Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Summary Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n=11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in periferal areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.     

Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n = 11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in peripheral areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.     

Proenzyme to urokinase-type plasminogen activator in the mouse in vivo

We have investigated whether urokinase-type plasminogen activator (u-PA) is present in the mouse in vivo as the proenzyme or as the active enzyme. u-PA in extracts of various murine tissues was of a one-polypeptide chain form with an electrophoretic mobility indistinguishable from purified proenzyme (pro-u-PA), as demonstrated by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblotting. No 2-chain u-PA was detected in any of the extracts (detection limit 10% of that of one-chain u-PA). In bladder urine more than half of the u-PA was of the one-chain form. Together with previous immunocytochemical studies of the normal murine tissues and studies of the Lewis lung carcinoma, the present results indicate that in these tissues the one-chain proenzyme is the predominant form of u-PA in intracellular stores and for the first time demonstrates that at least in some cases the one-chain form constitutes a sizeable fraction of the u-AP in extracellular fluids in the intact organism.     

Tissue-type plasminogen activator in rat adrenal medulla

Summary Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenalin. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Specific stimulation by estradiol of tissue-type plasminogen activator production in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor cells.

The hormonal regulation of two plasminogen activators, tissue-type plasminogen activator (t-PA) and urokinase (u-PA), was studied both in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma and in DMBA-induced rat mammary dysplasia. t-PA activity in DMBA-mammary carcinoma was decreased markedly by oophorectomy and recovered upon estradiol administration to reach the maximum level at 12 hr. In contrast to its effect on DMBA-mammary carcinoma, estradiol had no effect on t-PA activity in DMBA-mammary dysplasia. Furthermore, DMBA-mammary carcinoma cells in primary culture displayed similar estrogen-dependency in production of t-PA, while t-PA production in DMBA-mammary dysplasia cells was not under the control of estradiol in vitro. Moreover, estrogen-stimulated production of u-PA activity was not observed in DMBA-mammary carcinoma cells or DMBA-mammary dysplasia cells both in vivo and in vitro. Taken together, these results suggest that estrogen stimulates the production of t-PA but not u-PA and that this estrogen dependency of t-PA is limited to malignant DMBA-mammary tumor cells.     

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells

We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10(-6) M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PA-mediated focal proteolysis.     

Localization of urokinase-type plasminogen activator receptor on U937 cells: Phorbol ester PMA induces heterogeneity

The binding of human urokinase-type plasminogen activator (u-PA) to the surface of the human monocytic cell line U937 was studied by immunological detection of bound u-PA or binding of biotinylated diisopropyl fluorophosphate-inactivated human u-PA visualized by light or electron microscopy. Untreated U937 cells showed a characteristic binding pattern, with the majority of the u-PA bound to the microvillar-containing protruding pole of the cells. After treatment with the phorbol ester PMA, the resulting adherent cell population was very heterogeneous with respect to both cellular morphology and u-PA binding. The bound u-PA was distributed on both the dorsal and the substrate side of the cells, and the patches of bound u-PA could not be correlated to any typical membrane conformations or cell-cell or cell-substratum contacts. When a monoclonal antibody directed against the amino-terminal fragment (ATF) of u-PA was used, the results were identical regardless of whether intact u-PA or ATF was used for binding to the cells. In contrast, when a monoclonal antibody recognizing the non-receptor-binding protease domain of u-PA was used, bound ATF showed no staining, while bound intact u-PA was stained as efficiently as above. The alteration of u-PA receptor distribution following treatment with PMA could be related to the changes in glycosylation and ligand affinity of the purified u-PA receptor previously described following PMA treatment of U937 cells.     

