain A2 in TNC, termed TNIIIA2, has been shown to influence a variety of cellular processes, such as survival, proliferation, migration, and differentiation. In this study, we investigated the effect of TNC/TNIIIA2 on the invasion and metastasis of colon cancer cells, Colon26-M3.1, or PMF-Ko14, using an in vitro and in vivo experimental system. The degree of cell invasion was increased by the addition of TNC and TNIIIA2 in a dose-dependent manner. The invasion by TNC and TNIIIA2 were suppressed by an MMP inhibitor or TNIIIA2-blocking antibody. In an in vivo experiment, pulmonary metastasis was promoted conspicuously by the addition of TNIIIA2. In this study, we found that colon cancer cell invasion and metastasis was accelerated by TNC/TNIIIA2 via MMP induction. This result suggests the possibility of a new strategy targeting TNC/TNIIIA2 for colon cancer.

Tenascin-C-derived peptide TNIIIA2 highly enhances cell survival and platelet-derived growth factor (PDGF)-dependent cell proliferation through potentiated and sustained activation of integrin α5ß1.

Tenascin-C is an adhesion modulatory matrix protein that is highly expressed in tumors; however, its biochemical activity involved in tumorigenesis is not fully understood. On the other hand, increasing evidence indicates the importance of integrin α5ß1 in cancer development. We previously demonstrated that tenascin-C harbors a functional site that can be released as a proadhesive peptide such as TNIIIA2. Peptide TNIIIA2 is capable of inducing activation of ß1-integrins including α5ß1 via syndecan-4. In this study the proadhesive effect of TNIIIA2 was characterized by potentiated and sustained activation of integrin α5ß1. Based on this effect, TNIIIA2 rendered nontransformed fibroblasts (NIH3T3) resistant to serum deprivation-elicited anoikis through activation of the Akt/Bcl-2 pathway. Moreover, TNIIIA2 hyperstimulated PDGF-dependent proliferation of NIH3T3 by activating integrin α5ß1. Tenascin-C, a parental protein of TNIIIA2, also stimulated PDGF-dependent proliferation, which was blocked by a matrix metalloproteinase-2/9 inhibitor and an anti-TNIIIA2 function-blocking antibody, suggesting proteolytic exposure of the proadhesive effect of TNIIIA2. Mechanistic analyses revealed that TNIIIA2 induced a lateral association of PDGF receptor ß with the molecular complex of activated integrin α5ß1 and syndecan-4 in the membrane microdomains enriched with cholesterol/caveolin-1, resulting in prolonged activation of PDGF receptor ß and the subsequent Ras/mitogen-activated protein kinase pathway in a PDGF-dependent manner. Of note, TNIIIA2 induced continuous proliferation in NIH3T3 in an integrin α5ß1-dependent manner even after they formed a confluent monolayer. Thus, it was proposed that tenascin-C might be involved in deregulated cell growth through potentiated and sustained activation of integrin α5ß1 after exposure of the proadhesive effect of TNIIIA2.

Eukaryotic translation elongation factor 1A induces anoikis by triggering cell detachment.

Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing ß1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage.

[Biochemical markers of bone turnover. New aspect. Bone metabolic markers available in daily practice].

Changes of bone remodeling markers reflect bone growth and bone turnover. Information on bone metabolism can be attained by blood and urine laboratory tests. Recently developed bone specific markers are categorized by bone remodeling process, i.e. bone formation and resorption. The formation markers include bone-specific alkaline phosphatase (BAP), osteocalcin (OC), undercarboxylated osteocalcin (ucOC), procollagene type I C- and N-terminal peptides (P1CP and P1NP). Bone resorption markers include deoxypyridinoline, collagen I C- and N-terminal telopeptides (CTX and NTX) , and tartrate resistent acid phosphatase (TRACP) isoform 5b. These laboratory tests offer lots of advantages for the diagnosis of bone metabolic disorders and for the evaluation of clinical states of primary osteoporosis and other metabolic skeletal diseases.

Aggressive Progression in Glioblastoma Cells through Potentiated Activation of Integrin α5ß1 by the Tenascin-C-Derived Peptide TNIIIA2.

