Inhibitory Effect of a Synthetic Prostacyclin Analogue, Beraprost, on Urokinase-type Plasminogen Activator Expression in RC-K8 Human Lymphoma Cells

Summary

Plasminogen activation by urokinase-type plasminogen activator (uPA)is implicated in tumor invasion and metastasis by the breakdown of extracellular matrix. We have recently demonstrated the inhibitory effect of cAMP on uPA gene transcription in RC-K8 human lymphoma cells (Biochim Biophys Acta 1268: 293-9, 1995). Prostacyclin produced by endothelial cells is shown to increase cellular cAMP levels by activating adenylate cyclase. We, therefore, examined the effect of a stable analogue of prostacyclin, Beraprost, on uPA production in RC-K8 cells. uPA activity gradually increased in the conditioned medium with time. Beraprost (0.1 nM-1.0 μM) inhibited uPA accumulation in a dose-dependent manner without affecting cell viability. Fibrin-zymography demonstrated that high and low molecular forms of uPA were present in the conditioned medium and that after Beraprost-treat-ment all forms of uPA decreased and no PA/PA inhibitor complex was present. Northern blot analysis revealed that after exposure to Bera-prost, uPA mRNA levels increased transiently and then rapidly decreased to below control levels. Treatment with Beraprost resulted in a rapid activation of cellular cyclic AMP-dependent protein kinase (PKA). Beraprost completely negated uPA gene expression induced by phorbol myristate acetate, an activator of protein kinase C (PKC). These results suggest that Beraprost inhibits uPA production by suppressing uPA gene expression through the PKA pathway and that PKA-mediated signals are dominant in uPA gene expression as compared to those medicated by PKC. This inhibition of uPA expression by a prostacyclin analogue may be an important fact to explain the mechanism of anti-metastatic effects of prostacyclin.

