To examine sex- and menstrual cycle-related differences in thermoregulatory responses to heat exposure, ten young women and six young men were heated passively by immersing their legs in water heated to 42 degrees C for 60 min (in ambient conditions of 30 degrees C and 45% relative humidity). The women underwent heat exposure during the mid-follicular (F) and mid-luteal (L) phases of the menstrual cycle, which were confirmed by assaying plasma female reproductive hormones. The rectal and mean body (T(b)) temperatures of women in the L phase were significantly greater than those of women in the F phase and of men during a pre-heating equilibration period (28 degrees C) and during heat exposure. During heat exposure, the local sweat rates (m(sw)) on the forehead, chest, back, and forearm of women in either phase were significantly lower than those of men, but the thigh (m(sw)) was similar to that of men. The m(sw) did not change at any site during the different phases of the menstrual cycle. The cutaneous blood flow (%LDF) was significantly greater on the thigh for women in either phase compared with men, but no difference was found at any other site (forehead, chest, back, and forearm). The %LDF on the back was significantly greater for women in the L phase than in the F phase, but those at other sites were similar in both phases. We conclude that, compared with men, heat loss from women depends more on cutaneous vasodilation (especially on the thigh) than on sweating, irrespective of the phase of the menstrual cycle. This phenomenon was due to peripheral mechanisms, as reflected in the greater slope of the relationship between %LDF and T(b) lower slope of the relationship between m(sw)) and frequency of sweat expulsion, and lower sweat output per gland. The menstrual cycle modified the T(b) threshold for vasodilation and sweat onset in women. Therefore, the sex difference in the T(b) threshold was more marked for women during the L phase than during the F phase. Moreover, the menstrual cycle modified the slope of the relationship between %LDF on the back and T(b).

Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-␤1 induces a dose-and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-␤1, we investigated which signaling pathway transduced by TGF-␤1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-␤1 activated Src kinase; 2) TGF-␤1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-␤1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-␤1-mediated Akt stimulation, whereas TGF-␤1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-␤1 was almost totally blocked by PP2 and PD98059 and partially (ϳ55%) by LY294002; 6) TGF-␤1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-B by pyrrolidinedithiocarbamate reduced the TGF-␤1-induced activation of the uPA gene by ϳ65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-␤1 activation of two distinct pathways (Src-MAPK-PI3K-NF-B-dependent and Src-MAPK-AP-1-dependent) for TGF-␤1-dependent uPA up-regulation and promotion of invasion.The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) 1 could be involved in the pathogenesis of peritoneal dissemination (1). uPA is a serine protease associated with various pathological conditions including tumor invasion and metastasis (2). One of the factors regulating the metastatic process is considered to be transforming growth factor-␤ (TGF-␤), which is a multifunctional cytokine that elicits numerous cellular effects pertinent to the metastatic process (1). TGF-␤ regulates a wide range of physiological and pathological cellular processes, including cell growth, differentiation, invasion, migration, mesenchymal transition, extracellular matrix synthesis, and cell death in many cell types including ovarian cancer cells (3). Recent data demonstrated that TGF-␤ specifically stimulates up-regulation of uPA mRNA and protein in certain types of neoplastic cells (1). The cellular mechanism(s) of the TGF-␤-induced uPA-dependent tumor invasion and metastasis has been extensively studied. The identity of the signaling pathway(s) involved in the TGF-␤-induced uPA up-regulation (4) remains less known. In a well...

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik ؉ clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik ؉ clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik ؉ clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik ؉ clones. © 2003 Wiley-Liss, Inc. Key words: bikunin; invasion; transfection; urinary trypsin inhibitor; urokinase-type plasminogen activatorInvasion and metastasis by tumor cells have multifactorial steps, may require the action of proteolytic enzymes at each step and are complex processes in which many gene activities are involved. 1 Various proteins on the surfaces of tumor cells mediate attachment to and degradation of the basement membrane and components of the extracellular matrix. 2 Increased activity, membrane association and secretion of urokinase-type plasminogen activator (uPA), plasmin, certain cathepsins and matrix metalloproteinases (MMPs) have been shown to correlate positively with invasiveness and the metastatic properties of many tumor entities. [1][2][3][4] The penetrating tumor cell focuses proteolytic activity at its cell surface through receptors for uPA, plasmin and MMPs. 3,4 uPA and its receptor are instrumental in cell invasion and metastasis. 5 uPA has a pleiotropic effect on cell migration and spreading in vivo and in vitro through the activation of plasminogen or other protein factors at the cell surface or in the extracellular matrix. Thus, uPA is at the center of a complex system affecting cell invasion and metastasis. 5 Bikunin (bik), also known as urinary trypsin inhibitor, is a Kunitz-type protease inhibitor which presents in human amniotic fluid, urine and in...

Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. Tumor cell invasion is dependent on finely regulated extracellular proteolytic activity, which allows tumor cells to invade the extracellular matrix (1). Among the proteolytic enzymes involved in this process are PAs, 1 whose expression in cultured cells is regulated by several types of growth factors and cytokines (2). Invasive tumor cells not only express cell-associated proteases but also secrete anti-proteases, preventing the overdigestion of the extracellular matrix, which leads to a loss of cell attachment. The balance between proteolytic activity and inhibition is crucial in the invasive and metastatic event (3). Indeed, the increment of a specific PA inhibitor, PAI-1, could have an important regulatory role on the extracellular proteolysis and might explain the decrease of net PA and gelatinolytic activities measured in the medium (3, 4). Even in the high levels of PAI-1 in the medium, however, several growth factors, including TGF-␤1, induced the increase of net PA and gelatinolytic activities on the plasma membrane, which results in strong proteolytic activity in some specific sites, such as areas of cell-to-cell or cell-to-extracellular matrix contacts. For this reason, drugs that manipulate or suppress signal transduction on the PA system in malignant cells offer a potentially new approach to anti-cancer therapy. One prototype signal transduction therapy agent is bikunin, which is a Kunitz-type protease inhibitor with tumor-suppressive potential in several malignant cell types, acting at the level of tumor invasion and metastasis (5-7). It was subsequently demonstrated that bikunin inhibits tumor invasion, at least in part, by a direct inhibition of plasmin activity as well as by inhibiting uPA (5, 6) and uPAR (7) expression at the gene and protein levels. Interestingly, in cell-free solutions, bikunin does not inhibit uPA activity effectively. Mechanistic studies in several cell types demonstrated that bikunin interferes with an upstream target(s) of selected MAP kinase signaling processes such as phosphorylation of MEK and ERK, leading to overexpression of uPA (6). A recent study in our laboratory demonstrated that bikunin could interfere with selected calcium-sensitive signaling processes such as agonist-induced cytokine expression in several types of cells, including human umbilical vein endothelial cells, uterine myometrial cells, vascular endo...

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMAstimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC-and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.

Bikunin, a Kunitz-type protease inhibitor, could potentially suppress tumor cell invasion and metastasis. Our previous study revealed that overexpression of bikunin in a human ovarian cancer cell line, HRA, resulted in a down-regulation in uPA and uPAR gene expression. For identifying the full repertoire of bikuninregulated genes, a cDNA microarray hybridization screening was conducted using mRNA from bikunintreated or bikunin-transfected HRA cells. A number of bikunin-regulated genes were identified, and their regulation was confirmed by Northern blot analysis. Our screen identified 11 bikunin-stimulated genes and 29 bikunin-repressed genes. The identified genes can indeed be classified into distinct subsets. These include transcriptional regulators, oncogenes/tumor suppressor genes, signaling molecules, growth/cell cycle, invasion/ metastasis, cytokines, apoptosis, ion channels, extracellular matrix proteins, as well as some proteases. This screen identified suppression of several genes such as CDC-like kinase, LIM domain binding, Ets domain transcription factor, Rho GTPase-activating protein, tyrosine phosphorylation-regulated kinase, hyaluronanbinding protein, matriptase, and pregnancy-associated plasma protein-A (PAPP-A), which have previously been implicated in enhancing tumor promotion. Northern blot analysis confirmed that several genes including matriptase and PAPP-A were down-regulated by bikunin by ϳ9-fold. Further, genetic inhibition of matriptase or PAPP-A could lead to diminished invasion. These results show that bikunin alters the pattern of gene expression in HRA cells leading to a block in cell invasion.

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-␤1 (TGF-␤1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-␤1; 3) that exogenous TGF-␤1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a timeand concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.

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