Yasuko Nishizawa

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.

Effects of NG-nitro-l-arginine and/or l-arginine on experimental pulmonary metastasis in mice

Effects of NG-nitro-L-arginine methyl ester (L-NAME; an inhibitor of nitric oxide (NO) synthase) and/or L-arginine (substrate of NO synthase) on pulmonary metastasis of murine melanoma and Lewis lung carcinoma cells were investigated. L-NAME, L-arginine or both L-NAME and L-arginine was injected i.p. into mice 5, 3, and 1 h before and 1, 3, 5, and 7 h after the injection of tumor cells into mice via a tail vein. The administration of L-NAME (9.3 mumol/mouse) alone or L-arginine alone (46.5 or 186 mumol/mouse) potentiated pulmonary metastasis of highly and poorly metastatic B16 melanoma cells. L-NAME alone also increased the number of pulmonary metastasis of Lewis lung carcinoma cells, but L-arginine (185 mumol/mouse) did not. However, the combination of L-NAME and L-arginine increased the number of pulmonary metastasis of both the melanoma and Lewis lung carcinoma cells synergistically. L-NAME or L-arginine administration enhanced the retention of B16 melanoma cells in the lungs examined 24 h after the tumor cell injection. Synergistic effect of L-NAME and L-arginine was also seen in the tumor cell retention. The present results suggest that the metastatic potentials of the tumor cells do not simply correlate to NO production in vivo.

Increase in experimental pulmonary metastasis in mice by L‐arginine under inhibition of nitric oxide production by NG‐nitro‐L‐arginine methyl ester

As we have previously reported, intraperitoneal injections of NG‐nitro‐L‐arginine methyl ester [L‐NAME; a competitive inhibitor of nitric oxide (NO) synthase] before and after the injection of B16 melanoma cells through a tail vein increased experimental pulmonary metastasis, while simultaneous injections of L‐arginine (a substrate of NO synthase) at a 20‐fold higher dose synergistically increased pulmonary metastasis. Our present study was intended to elucidate the mechanisms by which L‐NAME alone or together with L‐arginine increases metastasis. Injections of L‐NAME decreased the serum concentration of nitrite plus nitrate (metabolites of NO) by about 50%, which was not reversed by simultaneous injections of L‐arginine. Injections of L‐NAME also decreased the diameter of arterioles and venules by 20–30%, while simultaneous injections of L‐arginine did not show any significant effect. When collagen‐ or ADP‐induced platelet aggregation was examined using platelet‐rich plasma, injections of L‐NAME showed little effects on platelet aggregation, while simultaneous injections of L‐arginine rather suppressed platelet aggregation. B16 melanoma cells produced NO in culture, and L‐NAME (0.2 mM) decreased NO production without effects on viability. Our results suggest that the increased experimental pulmonary metastasis induced by L‐NAME can be ascribed partly to the contraction of arterioles and venules, which is induced by the inhibition of endogenous NO production by L‐NAME, and that the synergistic effect of L‐arginine on metastasis is related to the inhibition of endogenous NO production through unknown mechanisms. Int. J. Cancer 75:140–144, 1998.© 1998 Wiley‐Liss, Inc.

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

Abstract To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E 2 ) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E 2 pellets were removed, but administration of progesterone (P), 5α-dihydrotestosterone (DHT), or continued implantation of E 2 pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E 2 , P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.

Clinicopathological study of livers from brain-dead patients treated with a combination of vasopressin and epinephrine

Studies were made on the pathological lesions and biochemical indices of the livers of 22 patients in whom normal hemodynamics was maintained for 0-48 days after brain death by administration of vasopressin and epinephrine. Thirty-one specimens of liver tissues were obtained by percutaneous biopsy or at autopsy. The degrees of central venous congestion, central fibrosis, focal fibrosis, fatty metamorphosis, piecemeal necrosis, periportal fibrosis, and intrahepatic cholangitis in livers on various days after brain death were compared with those on the day of brain death (day 0). Central venous congestion was extensive on days 0-4, significantly less on days 5-14, and then again extensive on days 15-48. Central fibrosis and focal fibrosis showed no remarkable change during the 48-day period. Fatty metamorphosis, piecemeal necrosis, and periportal fibrosis showed no significant changes until day 16, but spread extensively on days 40-48. Intrahepatic cholangitis was scarcely observed on day 0 but began to increase after day 3, and spread extensively after day 5. The level of serum glutamic pyruvic transaminase did not increase in most patients until day 15. The mean value of prothrombin activity also did not decrease until day 15. However, the mean value of serum alkaline phosphatase increased gradually after day 3, and was correlated with cholangitis. The present study showed that during prolonged hemodynamic maintenance of brain-dead patients, pathological lesions did not spread or diminished and that biochemical indices did not become worse, or improved, in the first 2 weeks, except for increases in cholangitis and the serum alkaline phosphatase level.

Cytotoxic actions of cytokines on cultured mouse luteal cells are independent of nitric oxide.

