Serum APOA4 Pharmacodynamically Represents Administered Recombinant Human Hepatocyte Growth Factor (E3112).

BACKGROUND: Hepatocyte growth factor (HGF) is an endogenously induced bioactive molecule that has strong anti-apoptotic and tissue repair activities. In this research, we identified APOA4 as a novel pharmacodynamic (PD) marker of the recombinant human HGF (rh-HGF), E3112. METHODS: rh-HGF was administered to mice, and their livers were investigated for the PD marker. Candidates were identified from soluble proteins and validated by using human hepatocytes in vitro and an animal disease model in vivo, in which its c-Met dependency was also ensured. RESULTS: Among the genes induced or highly enhanced after rh-HGF exposure in vivo, a soluble apolipoprotein, Apoa4, was found to be induced by rh-HGF in the murine liver. By using primary cultured human hepatocytes, the significant induction of human APOA4 was observed at the mRNA and protein levels, and it was inhibited in the presence of a c-Met inhibitor. Although mice constitutively expressed Apoa4 mRNA in the small intestine and the liver, the liver was the primary organ affected by administered rh-HGF to strongly induce APOA4 in a dose- and c-Met-dependent manner. Serum APOA4 levels were increased after rh-HGF administration, not only in normal mice but also in anti-Fas-induced murine acute liver failure (ALF), which confirmed the pharmacodynamic nature of APOA4. CONCLUSIONS: APOA4 was identified as a soluble PD marker of rh-HGF with c-Met dependency. It should be worthwhile to clinically validate its utility through clinical trials with healthy subjects and ALF patients.

Anti-Apoptotic Effects of Recombinant Human Hepatocyte Growth Factor on Hepatocytes Were Associated with Intrahepatic Hemorrhage Suppression Indicated by the Preservation of Prothrombin Time.

Hepatocyte growth factor (HGF) is an endogenously expressed bioactive substance that has a strong anti-apoptotic effect. In this study, we biochemically and histologically characterized the effects of rh-HGF on in vitro human hepatocyte injury and mouse acute liver failure (ALF) models, both of which were induced by antibody-mediated Fas signaling. rh-HGF inhibited intracellular caspase-3/7 activation and cytokeratin 18 (CK-18) fragment release in both models. Histologically, rh-HGF dramatically suppressed parenchymal damage and intrahepatic hemorrhage. Among the laboratory parameters, prothrombin time (PT) was strongly preserved by rh-HGF, and PT was well correlated with the degree of intrahepatic hemorrhage. These results showed that the anti-apoptotic effect of rh-HGF on hepatocytes coincided strikingly with the suppression of intrahepatic hemorrhage. PT was considered to be the best parameter that correlated with the intrahepatic hemorrhages associated with hepatocellular damage. The action of rh-HGF might derive not only from its anti-apoptosis effects on liver parenchymal cells but also from its stabilization of structural and vasculature integrity.

High-level expression of chemokine CXCL16 by tumor cells correlates with a good prognosis and increased tumor-infiltrating lymphocytes in colorectal cancer.

CXCL16 is a new member of the chemokine superfamily, which exists in a transmembrane as well as a soluble form. Its receptor CXCR6 is detected on CD4(+) T cells, CD8(+) T cells, and natural killer T cells. Here, we report a significant correlation of CXCL16 expression by tumor cells with the infiltration of T cells and prognosis in colorectal cancer (CRC). We first found that CXCL16 expression was consistently up-regulated more in tumor tissues than in normal mucosa derived from the same CRC patients. Four human CRC cell lines also expressed CXCL16 mRNA and secreted soluble CXCL16. We next examined the expression of CXCL16 and infiltration of lymphocytes in CRC specimens (n = 58) by immunohistochemistry. CRC patients with high levels of CXCL16 expression (n = 43) had higher levels of CD4(+) and CD8(+) tumor-infiltrating lymphocytes (TIL; P < 0.01) than those with low levels of CXCL16 expression (n = 15). Furthermore, the high CXCL16 expression group showed significantly better prognosis than the low CXCL16 expression group (P < 0.05). Collectively, our data suggest that the expression of CXCL16 by tumor cells enhances the recruitment of TILs, thereby bringing about a better prognosis in CRC. Thus, CXCL16 is a new prognostic biomarker and may be useful for the development of a more effective therapeutic strategy for CRC.

Role of the CXCL12/CXCR4 axis in peritoneal carcinomatosis of gastric cancer.

Peritoneal carcinomatosis is a frequent cause of death in patients with advanced gastric carcinoma. Because chemokines are now considered to play an important role in the metastasis of various malignancies, we hypothesized that they may be involved in the development of peritoneal carcinomatosis by gastric carcinoma. Human gastric carcinoma cell lines, which were all highly efficient in generating malignant ascites in nude mice upon i.p. inoculation, selectively expressed CXCR4 mRNA and protein. In particular, NUGC4 cells expressed CXCR4 mRNA at high levels and showed vigorous migratory responses to its ligand CXCL12. CXCL12 enhanced proliferation and rapid increases in phosphorylation of protein kinase B/Akt and extracellular signal-regulated kinase of NUGC4 cells. We also showed that AMD3100 (a specific CXCR4 antagonist) effectively reduced tumor growth and ascitic fluid formation in nude mice inoculated with NUGC4 cells. Additionally, we examined human clinical samples. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high concentrations of CXCL12 (4.67 ng/mL). Moreover, immunohistochemical analysis showed that 22 of 33 primary gastric tumors with peritoneal metastasis were positive for CXCR4 expression (67%), whereas only 4 of 16 with other distant metastasis were positive (25%). Notably, 22 of 26 CXCR4-expressing primary tumors developed peritoneal metastases (85%). CXCR4 positivity of primary gastric carcinomas significantly correlated with the development of peritoneal carcinomatosis (P < 0.001). Collectively, our results strongly suggest that the CXCR4/CXC12 axis plays an important role in the development of peritoneal carcinomatosis from gastric carcinoma. Thus, CXCR4 may be a potential therapeutic target for peritoneal carcinomatosis of gastric carcinoma.