Tenascin-C is a member of the matricellular protein family, and its expression level is correlated to poor prognosis in cancer, including glioblastoma, whereas its substantial role in tumor formation and malignant progression remains controversial. We reported previously that peptide TNIIIA2 derived from the cancer-associated alternative splicing domain of tenascin-C molecule has an ability to activate ß1-integrin strongly and to maintain it for a long time. Here, we demonstrate that ß1-integrin activation by TNIIIA2 causes acquisition of aggressive behavior, dysregulated proliferation, and migration, characteristic of glioblastoma cells. TNIIIA2 hyperstimulated the platelet-derived growth factor-dependent cell survival and proliferation in an anchorage-independent as well as -dependent manner in glioblastoma cells. TNIIIA2 also strongly promoted glioblastoma multiforme cell migration, which was accompanied by an epithelial-mesenchymal transition-like morphologic change on the fibronectin substrate. Notably, acquisition of these aggressive properties by TNIIIA2 in glioblastoma cells was abrogated by peptide FNIII14 that is capable of inducing inactivation in ß1-integrin activation. Moreover, FNIII14 significantly inhibited tumor growth in a mouse xenograft glioblastoma model. More importantly, FNIII14 sensitized glioblastoma cells to temozolomide via downregulation of O6-methylguanine-DNA methyltransferase expression. Consequently, FNIII14 augmented the antitumor activity of temozolomide in a mouse xenograft glioblastoma model. Taken altogether, the present study provides not only an interpretation for the critical role of tenascin-C/TNIIIA2 in aggressive behavior of glioblastoma cells, but also an important strategy for glioblastoma chemotherapy. Inhibition of the tenascin-C/ß1-integrin axis may be a therapeutic target for glioblastoma, and peptide FNIII14 may represent a new approach for glioblastoma chemotherapy. SIGNIFICANCE: These findings provide a proposal of new strategy for glioblastoma chemotherapy based on integrin inactivation.

[Bone quality changes in diabetes].

Diabetes-related bone fragility has recently drawn many researchers' attention. Diabetes would affect bone remodeling by various mechanisms, including deficiency of insulin actions, increased accumulation of advanced glycation end products and microangiopathy. The combination of poor bone quality of microstructure and nanoarchitecture (type I collagen and non-collageous proteins) would reduce bone strength. Bone mineral density is the best predictor for fractures of primary osteoporosis, but presumably not for type 2 diabetes. Quality changes of diabetic bone, therefore, should be more thoroughly studied.

[Newly developed drugs for osteoporosis in overseas and their future roles for the therapy].

Bisphosphonates are widely used, though gastrointestinal tolerance is a problem on daily administration. Intermittent regimen, from once weekly to once yearly, is now available in overseas and can overcome GI adverse events. New generation of anti-resorptive agents (anti-RANKL antibody and a new SERM, bazedoxifene) are promising and will be soon available for the treatment of osteoporosis. Anabolic agents such as teriparatide and strontium ranelate have marked effects on BMD and reduction on fracture risk. While none of these options is suitable for everyone, the range of future available therapies does mean that most patients can find an intervention that is effective and acceptable.

[Osteocalcin and bone].

Osteocalcin (OC) is a product of osteoblasts and accumulated in the extracellular matrix of bone. It has been recognized that serum OC is a marker of osteoblast activity, and the levels reflect the rate of bone formation. The present assay system was developed to assess the major circulating forms of intact and the large N-terminal fragments. OC binds to the crystal of hydroxyapatite, at least partly, through gamma-carboxylation of three residues. Increased rate of immature undercarboxylated osteocalcin, therefore, might display risks for osteoporotic fractures in clinical studies. However, at present, measurement of OC does not substitute for bone mass measurement and only provide limited values to evaluate the conditions of patients with primary osteoporosis.

[Parathyroid and bone. Evidence and perspective of parathyroid therapy for patients with osteoporosis].

Parathyroid hormone (PTH) is a new management option for patients with osteoporosis. As an anabolic agent that affects bone remodeling and modeling, a novel approach to reducing fracture risk could be considered for patients with severe conditions. A number of trials have shown that increases in spine and hip bone mineral density (BMD), and reduction of fracture risk in postmenopausal women. Although the combination of PTH and alendronate does not seem to be additive, PTH followed by alendronate would yield maximum increase in BMD. Treatment with PTH can change the course of osteoporosis by directly stimulating formation of new bone, and its application should be explored in daily clinical practice.

Involvement of hepatocyte growth factor in the development of bone metastasis of a mouse mammary cancer cell line, BALB/c-MC.

Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13+/-0.25 ng/ml), whereas HGF was undetectable (<0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10(-6) M PGE(2). Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALB/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling.

Potent antitumor activity of interleukin-27.