The murine receptor for urokinase-type plasminogen activator is primarily expressed in tissues actively undergoing remodeling.

uPAR is a cellular receptor for urokinase plasminogen activator, an enzyme involved in extracellular matrix degradation during processes involving tissue remodeling. We have expressed a recombinant soluble form of murine uPAR and raised rabbit polyclonal antibodies to study the expression of uPAR by immunohistochemistry. The immunohistochemical localization of uPAR was determined in normal mouse organs and in tumors formed by the highly metastatic Lewis lung carcinoma. uPAR immunoreactivity was found in the lungs, kidneys, and spleen, and in endothelial cells in the uterus, urinary bladder, thymus, heart, liver, and testis. No uPAR immunoreactivity was detected in muscle. In general, strong uPAR immunoreactivity was observed in organs undergoing extensive tissue remodeling, as exemplified by trophoblast cells in placenta, and in migrating, but not resting, keratinocytes at the edge of incisional wounds. Staining was not detected in any tissue sections derived from uPAR-deficient mice, thus confirming the specificity of the immunohistochemical staining of uPAR in normal mouse tissues. In Lewis lung carcinoma, uPAR immunoreactivity was found in the tumor cells of the primary tumor and in lung metastases. (J Histochem Cytochem 49:237-246, 2001)     

Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.     

Tissue-type plasminogen activator in rat adrenal medulla

Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenaline. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.     

Fibrinolytic components in nasal mucosa and nasal secretion

 We evaluated a possible role for fibrinolytic components in nasal secretion by tissue localization with immunohistochemical techniques and by measuring their antigen concentrations in nasal discharge by means of ELISA and fibrin autography. Nasal mucosa was obtained surgically from the inferior turbinate. Urokinase-type plasminogen activator (u-PA) specific staining was observed in pseudostratified ciliated epithelium and was predominant in mucous cells of the seromucinous gland, while serous cells were almost devoid of stain. The pattern of staining of plasminogen activator inhibitor-2 was similar to that of u-PA. In contrast, plasminogen activator inhibitor-1(PAI-1) immunoreactive material was localized exclusively in serous cells of seromucinous glands. Positive staining for tissue-type plasminogen activator (t-PA) was observed in endothelial cells and basal cells, which differentiate into either ciliated or goblet cells. Nasal secretions were partially fractionated by immunospecific antibody-immobilized Sepharose. Subsequent fibrin autography patterns indicated the presence of u-PA, PAI-1, and t-PA. After methacholine provocation, the level of t-PA increased transiently but decreased rapidly with subsequent challenges. These differential stainings of fibrinolytic components and the existence of PAs and PAI-1 in the nasal discharge suggest that the fibrinolytic system may play a role in the movement and fluidity of nasal secretion. Accepted: 25 May 1998     

Ultrastructural localization of plasma membrane-associated urokinase- type plasminogen activator at focal contacts

We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.     

Flavanone and 2'-OH flavanone inhibit metastasis of lung cancer cells via down-regulation of proteinases activities and MAPK pathway

Flavanones richly exist in citrus and have been well characterized to have various bioactive properties. However, the anti-metastasis properties of flavanones remain unclear. The anti-metastatic effects of six flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, naringin, and naringenin were investigated in lung cancer cells. Despite little influence on cell viability, flavanone and 2'-OH flavanone markedly inhibited the invasion, motility, and cell-matrix adhesion of A549 cells. This was associated with a reduced expression of matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (u-PA) in treated cells. Treatment with flavanone and 2'-OH flavanone also potently attenuated the phosphorylations of extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38(MAPK), as well as the activations of NF-kappaB and AP-1. The reduced expressions of MMP-2 and u-PA, as well as inhibition of cell invasion were obtained in the cultures treated with U0126 (ERK 1/2 inhibitor) and SB203580 (p38(MAPK) inhibitor). Thus, the inhibitory effects of flavanone and 2'-OH flavanone on the expression of MMP-2 and u-PA may be at least partly through inactivation of ERK 1/2 and p38(MAPK) signaling pathways. Finally, oral administration of flavanone and 2'-OH flavanone were evidenced by its inhibition on the metastasis of A549 cells and Lewis lung carcinoma (LLC) cells in vivo. In conclusion, flavanone and 2'-OH flavanone perturb the invasion and metastasis of lung cancer cells, thereby constituting an adjuvant treatment for metastasis control.     

