Inhibitory effects of malotilate on in vitro invasion of lung endothelial cell monolayer by human oral squamous cell carcinoma cells.

We have previously reported that malotilate (MT) inhibited the invasion and metastasis of rat mammary carcinoma cells through the modification of host endothelial cells. In this study, we examined the inhibitory effects of MT on invasion of human cancer, using five oral squamous cell carcinoma cells (SAS, Ca9-22 and HSC-2, -3 and -4). MT did not affect the growth of these tumor cells and the invasion of reconstituted basement membrane, Matrigel. In an in vitro invasion assay using rat lung endothelial (RLE) cells, invasion of tumor cells which had been treated with MT (10 ng/ml, 24 h) was not affected; however, when RLE cells had been treated with MT, invasion was significantly inhibited in three cell lines (SAS, Ca9-22 and HSC-4) and a tendency to inhibition was also observed in other cell lines. Electron-microscopical examination of the RLE monolayer treated with MT (MT-RLE) showed the development of gap and tight junction-like structures. Subsequently, junction-associated proteins, connexin 43, zonula occludin and desmoglein, were examined by Western blotting. Protein levels of connexin 43 and zonula occludin were elevated dose dependently, and connexin 43 was chronologically enhanced by MT whereas desmoglein was not. The enhanced gap junctional communication of MT-RLE cells was observed in the scrape-loading assay using lucifer yellow CH. These results suggest that MT promotes the development of cell-to-cell adhesion, e. g. gap junction and tight junction in endothelial cells, resulting in the inhibition of invasion by the tumor cells.

Unsaturated fatty acid feeding prevents the development of acute hepatitis in Long-Evans cinnamon (LEC) rats.

To investigate the effects of dietary alpha-linolenic acid (18:3, n-3; alpha-LNA) and linoleic acid (18:2, n-6; LA) on the development of hereditary hepatitis, we compared incidences and grades of acute hepatitis between the Long-Evans cinnamon (LEC) rats fed with safflower oil-supplemented diet and perilla oil-supplemented diet. Both safflower and perilla oil supplemented diets reduced the incidence of hepatitis and significantly prolonged its onset as compared to the non-supplemented conventional diet. No significant difference was observed between safflower and perilla oil diets in the rats of incidence of hepatitis. At the age of 16 weeks, just before the onset of hepatitis, serum levels of transaminase (AST, ALT) and concentration of copper in rats fed with both test diets were significantly reduced as compared with that of rats fed alpha-linolenate and linoleate have an inhibitory effect on the development of hepatitis in LEC rats due to the prevention of serum copper elevation.

Inhibitory effects of malotilate on invasion and metastasis of rat mammary carcinoma cells by modifying the functions of vascular endothelial cells.

Malotilate (diisopropyl,1,3-dithiol-2-ylidenemalonate, MT) is clinically used as a hepatoprotective agent. Because we noticed that MT induced the differentiation of cultured vascular endothelial cells, we have examined its effects on lung metastasis of the highly metastatic rat mammary carcinoma c-SST-2. MT was orally administered to syngeneic SHR rats from 7 days before or after s.c. inoculation of c-SST-2 cells to the end of the experiments. In the MT-treated rats, pulmonary metastasis was markedly suppressed compared with the non-treated rats. In the rats treated with MT for 19 days after i.v. inoculation of c-SST-2 cells, lung metastasis was also significantly suppressed. An in vitro invasion assay using a rat lung endothelial (RLE) cell monolayer revealed that pretreatment of the RLE cells with MT, but not c-SST-2 cells, significantly reduced the invasion of the RLE monolayer by c-SST-2 cells. An in vitro vascular permeability assay demonstrated that MT prevented the increase in permeability of the RLE monolayer by serum starvation. On the other hand, in vivo and in vitro growth, gelatinase production and adhesion to the RLE cell monolayer of c-SST-2 cells were not affected by MT treatment. These findings suggest that MT suppressed tumour metastasis by intensifying the cell-to-cell contact of endothelial cells, thus preventing tumour cells from invading vascular endothelium.