• References

• 1 Roldan AL, Cubellis MV, Masucci MT, Behrendt N, Lund LR, Danø K, Appella E, Blasi F. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis [published erratum appears in EMBO J 1990 May; 9 (5): 1674]. EMBO J 1990; 9: 467-474
• 2 Ellis V, Pyke C, Eriksen J, Solberg H, Danø K. The urokinase receptor: involvement in cell surface proteolysis and cancer invasion. [Review]. Ann NY Acad Sci 1992; 667: 13-31
• 3 Crowley CW, Cohen RL, Lucas BK, Liu G, Shuman MA, Levinson AD. Prevention of metastasis by inhibition of the urokinase receptor. Proc Natl Acad Sci USA 1993; 90: 5021-5025
• 4 Ganesh S, Sier C, Heerding MM, Griffioen G, Lamers C, Verspaget HW. Urokinase receptor and colorectal cancer survival. Lancet 1994; 344: 401-402
• 5 Wang H, Skibber J, Juarez J, Boyd D. Transcriptional activation of the urokinase receptor gene in invasive colon cancer. Int J Cancer 1994; 58: 650-657
• 6 Nekarda H, Schmitt M, Ulm K, Wenninger A, Vogelsang H, Becker K, Roder JD, Fink U, Siewert JR. Prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in completely resected gastric cancer. Cancer Res 1994; 54: 2900-2907
• 7 Wang SN, Miyauchi M, Koshikawa N, Maruyama K, Kubota T, Miura K, Kurosawa Y, Awaya A, Kanai Y. Antigen expression associated with lymph node metastasis in gastric adenocarcinomas. Pathology International 1994; 44: 844-849
• 8 Duffy MJ, Reilly D, Mcdermott E, Ohiggins N, Fennelly JJ, Andreasen PA. Urokinase plasminogen activator as a prognostic marker in different subgroups of patients with breast cancer. Cancer 1994; 74: 2276-2280
• 9 Pedersen H, Brunner N, Francis D, Osterlind K, Ronne E, Hansen HH, Danø K, Grondahlhansen J. Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue. Cancer Res 1994; 54: 4671-4675
• 10 Moncada S, Gryglewski R, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 1976; 263: 663-665
• 11 Moncada S, Herman AG, Higgs EA, Vane JR. Differential formation of prostacyclin (PGX or PGI2) by layers of the arterial wall. An explanation for the anti-thrombotic properties of vascular endothelium. Thromb Res 1977; 11: 323-344
• 12 Moncada S, Korbut R, Bunting S, Vane JR. Prostacyclin is a circulating hormone. Nature 1978; 273: 767-768
• 13 Armstrong JM, Dusting GJ, Moncada S, Vane JR. Cardiovascular actions of prostacyclin (PGI2), a metabolite of arachidonic acid which is synthesized by blood vessels. Circ Res 1978; 43 (Suppl. 01) 112-119
• 14 Kadowitz PJ, Chapnick BM, Feigen LP, Hyman AL, Nelson PK, Spannhake EW. Pulmonary and systemic vasodilator effects of the newly discovered prostaglandin, PGI2. J Applied Physiol: Respiratory Environ Exercise Physiol 1978; 45: 408-413
• 15 Honn KV, Tang DG, Chen YQ. Platelets and cancer metastasis: more than an epiphenomenon. [Review]. Semin Thromb Hemost 1992; 18: 392-415
• 16 Schneider MR, Tang DG, Schimer M, Honn KV. Prostacyclin and its analogues: antimetastatic effects and mechanisms of action. [Review]. Cancer Metastasis Rev 1994; 13: 349-364
• 17 Niiya K, Nsimba M, Hayashi T, Sakuragawa N. Down-regulation of urokinase secretion from a human lymphoma cell line RC-K8 by dexa-methasone without inducing plasminogen activator inhibitors. Thromb Res 1992; 65: 311-321
• 18 Kubonishi I, Niiya K, Yamashita M, Yano S, Abe T, Ohtsuki Y, Miyoshi I. Characterization of a new human lymphoma cell line (RC-K8) with t (11; 14) chromosome abnormality. Cancer 1986; 58: 1453-1460
• 19 Shinbo M, Niiya K, Al-mokdad M, Hayakawa Y, Hiraga K, Fujimaki M, Sakuragawa N. Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8. Biochim Biophys Acta 1995; 1268: 293-299
• 20 Degen JL, Estensen RD, Nagamine Y, Reich E. Induction and desensitization of plasminogen activator gene expression by tumor promoters. J Biol Chem 1985; 260: 12426-12433
• 21 Canipari R, O’Connell ML, Meyer G, Strickland S. Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator. J Cell Biol 1987; 105: 977-981
• 22 Queenan Jr J, Kao LC, Arboleda CE, Ulloa-Aguirre A, Golos TG, Cines DB, Strauss J. Regulation of urokinase-type plasminogen activator production by cultured human cytotrophoblasts. J Biol Chem 1987; 262: 10903-10906
• 23 Mira-y-Lopez R. Retinoic acid priming potentiates the induction of urokinase-type plasminogen activator by cyclic adenosine monophosphate in mouse mammary carcinoma cells. J Cell Physiol 1991; 147: 46-54
• 24 Takeuchi T, Niiya K, Kubonishi I, Miyoshi I. A plasminogen activator inhibitor-2 from a promyelocytic leukemia cell line, PL-21, binds to the carboxy-terminal chain of plasminogen activators. Thromb Haemost 1990; 63: 259-264
• 25 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63
• 26 Pearson D, Nigg EA, Nagamine Y, Jans DA, Hemmings BA. Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase. Exp Cell Res 1991; 192: 315-318
• 27 Granelli-Pipemo A, Reich E. A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med 1978; 148: 223-234
• 28 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-159
• 29 Sambrook J, Fritsh EF, Maniatis T. Molecular Cloning, A Laboratory Manual 2nd Ed. (2nd Ed. ed.). Cold Spring Harber Laboratory; 1989: 7.39
• 30 White WF, Barlow GH, Mozen MM. The isolation and characterization of plasminogen activators (urokinase) from human urine. Biochemistry 1966; 5: 2160-2169
• 31 Jans DA, Hemmings BA. LLC-PK1 cell mutants in cAMP metabolism respond normally to phorbol esters. FEBS Lett 1986; 205: 127-131
• 32 Rondeau E, Ochi S, Lacave R, He CJ, Medcalf R, Delarue F, Sraer JD. Urokinase synthesis and binding by glomerular epithelial cells in culture. Kidney Int 1989; 36: 593-600
• 33 Medcalf RL. Cell- and gene-specific interactions between signal transduction pathways revealed by okadaic acid. Studies on the plasminogen activating system. J Biol Chem 1992; 267: 12220-12226
• 34 von der Ahe D, Pearson D, Nagamine Y. Macromolecular interaction on a cAMP responsive region in the urokinase-type plasminogen activator gene: a role of protein phosphorylation. Nucleic Acids Res 1990; 18: 1991-1999
• 35 Ziegler A, Knesel J, Fabbro D, Nagamine Y. Protein kinase C down-regu-lation enhances cAMP-mediated induction of urokinase-type plasminogen activator mRNA in LLC-PK1 cells. J Biol Chem 1991; 266: 21067-21074
• 36 Foulkes NS, Borrelli E, Sassone-Corsi P. CREM gene: use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcription. Cell 1991; 64: 739-749
• 37 Hayashi T, Niiya K, Hirokawa S, Sakuragawa N. Synergistic stimulating effect between cyclic AMP and phorbol ester on plasminogen activator inhibitor type 2 production in human promyelocytic leukemia cell line PL-21. Biochim Biophys Acta 1992; 1134: 273-277
• 38 Niiya K, Taniguchi T, Shinbo M, Ishikawa T, Tazawa S, Hayakawa Y, Sakuragawa N. Different regulation of plasminogen activator inhibitor 2 gene expression by phorbol ester and cAMP in human myeloid leukemia cell line PL-21. Thromb Haemost 1994; 72: 92-97