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In vitro decreases of the fibrinolytic potential of cultured human fibrosarcoma cell line, HT1080, by Nigella sativa oil.

Abstract

It is generally accepted that the fibrinolytic potential of tumor cells is related to their malignant phenotype. In the present study, Nigella sativa oil (NSO) was studied to evaluate its effect on the fibrinolytic potential of the fibosarcoma cell line HT1080 to elucidate whether this oil might have an antitumor activity through its modulation of the fibrinolytic potential of such cells. NSO produced a concentration-dependent inhibition of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1). When subconfluent HT1080 cells were conditioned with oil, a concentration (0.0-200 [micro]g oil/ml)-dependent decrease in t-PA, u-PA and PAI-1 antigen was observed. There was also a concentration-dependent decrease (from 0.0 to 112.5 [micro]g oil/ml) in the confluent cultures. The results showed that blackseed oil decreases the fibrinolytic potential of the human fibrosarcoma cell line (HT1080) in vitro, implying that inhibition of local tumor invasion and metastasis may be one such mechanism.

[c] 2004 Published by Elsevier GmbH.

Keywords: Nigella sativa; HT1080 cells; Fibrinolytic system; t-PA; u-PA; PAI-1

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Introduction

Blackseed, the seed of Nigella sativa L. (Ranunculaceae), has been employed for thousands of years as a spice, food preservative and curative remedy for numerous disorders (Chopra et al., 1956; Nadakarni, 1976; Yesilada et al., 1995). The historical tradition of blackseed use in medicine is substantial. It is referred to by the prophet Mohammed as having healing powers; it is also identified as the curative black cumin in the Holy Bible, and is described as the Melanthion of Hippocrates and Dioscorides and as the Gith of Pliny (Atta-ur Rahman et al., 1985).

The methanollic crude extract of N. sativa seed has shown in vitro cytotoxic effect on Erlich ascites carcinoma, Dalton's ascites lymphoma and sarcoma (Salomi et al., 1991, 1992). This extract was also active in vivo; the growth of Erlich ascites carcinoma was completely inhibited in mice. Exposure to the volatile oil obtained from blackseed altered the cellular expression of specific polypeptides in Jurkart T lymphoma cells, suggesting that changes in polypeptide expression might play a role in the biological activities attributed to blackseed (Hailat et al., 1995). In addition to these direct anti-tumor effects, blackseed preparations may have potential for cancer chemoprevention, as well as for reducing the toxicity of standard antineoplastic drugs (Salomi et al., 1991; Nair et al., 1991; Jang et al., 1997; Mabrouk et al., 2002). The anticancer activity of specific blackseed components, in particular that of the quinones thymoquinone (TQ) and dithymoquinone (DIM), has also been examined (Salomi et al., 1992; Worthen et al., 1998; Badary, 1999; Badary et al., 1999; Swamy et al., 2000).

A free vascular blood flow is maintained by a dynamic equilibrium between blood coagulation and fibrinolysis. The fibrinolytic system, the main function of which is to dissolve fibrin clots in the circulatory system, is composed of the inactive precursor plasminogen (plg), which can be converted into the proteolytic enzyme plasmin by the plasminogen activators (PAs), tissue-type PA (t-PA), and urokinase-type PA (u-PA). Fibrinolytic activity can be inhibited at both the level of the PAs and plasmin by PA inhibitors (PAI-1 and PAI-2) and [[alpha].sub.2]-antiplasmin, respectively, which are members of the serpin (serine proteinase inhibitor) family (Collen and Lijnen, 1991; Bulens et al., 1997).

