Teruo Iwasaki

Interleukin-18 and Interleukin-12 Synergistically Inhibit Osteoclastic Bone-resorbing Activity

The effect of interleukin (IL)-18 on osteoclastic bone-resorbing activity was investigated in vitro. Osteoclast-enriched cells, about 70% of which were tartrate-resistant acid phosphatase (TRAP)-positive, were cultured on dentine slices, and then the total volume of resorption pits on each dentine slice was measured as bone-resorbing activity. When the effects of IL-18 alone at 1, 10, 100, and 1000 ng/mL were examined, bone-resorbing activity was significantly reduced only at 1000 ng/mL, by about 50%. However, IL-18 plus IL-12 (10 ng/mL each) reduced bone-resorbing activity by about 70%, whereas IL-12 alone had no significant effect. When the concentration of interferon (IFN)-gamma in the medium was measured, IL-18 or IL-12 was found to increase it slightly, and the combination of these two cytokines synergistically increased it. The inhibitory effect of the combination of the two cytokines was completely abolished by the addition of an anti-IFN-gamma neutralizing antibody to the medium, but IFN-gamma by itself did not inhibit osteoclastic bone resorption. IL-18 alone or in combination with IL-12 did not affect the number of TRAP-positive cells in culture of osteoclast-enriched cells. Osteoclasts prepared from osteoclast-enriched cells expressed mRNAs of IL-18 receptor, MyD88, and cathepsin K. Furthermore, IL-18 receptor protein was detected on the cell surface of osteoclasts. The present results indicate that the combination of IL-18 and IL-12 synergistically inhibits osteoclastic bone-resorbing activity, suggesting that IFN-gamma participates in the mechanism underlying this inhibition.

Inhibition of tumor invasion and metastasis by a novel lysophosphatidic acid (cyclic LPA).

Fetal calf serum (FCS) and 1‐oleoyl lysophosphatidic acid (LPA) were previously found to be potent inducers of invasion (transcellular migration) in an in vitro system. A novel LPA, composed of cyclic phosphate and cyclopropane‐containing hexadecanoic acid (PHYLPA), first isolated from myxoamoebae of Physarum polycephalum, and its synthetic derivatives (cLPA) were tested for their ability to inhibit tumor cell invasion and metastasis. Amoung these, Pal‐cLPA, which has a palmitoyl moiety, was most potent in inhibiting invasion, with 93.8% inhibition at the concentration of 25 μM. Invasion in vitro by mouse melanoma cells (B16), human pancreatic adenocarcinoma cells (PSN‐1), human lung cancer cells (OC‐10) and human fibrosarcoma cells (HT‐1080) was also inhibited by Pal‐cLPA. The stimulation of MM1 cells with LPA triggered F‐actin formation, which was impaired by the addition of Pal‐cLPA at invasion‐inhibitory concentration. Pal‐cLPA induced a rapid increase in adenosine 3′,5′‐cyclic monophosphate (cAMP) concentration in MM1 cells. The addition of dibutyryl cAMP significantly abrogated LPA‐induced invasion by MM1 cells and actin polymerization in the cells. The inhibition of MM1 cell invasion by Pal‐cLPA may be ascribed to an increased level of cAMP. Pal‐cLPA also suppressed invasion in vitro by MM1 cells induced by FCS dose dependently, without affecting proliferation. It also suppressed the pulmonary metastasis of B16 mouse melanoma cells injected into the tail vein of C57BL/6 mice. Thus, Pal‐cLPA is effective in inhibiting invasion and metastasis of a variety of tumor cells. Int. J. Cancer 81:918–922, 1999.

Expression of the intermediate filament nestin in gastrointestinal stromal tumors and interstitial cells of Cajal.

It has recently been proposed that gastrointestinal stromal tumors (GISTs) originate from stem cells that differentiate toward a phenotype of interstitial cells of Cajal (ICCs). Nestin is a newly identified intermediate filament protein, and is predominantly expressed in immature cells, such as neuroectodermal stem cells and skeletal muscle progenitor cells, and tumors originating from these cells. In this study, we examined, using immunohistochemistry, the nestin expression in GISTs and ICCs to clarify the origin of GISTs. Strong immunoreactivity for nestin was observed in all 18 GISTs, and its expression was confirmed by Western blot and Northern blot analyses. In contrast, three leiomyomas and a schwannoma that developed in the gastrointestinal tract showed no apparent immunoreactivity for nestin. Among 17 mesenchymal tumors (seven leiomyosarcomas, five malignant peripheral nerve sheath tumors, and five fibrosarcomas) that occurred in sites other than the gastrointestinal tract, only two malignant peripheral nerve sheath tumors were moderately immunoreactive for nestin. Furthermore, with fluorescence double immunostaining of the normal small intestine, nestin expression was demonstrated in ICCs. These results show that nestin may be a useful marker for diagnosis of GISTs, and support the current hypothesis that GISTs are tumors of stem cells that differentiate toward an ICC phenotype.