一些抗癌药物对荷瘤小鼠血清中唾液酸含量的影响

测定荷六种小鼠肿瘤S180肉瘤(实体型和腹水型),腹水肝癌(HepA),艾氏腹水瘤(EC),白血病P388和Lewis肺癌的小鼠腹水和血清中唾液酸含量,结果显示血清中唾液酸含量与肿瘤生长、肿瘤类型有关。腹水中唾液酸含量高,推测肿瘤能比正常组织产生更多唾液酸。对四种腹水肿瘤用阴离子交换树脂层析鉴定,发现HepA腹水中葡萄糖代唾液酸(NcuGc)含量明显低于其它三种腹水瘤。还研究了十几种抗癌药物对荷S180和Lewis肺癌小鼠血清中唾液酸含量的影响。发现吗丙嗪(probimane)和顺铂(DDP)能降低荷瘤小鼠血清中唾液酸含量,提示此二药物在肿瘤治疗中更具选择性。     

Suppression of tumor growth and invasion in 9,10 dimethyl benz(a) anthracene induced mammary carcinoma by the plant bioflavonoid quercetin

Administration of quercetin, a common polyphenolic component of many vascular and edible plants including vegetables, fruits and tea significantly reduced the tumor volume in rats induced for mammary carcinoma using dimethyl benz (a) anthracene (DMBA). Dose response was assessed, by treating the animals with different doses (15-45 mg/kgbw) of quercetin and 25 mg/kgbw was taken as effective dose. Quercetin was administered as an intra tumoral injection once a week for 4 weeks. Serum levels of carcino embryonic antigen (CEA), a potent marker for tumor growth and invasion was significantly decreased on quercetin treatment. Quercetin caused a significant decrease in the activities of acid phosphatase and Cathepsin D in serum of experimental animals. Activities of lysosomal enzymes- (beta-D galactosidase, beta-D glucuronidase, beta-D glucosidase and sialidase), in serum and tissue were significantly altered in DMBA animals compared to control animals. However, quercetin treatment caused no significant change in lysosomal enzyme activities in tissues, whereas the activities were significantly lowered in serum. Partial purification of tissue type plasminogen activator (t-PA) from the tumor and kidney showed increased activity in the DMBA induced animals. Serum urokinase, -like plasminogen activator (u-PA) was also increased in animals with tumor, indicating tumor invasion. Administration of quercetin caused a significant decrease of both t-PA and u-PA. In conclusion, the present study suggests the possible role of quercetin in primary and invasive mammary tumor treatment. The above observations in vivo warrant further studies, due to the easy availability, common occurrence and low toxicity of this dietary bioflavonoid.     

IL-10 promotes resistance to apoptosis and metastatic potential in lung tumor cell lines

Treatment of non-small cell lung carcinoma (NSCLC) remains at a disappointingly low success rate. Not only is metastatic spread common in NSCLC, but therapeutic success decreases dramatically once metastases are present. Understanding factors which contribute to poor prognosis in NSCLC is critical for development of more successful therapeutic approaches. Interleukin-10 (IL-10) expression has been shown in several studies to correlate with a poorer prognosis in NSCLC; however, the mechanisms by which IL-10 affects lung tumor growth and metastases are unclear. The goal of this study was to evaluate the effects of tumor-derived IL-10 on the growth and metastasis of lung cancer cells in a murine model. Lewis lung carcinoma cells were stably transfected with the chicken ovalbumin gene (cOVA) as a model tumor antigen (LL43 tumor cells) and subsequently transfected with the murine IL-10 gene (LL43-10 tumor cells). Subcutaneous growth of the LL43 tumor cells was not affected by expression of IL-10. However, LL43-10 tumors had a fourfold increase in tumor microvessel density, as indicated by CD31 staining. Metastatic potential was also increased in IL-10-expressing lung tumor cells, leading to a greater number of tumor cells in lymph nodes draining the primary tumor site. Finally, exposure of Lewis lung tumor cells in vitro to exogenous IL-10 dramatically increased their resistance to UV-induced apoptosis. These results indicate that a primary effect of IL-10 on lung cancer cells may be to increase their metastatic potential by promoting angiogenesis and resistance to apoptosis.