Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death and its prognosis remains poor due to the high risk of tumor recurrence and metastasis. Berberine (BBR) is a natural compound derived from some medicinal plants, and accumulating evidence has shown its potent anti-tumor activity with diverse action on tumor cells, including inducing cancer cell death and blocking cell cycle and migration. Molecular targets of berberine involved in its inhibitory effect on the invasiveness remains not yet clear. In this study, we identified that berberine exhibits a potent inhibition on the invasion and migration of HCC cells. This was accompanied by a dose-dependent down-regulation of expression of Cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 in berberine-treated HCC cells. Furthermore, berberine inactivated p38 and Erk1/2 signaling pathway in HCC cells. Primarily, this may be attributed to the up-regulation of plasminogen activator inhibitor-1 (PAI-1), a tumor suppressor that can antagonize uPA receptor and down-regulation of uPA. Blockade of uPA receptor-associated pathways leads to reduced invasiveness and motility of berberine-treated HCC cells. In conclusion, our findings identified for the first time that inactivation of uPA receptor by up-regulation of PAI-1 and down-regulation of uPA is involved in the inhibitory effect of berberine on HCC cell invasion and migration.

Targeting of plasminogen activator inhibitor 1 improves fibrinolytic therapy for tetracycline-induced pleural injury in rabbits.

Endogenous active plasminogen activator inhibitor 1 (PAI-1) was targeted in vivo with monoclonal antibodies (mAbs) that redirect its reaction with proteinases to the substrate branch. mAbs were used as an adjunct to prourokinase (single-chain [sc] urokinase [uPA]) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. Outcomes of scuPA IPFT (0.25 or 0.0625 mg/kg) with 0.5 mg/kg of mouse IgG or mAbs (MA-33H1F7 and MA-8H9D4) were assessed at 24 hours. Pleural fluid (PF) was collected at 0, 10, 20, and 40 minutes and 24 hours after IPFT and analyzed for plasminogen activating (PA), uPA, fibrinolytic activities, levels of total plasmin/plasminogen, α-macroglobulin (αM), mAbs/IgG antigens, free active uPA, and αM/uPA complexes. Anti-PAI-1 mAbs, but not mouse IgG, delivered with an eightfold reduction in the minimal effective dose of scuPA (from 0.5 to 0.0625 mg/kg), improved the outcome of IPFT (P < 0.05). mAbs and IgG were detectable in PFs at 24 hours. Compared with identical doses of scuPA alone or with IgG, treatment with scuPA and anti-PAI-1 mAbs generated higher PF uPA amidolytic and PA activities, faster formation of αM/uPA complexes, and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and αM antigens. Targeting PAI-1 did not induce bleeding, and rendered otherwise ineffective doses of scuPA able to improve outcomes in tetracycline-induced pleural injury. PAI-1-neutralizing mAbs improved IPFT by increasing the durability of intrapleural PA activity. These results suggest a novel, well-tolerated IPFT strategy that is tractable for clinical development.

Hyperthermia inhibits angiogenesis by a plasminogen activator inhibitor 1-dependent mechanism.

Hyperthermia (HT) associated with radiotherapy or chemotherapy is a promising method for cancer treatment, although the molecular mechanisms of this process are not well understood. HT exhibits various antitumor effects, including damage of tumor vasculature. Here, we investigate the effect of HT on in vitro and in vivo angiogenesis. We show that heat treatment of endothelial cells (ECs) affect their differentiation into capillary-like structures in two models of in vitro angiogenesis. Furthermore, the formation of new vessels promoted by angiogenic inducers in the chick embryo chorioallantoic membrane assay is impaired after heat treatment. These effects cannot be explained by direct cytotoxicity but are dependent on modulation of angiogenesis-involved genes. Gene expression profile of ECs subjected to heat shock demonstrates that plasminogen activator inhibitor 1 (PAI-1), a protein involved in the control of extracellular matrix degradation, is specifically up-regulated. The use of anti-PAI-1-neutralizing antibodies reverts the effect of HT on the in vitro EC morphogenesis and in vivo vessel formation. Moreover, microvessel outgrowth from PAI-1(-/-) aortic rings was not affected by HT compared with aortic rings from PAI-1(+/+) mice. Heat treatment of murine mammary adenocarcinomas results in inhibition of tumor growth, associated with a reduction of microvessel number and an increase of PAI-1 expression. These results indicate that heat-mediated PAI-1 induction is an important pathway by which HT exerts its antitumor activity and may represent a rationale for a combined cancer therapy based on HT associated with antiangiogenic molecules.