Although much promising data that interleukin (IL)-12 could be a powerful therapeutic agent against cancer were reported in animal models, its excessive toxicity has become a problem for its clinical application. IL-27 is a novel IL-12 family member that plays a role in the early regulation of T helper cell 1 initiation, including induction of T-bet and IL-12 receptor beta 2 expression. In the present study, we have evaluated the antitumor activity of IL-27 against a murine tumor model of colon carcinoma C26. C26 cells, which were transduced with the single-chain IL-27 cDNA and became secreting IL-27 (C26-IL-27), exhibited minimal tumor growth in vivo, and all of the mice inoculated with these cells survived healthily with complete tumor remission. Inoculation of mice with C26-IL-27 induced enhanced IFN-gamma production and cytotoxic T-lymphocyte activity against C26 tumor in spleen cells. Recovered mice from the inoculation showed a tumor-specific protective immunity to the following challenge with parental C26 tumor. The antitumor activity of IL-27 was almost diminished in nude mice, and depletion of CD8(+) T cells and neutralization of IFN-gamma in immunocompetent mice reduced greatly the antitumor activity. Moreover, the antitumor activity was abolished in T-bet-deficient mice, whereas it was observed unexpectedly in mice deficient of signal transducer and activator of transcription (STAT) 4. These results suggest that IL-27 has potent abilities to induce tumor-specific antitumor activity and protective immunity and that the antitumor activity is mediated mainly through CD8(+) T cells, IFN-gamma, and T-bet but not through STAT4.

[Bone and bone related biochemical examinations. Bone and collagen related metabolites. Osteocalcin (OC)].

Osteocalcin is produced by mature osteoblasts and primarily deposited in the extracellular matrix of skeletal tissue. It has been shown that serum osteocalcin is a marker of osteoblastic activity, and the levels reflect the rate of bone formation. The first osteocalcin assays were competitive radioimmunoassays using bovine osteocalcin as a standard, and a new generation of assays was developed to measure the major circulating forms of the protein, which is the intact and the large N-terminal fragment. Since osteocalcin binds to hydroxyapatite crystal after gamma-carboxylation at three residues, increased amounts of immature undercarboxylated osteocalcin might reflect risks for fractures. However, at present, measurement of osteocalcin does not substitute for bone mass measurement and only provide limited clinical values on evaluation of patients with osteoporosis or metabolic bone diseases.

[Management of osteoporosis in patients with inhaled glucocorticoids].

Inhaled glucocorticoids are the standard of therapy in asthma and are commonly prescribed for chronic obstructive pulmonary disease. Accumulating evidence suggests that the effect of inhaled glucocorticoids on bone is not small, especially in patients taking moderate or high doses for long periods of time. The risk of adverse events is likely to differ between inhaled glucocorticoids. Inhaled glucocorticoids should be used widely, since they reduce the need of oral corticosteroids and improve respiratory function, but that they need to be managed carefully to minimize the risk of fracture with long-term use. This article described the effects of inhaled glucocorticoids on bone and fracture risk.

A fibronectin fragment induces tumor necrosis factor production of rat basophilic leukemia cells.

Proteolytic digest of fibronectin (FN), but not intact FN, induced TNF-alpha secretion of rat basophilic leukemia (RBL-2H3) cells. As a result of the identification of FN fragment responsible for TNF-alpha secretion, a 30-kDa fragment derived from the carboxyl-terminal heparin-binding (Hep 2) domain of FN was isolated from the FN digest. The TNF-alpha secretion was abrogated by treatment of RBL-2H3 cells with cycloheximide, indicating the de novo synthesis of TNF-alpha, but not with polymyxin B, excluding the possible TNF-alpha induction by some contaminated lipopolysaccharides. A 22-mer synthetic peptide originated from the Hep 2 domain, termed FNIII14, which has been found to negatively modulate the beta1 integrin activation, had the ability to induce TNF-alpha production, whereas this activity of FNIII14 disappeared by shuffling a YTIYVIAL sequence essential for the integrin-inactivating activity. FNIII14 suppressed the spreading of RBL-2H3 cells on FN substrate, wherein RBL-2H3 cell proliferation was inhibited with FNIII14 in a dose-dependent manner. Thus, it appears that FN fragments containing the YTIYVIAL anti-adhesive site affect the activation status of RBL-2H3 mast cells, characterized by the stimulation of TNF-alpha production and growth suppression, probably due to negative regulation of beta1 integrin activity.

A new type of antimetastatic peptide derived from fibronectin.