Expression and prognostic significance in lung cancer of human tumor‐associated antigen RCAS1

A new monoclonal antibody (MAb), 22‐1‐1, acts against a novel tumor‐associated antigen (Ag) strongly expressed in human uterine cervical adenocarcinomas. A cDNA encoding the Ag recognized by the 22‐1‐1 MAb has been isolated and called RCAS1 (receptor‐binding cancer antigen expressed on SiSo cells). RCAS1 can induce growth arrest and apoptosis in RCAS1 receptor‐positive cells including T cells and natural killer cells in vitro. These results suggest that RCAS1 is involved in tumor escape from the immune system. Immunohistochemical analysis revealed the relationship between RCAS1 expression and clinicopathological variables (age, sex, smoking, histology, differentiation grade, pathological T factor, N factor and stage) and the prognostic significance of RCAS1 in 66 lung‐cancer patients who underwent curative operations: 33 adenocarcinomas, 24 squamous‐cell carcinomas, 3 large‐cell carcinomas, 4 adenosquamous carcinomas and 2 small‐cell carcinomas. Median follow‐up period of 64 non‐small‐cell carcinomas (NSCLCs) was 67.4 months. RCAS1 was expressed in 74.2% of lung cancers. RCAS1 in NSCLC cases with advanced T factor or pathological stage or in poorly differentiated adenocarcinomas was highly expressed. Furthermore, RCAS1 expression inducing apoptosis of tumor‐infiltrating lymphocytes was a significant prognostic factor in NSCLC (p < 0.03). Int. J. Cancer 89:488–493, 2000.

Hepatoma cell migration through a mesothelial cell monolayer is inhibited by cyclic AMP-elevating agents via a Rho-dependent pathway

1‐Oleoyl lysophosphatidic acid (LPA) induces transmonolayer migration (in vitro invasion) of rat ascites hepatoma MM1 cells and their morphological changes leading to the migration. We have previously shown that an LPA analog, palmitoyl cyclic phosphatidic acid (Pal‐cPA), suppresses transmonolayer migration of MM1 cells by rapidly increasing the intracellular cyclic AMP (cAMP) concentration. We report here that various cAMP‐elevating agents, including dibutyryl cAMP, forskolin, cholera toxin and 3‐isobutyl‐1‐methylxanthine, consistently inhibited LPA‐induced transmonolayer migration of MM1 cells. Moreover, pull‐down assays for GTP‐bound, active RhoA demonstrated that the blockage by cAMP‐elevating agents of morphological changes leading to the migration was probably mediated through inhibiting RhoA activation.

Mouse Uterine Epithelial Apoptosis Is Associated with Expression of Mitochondrial Voltage-Dependent Anion Channels, Release of Cytochrome C from Mitochondria, and the Ratio of Bax to Bcl-2 or Bcl-X

Abstract The release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members and is considered to take place through voltage-dependent anion channels (VDACs) on the outer membranes of mitochondria, results in activation of effector caspases, such as caspase-3, which induce apoptosis. We studied the involvement of the mitochondrial apoptosis pathway in uterine epithelial apoptosis. Estradiol-17β pellets were implanted into ovariectomized mice and removed 4 days later (Day 0). The apoptotic index (percentage of apoptotic cells) of the luminal epithelium increased markedly, peaking on Day 2, whereas that of the glandular epithelium increased much less. Expression of VDAC1, 2, and 3 mRNAs increased in the luminal epithelium in correlation with the apoptotic index of the luminal epithelium. No increases in VDAC1, 2, and 3 mRNA levels were observed in the stroma or muscle, where no apoptosis occurs. VDAC1 protein levels in the uterus also correlated well with the apoptotic index of the luminal epithelium. In addition, the apoptotic index showed good correlation with the release of cytochrome c from mitochondria, activation of caspase-3, which was immunohistochemically detected only in the epithelium, and the mRNA and protein ratios of Bax:Bcl-2 and Bax:Bcl-X in the uterus. The present results suggest that the release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members, plays a role in uterine epithelial apoptosis after estrogen deprivation. The increase in VDAC expression may facilitate the release of cytochrome c during apoptosis.

Involvement of phosphorylation of tyr-31 and tyr-118 of paxillin in MM1 cancer cell migration

We demonstrated previously that rat ascites hepatoma MM1 cells require both lysophosphatidic acid (LPA) and fibronectin (FN) for phagokinetic motility and transcellular migration and that these events are regulated through the RhoA‐ROCK pathway. It remains to be elucidated, however, how the signals from both LPA and FN are integrated into cell migration. To examine this, total cellular lysates after stimulation with LPA or FN were subjected to time‐course immunoblot analysis with anti‐phosphotyrosine antibodies (Abs). Consequently, tyrosine‐phosphorylation of paxillin was obviously persistent after stimulation with FN + LPA as compared to after stimulation with either alone. Tyrosine‐phosphorylated paxillin comprised 2 components; slowly and fast migrating ones. Immunoblotting of anti‐paxillin immunoprecipitates with phosphorylation site‐specific Abs revealed the following: tyrosine‐phosphorylation was enhanced preferentially on a slowly migrating component after stimulation with FN + LPA; this component contained phosphorylation at both tyrosine residue (Y) 31 and Y118; and phosphorylation of paxillin at Y181 was constitutive and not augmented by stimulation with either FN or LPA. Amiloride, an inhibitor of the Na+/H+ antiporter downstream of ROCK, suppressed cell motility and correspondingly paxillin tyrosine‐phosphorylation at both Y31 and Y118. Paxillin phosphorylation weakly induced by FN alone, insufficient for cell migration, was not inhibited by amiloride. These results demonstrate that LPA collaborates with FN for persistent tyrosine phosphorylation of paxillin at both Y31 and Y118, regulated by the Na+/H+ antiporter downstream of ROCK and that this phosphorylated paxillin is essential for MM1 cancer cell migration.