Prevention of renal fibrin deposition in endotoxin-induced DIC through inhibition of PAI-1.

Plasminogen activator inhibitor-1 (PAI-1) increases in endotoxemia thus possibly cooperating in altering the hemostatic balance in a prothrombotic direction. The effect of the inhibition of PAI-1 with the monoclonal antibody MA-33B8 was studied systemically and in kidneys in a lapine model of endotoxin-induced disseminated intravascular coagulation (DIC). The increase in plasmatic PAI activity in the control group (n = 9) was inhibited in the MA-33B8 treated rabbits (n = 5). Control rabbits showed renal fibrin deposits, whereas only one of the MA-33B8 rabbits did so. These results were confirmed immunohistochemically in kidneys as PAI-1 immunostaining was seen inside the glomeruli and larger vessels in the control group, whereas MA-33B8 rabbits showed a remarkable decrease, demonstrating that MA-33B8 successfully inhibited PAI-1 in the kidneys as well. Therefore evidence for the important role of PAI-1 in fibrin generation in endotoxin-induced DIC is presented, suggesting that strategies aiming at its reduction can be useful in this pathology.

Alterations of fibrinolytic activity in human during and after hyperbaric oxygen exposure.

To clarify the stage of fibrinolytic activation by hyperbaric oxygen (HBO) exposure, we examined its alterations in human during and after the HBO exposure. Eight healthy female volunteers breathed oxygen at 284 kPa (2.8 atmospheres absolute). Blood samples were collected before compression, shortly after compression to the pressure 284 kPa, shortly before the start of decompression, shortly after decompression, and then again 3 hours after decompression. We estimated the euglobulin fibrinolytic activity (EFA) and, the activities and antigens of both tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). The PAI-1 activity and PAI-1 antigen showed significant decrease after compression to a pressure 284 kPa, before the start of decompression, and after decompression. The EFA level and t-PA activity rose significantly shortly after decompression, and 3 hours later returned on baseline. These findings suggest that fibrinolytic activity is elicited after HBO rather than during HBO.

Preliminary studies on the role of plasminogen activator in seminal plasma of human and rhesus monkey.

Two types of plasminogen activators (PA), tissue type (tPA) and urokinase type (uPA), were identified in the seminal plasma of both the human and the rhesus monkey. We studied the possible relationship between PA activities in the seminal plasma and the sperm counts and motility and demonstrated that: (i) PA activity in human seminal plasma from infertile patients was associated with immotile spermatozoa; (ii) the treatment of fertile men with testosterone enanthate (TE) to induce azoospermia was accompanied by an increase in seminal PA activity; (iii) when monomer T4 (isolated from multiglycosides of Tripterygium wilforddi) was administered to fertile male rhesus monkeys to induce azoospermia, PA activities in seminal plasma increased considerably; and (iv) immunocytochemistry studies showed that both uPA and PAI-1 antigens were localized on the surface of human spermatozoa, indicating that human spermatozoa were capable of binding uPA and PAI-1 through their receptors or forming a complex. These data demonstrate that seminal PA activity may be related to azoospermia, and possibly, to the fertilizing capability of spermatozoa in primates.

Separation of active and inactive forms of recombinant human plasminogen activator inhibitor type 1 (PAI-1) expressed in Chinese hamster ovary cells: comparison with native human PAI-1.

Human plasminogen activator inhibitor type 1, PAI-1, was expressed in Chinese hamster ovary cells. A production level of 10-15 mg latent PAI-1 per liter of media was achieved after methotrexate amplification. Latent recombinant PAI-1 was purified by two chromatographic steps, cation exchange chromatography on CM-Sepharose and affinity chromatography on heparin-Sepharose. The obtained latent PAI-1 was approximately 90-95% pure showing one homogenous peak upon size-exclusion chromatography. However, four different isoforms due to different degrees of sialylation could be seen upon isoelectric focusing. Purified latent PAI-1 was activated by incubation in 6 M guanidine-HCl. By this method, 40-60% of PAI-1 was converted to an active form after removing the denaturant. The active fraction of PAI-1 was separated from inactive material by size exclusion chromatography on Superdex 200. Active PAI-1 migrated as expected for a 43-kDa large protein, while inactive PAI-1 migrated as larger protein complexes, suggesting that the remaining inactive PAI-1 was in the form of aggregates. This method for the separation of active and inactive PAI-1 could also be used for activated native PAI-1 prepared from human endothelial cells. Active recombinant PAI-1 was remarkably stable at pH 5.5, both when stored on ice and when stored at room temperature.