Characterization of the progressive sublines derived from a weakly malignant cloned cell line, ER-1, co-inoculated subcutaneously with a foreign body.

We previously established an experimental model of tumor progression using a weakly malignant rat mammary carcinoma cell line, ER-1. Using this model, we demonstrated that ER-1 cells converted into highly tumorigenic and metastatic cells, ERpP, by s.c. co-inoculation with plastic plates. We here compared in vitro biological properties associated with malignancy of ER-1 cells with those of ERpP cells which were highly malignant when inoculated into syngeneic rats. In vitro growth rate of ERpP cells was higher than that of ER-1 cells under a low nutrient condition. Invasion capacity of ERpP cells to rat lung endothelial cell monolayer or reconstituted basement membrane, Matrigel, was higher than that of ER-1 cells. Migration of ERpP cells toward fibronectin or laminin was also significantly higher than that of ER-1 cells. There was no difference in gelatinolytic or plasminogen activator activity detected in conditioned media between ER-1 and ERpP cells. Furthermore, we found that ER-1 cells communicated better among themselves and with normal fibroblasts through gap junctions compared to ERpP cells. These results suggest that growth advantage in a poor nutrient condition, enhancement of cell motility, and loss or decrease of junctional communication may be associated with tumor progression of ER-1 cells.

Reversible and irreversible tumor progression of a weakly malignant rat mammary carcinoma cell line by in vitro exposure to epidermal growth factor.

We examined the effects of epidermal growth factor (EGF) on tumor progression of a weakly malignant rat mammary carcinoma cell line, ER-1. In vitro treatment with EGF enhanced tumorigenicity, metastatic capacity and in vitro invasive capacity of ER-1 cells. The increased malignancy of ER-1 cells was reversible, when the cells were pretreated with EGF for 24 h, whereas it was irreversible when pretreated with EGF for 1 month. EGF treatment elevated the intracellular peroxide level in ER-1 cells. When ER-1 cells were treated with EGF in the presence of N-acetylcysteine, a chemical antioxidant, the reversible or irreversible EGF-induced progression was inhibited. These results suggest that the reversible or irreversible tumor progression in ER-1 cells occur in accordance with the duration of exposure to EGF, and that reactive oxygen species may be involved in the progression.

Enhancing effects of epidermal growth factor on human squamous cell carcinoma motility and matrix degradation but not growth.

In order to ascertain the effects of epidermal growth factor (EGF) on human cancer invasion abilities, three cell lines of human oral squamous cell carcinoma were studied using a phagokinetic track assay and zymography. EGF (1-100 ng/ml) was found to inhibit the growth but enhance the random motility of all three cell lines in a concentration-dependent fashion. Exposure to EGF, dose-dependently, led to an increased production of urokinase-type plasminogen activator and M(r) 92 kD matrix metalloproteinase by the same cells. These results strongly suggest that EGF may promote human squamous cell carcinoma invasion and metastasis.

Enhanced effect of epidermal growth factor on pulmonary metastasis and in vitro invasion of rat mammary carcinoma cells.

We examined the effects of epidermal growth factor (EGF) on metastatic and in vitro invasive capacity of weakly malignant ER-1 cells derived from a rat mammary carcinoma cell line, c-SST-2. EGF enhanced the metastatic capacity and in vitro invasiveness to reconstituted basement membrane, Matrigel, of ER-1 cells in a dose-dependent fashion. EGF-stimulated invasiveness was inhibited by anti-EGF antibody, which is able to neutralize the binding of EGF to EGF receptor, in the invasion assay system. EGF stimulated chemotactic migration toward fibronectin, laminin or newborn rat fibroblast-conditioned medium which was used as a chemoattractant in the in vitro invasion assay, but showed neither adhesion to Matrigel nor production of gelatinase and plasminogen activators. These results suggested that the increased metastatic and invasive capacity of ER-1 cells by EGF might be due to the increase in cell motility.

Establishment of a retrodifferentiated cell line from a single differentiated rat myelomonocytic leukemia cell: possible roles of retrodifferentiation in relapses of leukemia after differentiation-inducing therapy.