Predictive factors for postoperative acute exacerbation of interstitial pneumonia combined with lung cancer

PurposePostoperative acute exacerbation (AE) of usual interstitial pneumonia (UIP) is a serious complication in the surgical treatment for primary lung cancer combined with UIP. The purpose of this study was to determine the predictors of AE of UIP after a major lung resection.MethodsWe retrospectively collected data for 40 patients who had been operated on for lung cancer and were diagnosed as UIP based on postoperative histopathological diagnosis. We then evaluated some predictive factors related to the AE of UIP.ResultsThe incidence of postoperative AE of UIP was 15% (6/40 patients). No correlation between patients who developed AE of UIP and those who did not, in terms of preoperative C-reactive protein, white blood cell count, percentage lymphocytes, forced expiratory volume in 1 s, percentage total lung capacity, percentage diffusing capacity of lung for carbon monoxide, and the alveolar partial pressures of oxygen and carbon dioxide. Preoperative serum lactate dehydrogenase (LDH) and serum KL-6 were significantly higher and the percent vital capacity (%VC) was significantly lower in patients who developed AE of UIP than in those who did not. Furthermore, recursive descent partition analysis revealed that %VC (<80.6%) and LDH (≥241 IU/l) could distinguish patients with AE from those without AE.ConclusionPreoperative %VC plus serum LDH values were considered the predictive factors for AE of UIP after surgery for lung cancer.

Interleukin-18 Inhibits Osteolytic Bone Metastasis by Human Lung Cancer Cells Possibly Through Suppression of Osteoclastic Bone-resorption in Nude Mice

Interleukin (IL)-18 exhibits antitumor as well as antiosteoclastogenic activities. These findings suggest that IL-18 is a potential tool for the treatment of cancers with associated osteolytic bone metastasis. We have previously shown that systemic daily administration of recombinant (r) IL-18 inhibits the development of osteolytic bone metastasis by human breast cancer cells. Here we demonstrate that systemic daily administration of rIL-18 (1 &mgr;g/mouse/d) for 21 days significantly inhibited the number and the total area of osteolytic bone metastasis by RWGT2 human lung cancer cells in nude mice. No severe adverse effects were observed. Natural killer (NK) cells did not increase in splenocytes from rIL-18-treated mice, and the in vitro NK activity of splenocytes against RWGT2 cells was only weakly enhanced in the presence of IL-18. The administration of rIL-18 made no difference in the growth of subcutaneous tumors, histologic indices (mitotic index, apoptotic index, and Ki-67-labeling index) of subcutaneous tumors or metastatic bone foci, or in the number of osteoclasts along the bone surface adjacent to tumors. Moreover, serum levels of cytokines including interferon-&ggr;, IL-1&agr;, IL-6, tumor necrosis factor-&agr;, and granulocyte/macrophage colony-stimulating factor, which regulate bone-resorbing activity of osteoclasts, were evaluated. Among them, IL-6 was remarkably downregulated in rIL-18-treated mice. These findings suggest that IL-18 inhibits osteolytic bone metastasis possibly through suppression of osteoclastic bone-resorption mediated in part by IL-6.

Cooperation of fibronectin with lysophosphatidic acid induces motility and transcellular migration of rat ascites hepatoma cells.

We have previously shown that the transcellular migration of rat ascites hepatoma (AH130-MM1) cells through a cultured mesothelial cell monolayer (MCL) is triggered with lysophosphatidic acid (LPA) that stimulates actin polymerization and myosin light chain phosphorylation through the activation of Rho-ROCK (Rho-kinase) cascade. When, however, the motility of MM1 cells on a glass surface was tested by phagokinetic track motility assay, LPA failed to induce the motility. Nevertheless, when the glass had been coated with fibronectin (FN), LPA could induce phagokinetic motility which was accompanied by transformation of MM1 cells to fusiform-shape and assembly of focal adhesion. beta1 integrin, the counter receptor of FN, was expressed on MM1 cells. Anti-FN antibody, anti-beta1 integrin antibody and cyclo-GRGDSPA remarkably suppressed LPA-induced phagokinetic motility. These antibodies suppressed LPA-induced transcellular migration through MCL, as well. These results indicate that actin polymerization and phosphorylation of myosin light chain through Rho activation are insufficient for inducing motility but the cooperative FN/beta1 integrin-mediated adhesion is necessary for both the phagokinetic motility and transcellular migration of MM1 cells.