We investigated the cytotoxic effects of various cytokines secreted by macrophages or T lymphocytes on luteal cells, and the role of nitric oxide (NO) produced by luteal cells in cytotoxic actions of cytokines. Mouse luteal cells were cultured in serum-free medium with interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta) alone, or with various combinations of these cytokines for 6 days. Cytotoxic actions of cytokines and NO production by luteal cells were evaluated by number of viable cells and the amount of nitrite and nitrate (stable metabolites of NO) in medium, respectively. IFN-gamma (1000 U/ml), TNF-alpha (3000 U/ml), or IL-1 beta (30 U/ml) alone, and the combination of TFN-alpha and IL-1 beta (10 U/ml) did not decrease number of viable cells and was without effects on NO production. The combination of IFN-gamma and IL-1 beta (10 U/ml) also did not decrease the number of viable cells, while it increased NO production a little but significantly. Combinations of INF-gamma and TNF-alpha, and IFN-gamma, TNF-alpha and IL-1 beta (10 U/ml) markedly decreased number of viable cells. The combination of IFN-gamma and TNF-alpha increased NO production a little but significantly, and the combination of three cytokines (IFN-gamma, TNF-alpha, and IL-1 beta) caused a greater increase in NO production. An NO synthase inhibitor, L-NG-monomethy-L-arginine (0.5 mM) or aminoguanidine (0.5 mM) abolished increases in NO production induced by combinations of IFN-gamma and TNF-alpha, and IFN-gamma, TNF-alpha and IL-1 beta completely without effects on number of viable cells. The present results indicate that combinations of cytokines including IFN-gamma and TNF-alpha induce death of cultured mouse luteal cells, and that the cytotoxic actions of these cytokines are independent of NO production by luteal cells.

Establishment and further characterization of a line of transgenic mice showing testicular tumorigenesis at 100% incidence.

We have reported production of transgenic mice containing human papillomavirus type 16 (HPV16) E6 and E7 oncogenes in which a characteristic testicular tumor develops at a very high incidence. Three transgenic mice transmitted the transgene to their siblings, in which the same type of tumor developed. In one line, named line 181, this testicular tumor developed in all the 93 males obtained for 10 generations. In most cases, this tumor was detectable bilaterally in the testes 9 to 10 months postdelivery. On cross-matings with other inbred strains, the HPV transgene was dominant in all the genetic backgrounds examined. In the condition of experimental cryptorchidism, obvious delay of tumor formation was observed. In these testes, the tumor cells were seen to arise from the interstitium. Moreover, this tumor also manifested obvious expression of gonadal specific 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and other enzymes for androgen metabolism. These observations strongly suggest that this tumor has originated from Leydig cells. This transgenic mouse line, therefore, provides a novel system for investigating in vivo carcinogenesis and the mechanism of transformation of male gonadal cells.

Concomitant hepatocellular carcinoma and non-Hodgkin's lymphoma in a patient with nodular regenerative hyperplasia

Hepatocellular carcinoma (HCC) is the most common primary cancer in the liver. Liver invasion of non‐Hodgkins lymphoma (NHL) is also often observed. But simultaneous existence of HCC and NHL in a liver is extremely rare. Such patients reported previously had cirrhotic livers. Herein is reported a patient who simultaneously had HCC and NHL in a liver without cirrhosis, but with nodular regenerative hyperplasia (NRH). NHL was of the diffuse large B‐cell type. Lymphoma cells invaded the portal vein, and formed thrombi. These thrombi would contribute to the development of NRH by decreasing portal vein blood flow. HCC was of the well‐differentiated type and there was a 2 cm‐sized nodule at the lateral segment. There is the possibility that NRH was associated with the HCC because NRH is reported as a premalignant lesion. HCC and NHL were colocalized in the liver without hepatic virus infection or cirrhosis, although common cause(s) of development of these malignancies remain unclear in the present case.

In vivo inhibition of tumour growth by dexamethasone in murine osteosarcomas

This study was performed to determine whether glucocorticoid (GC) is an effective inhibitor of tumour growth in murine osteosarcoma (OS) in vivo. The effects of dexamethasone (DEX) on the growth of this tumour were studied in male C3H/He mice. The animals received a dose of 1.25 or 5 microg/g of DEX in 0.1 ml of steroid solution daily intraperitoneally (i.p.) for 14 days. In each DEX-treated group, significant inhibition of the tumour growth curve was seen in a dose- dependent manner compared with the control group (P<0.0001). The percentage of proliferative cell nuclear antigen (PCNA)-positive cells was 22.7% in the 5 microg/g DEX treatment group compared with 67.6% in the control group (P=0.009). Furthermore, mifepristone, a GC receptor antagonist, blocked the inhibition of tumour growth induced by DEX. In the control group, tumour cells showed positive reactivity for nuclear glucocorticoid receptors (GR) by immunohistochemistry. The results of this study indicate that tumour growth inhibition by DEX in murine osteosarcoma may be via GR.

Effects of various growth factors on growth of a cloned human esophageal squamous cancer cell line in a protein-free medium

In order to investigate molecular mechanisms of the growth of human esophageal cancer in relation to growth factors, we have recently established a protein-free culture system [Hams F-12: Eagles minimum essential medium (1:1, v/v)] of TE-3-OS cells (a cloned cell line from human esophageal squamous cancer, TE-3). In the present study, we first examined effects of exogenous growth factors on the growth of TE-3-OS cells. The growth of TE-3-OS cells in the protein-free medium was significantly stimulated by insulin and insulin-like growth factor (IGF)-I or IGF-II, and less effectively stimulated by epidermal growth factor (EGF) or transforming growth factor (TGF)-alpha; platelet-derived growth factor, TFG-beta, acidic fibroblast growth factor (FGF) or basic FGF had no effects. TE-3-OS cells contained specific IGF-I binding sites (110,000 sites/cell), with a Kd value of 800 pM. Moreover, the growth induced by IGF-I, IGF-II or insulin was markedly and similarly (70-80%) inhibited by anti-IGF-I receptor antibody IgG. These data suggest that IGF-I, IGF-II and insulin, as well as EGF and TGF-alpha, are important mitogens for human esophageal cancer cells and that effects of IGFs and insulin are mediated predominantly via IGF-I receptors.