In order to demonstrate possible roles of retrodifferentiation in relapses after differentiation therapies, we have established a retrodifferentiated cell line (RD-1) from a single rat myelomonocytic leukemia cell which differentiated into a macrophage-like cell by treatment with lipopolysaccharide (LPS). The established RD-1 cells showed microscopic features slightly maturer than their parent cells. The RD-1 cells had the ability to differentiate into macrophage-like cells by treatment with fewer doses of LPS than those for parent cells. All rats inoculated with the parent cells (more than 10(2)/rat) died within 50 days. Rats inoculated with 10(4) RD-1 cells survived for more than 120 days, whereas two out of four rats inoculated with 10(5) cells and all the rats inoculated with 5 x 10(5) cells died of leukemia. These results suggest that RD-1 cells are retrodifferentiated cells from a single rat myelomonocytic leukemia cell which differentiated into a macrophage-like cell; they have similar phenotypes and lower tumorigenicity than the parent cells and they also suggest that the appearance of retrodifferentiated leukemia cells may be responsible for relapse after differentiation therapy for leukemia in some cases.

ONO-4007, a new synthetic lipid A derivative, induces differentiation of rat myelomonocytic leukemia cells in vitro and in vivo.

We have examined the differentiation-inducing effects of ONO-4007, a new synthetic lipid A derivative with low endotoxic activities, on a rat myelomonocytic cell line, c-WRT-7, in vitro and in vivo. ONO-4007 induced the differentiation of c-WRT-7 cells into macrophage-like cells and inhibited the proliferation of c-WRT-7 cells in vitro. Stimulation with ONO-4007 induced messenger RNA expression of interleukin-1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha (TNF-alpha), which have been reported to induce differentiation of several leukemia cell lines. However, autocrine production of these cytokines may not be involved in the mechanisms of differentiation induced by ONO-4007, because the treatment with IL-1 alpha, IL-6, or TNF-alpha does not induce the differentiation of c-WRT-7 cells. In vivo treatment by intravenous administration of ONO-4007 resulted in a significant prolongation of survival time of the rats inoculated intravenously with c-WRT-7 cells compared with that of untreated rats. These results suggest that ONO-4007 can be therapeutically useful for the treatment of leukemia.

Enhancement of tumorigenicity and invasion capacity of rat mammary adenocarcinoma cells by epidermal growth factor and transforming growth factor-beta.

We have studied the effects of growth factors and cytokines on the tumorigenicity and invasion capacity of tumor cells by using regressor and progressor tumor cell lines (ER-1 and ERpP, respectively) derived from an SHR rat mammary adenocarcinoma. ER-1 cells regress spontaneously whereas ERpP cells show invasive growth and high metastasis to lung and other organs in syngeneic SHR rats. When ER-1 cells were pretreated with either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) for 24 h in vitro, and intraperitoneally transplanted into SHR rats, they grew and killed the host, whereas ER-1 cells pretreated with tumor necrosis factor-alpha did not. Tumorigenicity and invasion capacity of ERpP cells were also enhanced by treatment with EGF and TGF-beta. The ER-1 cells pretreated with EGF, once grown in vivo, had acquired irreversible tumorigenicity and invasion capacity without requiring further EGF treatment, and the enhanced malignancy was irreversible. These findings suggest that growth factors play an important role in acquisition of malignancy of tumor cells.

[Enhanced malignancy of tumor cells by the interaction with host cells reactive to foreign body].

We examined factors promoting malignant progression using a weakly malignant variant cell line, ER-1, derived from c-SST-2, a rat mammary carcinoma. ER-1 cells were converted to a highly malignant phenotype (highly tumorigenic, metastatic, invasive in vitro) by the in vitro/in vivo interaction with host cells reactive to foreign body. Epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) produced by host reactive cells, transiently enhanced the tumorigenicity and in vitro invasiveness of ER-1 cells into an endothelial cell monolayer. The host reactive cells also produced oxygen radicals and induced mutations in ER-1 cells. It is speculated that mutations induced by host reactive cells cause cellular diversification, including the emergence of highly malignant variant cells whose growth is selectively promoted by growth factors such as EGF and TGF-beta.