RANKL-induced CCL22/macrophage-derived chemokine produced from osteoclasts potentially promotes the bone metastasis of lung cancer expressing its receptor CCR4.

Chemokines are now known to play an important role in cancer growth and metastasis. Here we report that differentiating osteoclasts constitutively produce CCL22 (also called macrophage-derived chemokine) and potentially promote bone metastasis of lung cancer expressing its receptor CCR4. We first examined expression of chemokines by differentiating osteoclasts. CCL22 was selectively upregulated in osteoclast-like cells derived from RAW264.7 cells and mouse bone marrow cells upon stimulation with RANKL (receptor activator of nuclear factor-kappaB ligand). In addition, a human lung cancer cell line SBC-5 that efficiently metastasized to bone when intravenously injected into NK cell-depleted SCID mice was found to express CCR4. Stimulation of SBC-5 cells with CCL22 induced cell migration and also enhanced phosphorylation of protein kinase B/Akt and extracellular signal-regulated kinase (ERK). Furthermore, immunohistochemical analysis of bone metastasis lesions demonstrated close co-localization of tartrate-resistant alkaline phosphatase (TRAP)-positive osteoclasts expressing CCL22 and SBC-5 cells expressing CCR4. Collectively, these results suggest that osteoclasts may promote bone metastasis of cancer cells expressing CCR4 in the bone marrow by producing its ligand CCL22.

Antitumor immune activity by chemokine CX3CL1 in an orthotopic implantation of lung cancer model in vivo.

Due to their chemoattractant properties stimulating the accumulation of infiltrating immune cells in tumors, chemokines are known to have antitumor effects. Fractalkine, a unique CX3C chemokine, is expressed in activated endothelial cells, while its receptor, CX3CR1, is expressed in cytolytic immune cells, such as natural killer cells, monocytes and some CD8+ T cells. The biological properties of cancer cells are affected by the implantation organ and differences in immune systems, requiring cancer implantation in orthotopic organs in an in vivo experiment. To develop new therapy strategies for lung cancer, an animal model reflecting the clinical features of lung cancer was previously established. This study aimed to determine whether CX3CL1-induced biological functions should be used for immune cell-based gene therapy of lung cancer in the orthotopic implantation model. An orthotopic intrapulmonary implantation of CX3CL1-stable expression in mouse lung cancer (LLC-CX3CL1) was performed to analyze growth. Results showed a significant decrease in tumor growth in the lung compared to the control cells (LLC-mock). Furthermore, the antitumor effects of CX3CL1 were derived from natural killer cell activities in the depletion experiment in vivo. Therefore, CX3CL1 has the potential of a useful therapeutic target in lung cancer.

Inhibition of CCL20 increases mortality in models of mouse sepsis with intestinal apoptosis.

BACKGROUND: CC chemokine ligand 20 (CCL20) and CC chemokine receptor 6 are believed to stimulate the recruitment of neutrophils and activation of macrophages against bacterial pathogens through the activation of T helper cells. We analyzed the role of CCL20 in the acute phase of sepsis. METHODS: The effect of a neutralizing, anti-mouse CCL20 monoclonal antibody (mAb) was examined in 2 murine models of sepsis: Cecal ligation and puncture (CLP) and Escherichia coli peritonitis. Immune cell migration, bacterial clearance, and expression of 17 cytokines and 5 chemokines were quantified in E coli-induced peritonitis. Expression of CCL20 in various tissues was determined, and apoptotic cells in jejunum were measured. RESULTS: Anti-CCL20 mAb increased mortality in CLP and E coli peritonitis (P = .029 and .024, respectively by Kaplan-Meier method and log-rank test). The 48-hour survival rate in anti-CCL20 mAb- and control immunoglobulin (Ig)G-treated mice was 37% (11/30) vs 62% (18/29) in CLP and 28% (11/40) vs 48% (19/40) in bacterial peritonitis. Neutralization of CCL20 showed no effect on leukocyte infiltration into the peritoneal cavity or bacterial clearance at 24 hours. CCL20 was induced strongly and predominantly in jejunum after bacterial infection, and neutralizing CCL20 increased apoptosis of epithelial cells in jejunum crypt. Inhibition of CCL20 increased serum tumor necrosis factor (TNF)-α (3.3-fold greater than control mice) and decreased serum interleukin (IL)-1α and IL-6. CONCLUSION: Neutralization of CCL20 before induction of sepsis increased mortality during sepsis accompanied with increasing epithelial apoptosis in the jejunum and augmenting serum TNF-α.