PURPOSE: We found previously that fibronectin (FN) has a cryptic functional site (YTIYVIAL sequence within the 14th type III repeat) opposing cell adhesion to extracellular matrix. A 22-mer FN peptide containing this site, termed FNIII14, inhibits beta1 integrin-mediated adhesion without binding to integrins. The present study shows that FNIII14 has the potential to prevent lymphoma cell metastasis. EXPERIMENTAL DESIGN: Antimetastatic effect of FNIII14 has been evaluated through in vitro or in vivo experiments. RESULTS: FNIII14 inhibited the integrin alpha4beta1-mediated B lymphoma Ramos cell adhesion to VCAM-1 on venule endothelial cells, as well as to FN. Murine T lymphoma L5178Y-ML25 cells, which are known to metastasize to liver and spleen, preferentially adhered to vitronectin (VN) and migrated toward VN concentration gradients. FNIII14 abrogated both the integrin alphavbeta3-mediated adhesion and migration of L5178Y-ML25 cells. Inhibition of the alphavbeta3mediated L5178Y-ML25 cell adhesion by FNIII14 was reversed by phenylarsine oxide, a protein tyrosine phosphatase inhibitor. In addition, FNIII14 abrogated the VN-stimulated tyrosine phosphorylation of intracellular signaling proteins, including focal adhesion kinase (p125(FAK)) and paxillin, suggesting that such a diversity of FNIII14 effects might be because of the negative regulation of p125(FAK) and paxillin tyrosine phosphorylation, which has been involved in adhesion signals transduced by different integrins. The in vivo experiment using a murine metastasis model showed that FNIII14 would inhibit liver and spleen metastases of L5178Y-ML25 cells at a dose much lower than that of RGDS. CONCLUSIONS: FNIII14 might be applicable as a new type of antimetastatic agent distinct from integrin-binding peptides.

[Bisphosphonates: the molecular targets and mechanisms of action].

Bisphosphonates directly act on osteoclasts to inhibit bone resorption and used most widely to treat osteoporosis. The compounds can be classified into two groups with different modes of action. Nitrogen containing bisphosphonates exert their effects by inhibiting a key enzyme in the mevalonate pathway. The specific target is the isoprenoid biosynthetic enzyme, farnesyl pyrophosphate synthase, which is indispensable for protein prenylation and activation of intracellular signalling proteins, including the small GTPases Rho, Rac, Cdc42 and Ras. The disruption of the function of these key enzymes may explain the loss of osteoclast activity and induction of apoptosis. Whereas the first generation of bisphosphonates such as etidronate and clodronate (nitrogen deficient bisphosphonates) can be incorporated into nonhydrolysable analogues of ATP that may inhibit ATP-dependent intracellular events. Bisphosphonates are highly effective to inhibit bone resorption and increase bone mineral density, although their precise mechanisms of molecular action are not completely understood.

Molecular cloning and characterization of the mouse thymocyte protein gene.

Chicken thymocyte protein (cThy28) has recently been identified and implicated to be involved in apoptosis of avian lymphocytes, while its functional role remains undefined. To elucidate the role of Thy28, we have molecularly cloned the mouse Thy28 (mThy28) cDNA and clarified its genomic organization in the present study. The mThy28 cDNA encodes a 226 amino acid protein, whose sequence is highly conserved among bacteria, yeast and plants as well as vertebrates. Northern blot analysis revealed abundant expression of approximately 1 kb mRNA in testis, liver, brain and kidney with lower levels of the expression in thymus, spleen, heart and stomach. The mThy28 gene spans at least 7 kb of genomic DNA, and contains eight exons and seven introns. The 5'-flanking region of the mThy28 gene does not contain a typical TATA box, but contains a putative CCAAT box and presumably multiple transcription initiation sites. Potential Sp1, GATA-1, -2, -3, AP-1, and p300 binding sites were found in the 5'-flanking region of the mThy28 gene. The gene was mapped to mouse chromosome 9.

Synergistic antitumor effect by coexpression of chemokine CCL21/SLC and costimulatory molecule LIGHT.

To establish a more efficient treatment for immunotherapy against solid tumors, we have evaluated the antitumor effect by coexpression of a chemokine CCL21/secondary lymphoid tissue chemokine and a costimulatory molecule LIGHT in colon carcinoma C26. C26 cells expressing either CCL21 or LIGHT exhibited a significantly reduced tumor growth in vivo, and mice inoculated with these cells showed a prolonged survival, but eventually all these mice died. In contrast, C26 cells expressing both CCL21 and LIGHT exhibited a minimal tumor growth in vivo, and all these mice survived healthily with a tumor remission and consequently acquired a strong protective immunity. A markedly increased infiltration of mature dendritic cells (DCs), and CD8(+) T cells was observed in the tumor mass, and their spleen cells showed a greatly enhanced cytotoxic T lymphocyte (CTL) activity against C26 tumor and interferon (IFN)-gamma production. Neutralization of IFN-gamma or depletion of CD8(+) or CD4(+) T cells significantly reduced the antitumor activity. These results suggest that the combined treatment with CCL21 and LIGHT is able to induce a synergistic antitumor effect to eradicate tumor completely by greatly enhancing tumor-infiltration of lymphocytes including mature DCs and CD8(+) T cells, resulting in markedly augmented CTL activity and IFN-gamma production.