There are many examples of increased plasminogen activator activity, especially of u-PA, in transformed cells (Unkeless et al., 1973; Christman et al., 1975) or in malignant tissues, as compared to their normal counterparts (Markus et al., 1983; Kirchheimer et al., 1985a). There are also some reports which indicate that urokinase antigen in plasma is often increased in malignant diseases (Huber et al., 1983; Kirchheimer et al., 1985b, 1987, 1990). In general, a high u-PA level and/or its receptor expression is correlated with invasive and metastatic behavior in various cancers (Kwaan, 1992). The PAs production by metastatic breast cancer cells and HT1080 fibrosarcoma cells can be modulated through interactions with basement membrane components. Researchers have reported that the marked increase in cell-bound u-PA through the encounter of MDA-MB-231 cells with the laminin and fibronectin (to a lesser extent) may set in motion a proteolytic cascade via plasmin, causing tissue degradation and facilitating tumor cell invasion (Pourreau-Schneider et al., 1989; Pollanen et al., 1987).

It is generally believed that u-PA, rather than t-PA, plays a crucial role in the metastatic spread of tumor cells (Dano et al., 1985). This belief is supported by the fact that in the tissues of tumors of diverse origin t-PA levels are often much lower than in benign tissue, whereas u-PA levels are consistently increased in tumors of diverse origin (De Vries et al., 1996a). Various reports suggest that t-PA can be involved in the matrix degradation and invasive processes of melanoma cells (Meissauer et al., 1991, 1992; Quax et al., 1991; Bizik et al., 1993; Qian et al., 1993; Stack et al., 1993; De Vries et al., 1996b). Kirchheimer et al. (1987) reported that it is unlikely that basal t-PA levels contribute significantly to changes in the activity of the fibrinolytic system of gastrointestinal carcinomas. In contrast to t-PA, PAI levels were elevated in these patients, an effect which was also reported by others in several groups of patients suffering from different diseases (Colucci et al., 1985; Kluft et al., 1985; Paramo et al., 1985), regardless of whether these patients suffered from deep venous thrombosis or not (Wiman et al., 1985). A positive correlation between PAI-1 experssion and pulmonary metastatic potential in human fibrosarcoma cells (HT1080 and HT1080-P4) has also been reported (Matsuda et al., 1993; Tsuchiya et al., 1995). Increased levels of PAI, on the other hand, might contribute to a thrombotic tendency (Wiman et al., 1985). The increase of PAI, however, cannot be used as an indicator of a carcinomatous disease, but rather of a diseased state in the patient (Kirchheimer et al., 1987).

Although the effect of NS on several processes has been investigated, there is a lack of information on the effect of NS on fibrinolysis of tumor cells. This study was therefore planned to elucidate the effect of N. sativa on the fibrinolytic potential of the fibrosarcoma cell line HT1080.

Materials and methods

Materials

Human HT1080 fibrosarcoma cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). N. sativa L. seed oil (El-Baraka seed oil, 450 mg/cap) was purchased as soft gelatin capsules from Pharco-pharmaceuticals (Alexandria, Egypt). This natural oil in Baraka capsules contains various constituents, including nigellone, fatty acids, glycosides, phenolic components, carotene, minerals such as phosphorus and iron, and some digestive enzymes. The oil was dissolved in dimethylsulfoxide (DMSO) and then diluted in incubation medium to yield 450 [micro]g/ml. Dulbeco's Modified Eagle's Medium (DME) and Ham's F-12 (F-12) culture media, Hank's Balanced Salt Solution (HBSS), and normal bovine, calf, and horse sera were purchased from GIBCO (Grand Island, NY). Serum-free medium supplement (ITS) was purchased from Collaborative Research, Inc. (Bedford, MA). Culture flasks, plates, and sterile plasticware were purchased from Costar (Cambridge, MA). Other materials used in the methods described below have been specified in detail in the pertinent references.

Cell culture

HT1080 cells were grown in supplemented DME medium containing glutamine (1 mM), penicillin (100 IU/ml), streptomycin, (100 [micro]g/ml), and 10% heatinactivated fetal calf serum. Cells were seeded at a density of 2-4 X [10.sup.4] cells/[cm.sup.2] and grown overnight at 37[degrees]C in a humidified 95% air/5% C[O.sub.2] atmosphere in DME medium with 5% heat-inactivated, charcoal-stripped fetal calf serum.

For experiments, cells were subcultured into gelatin-coated 12-well plates in DMEM medium supplemented with glutamine (1 mM), penicillin (100 IU/ml), 10% fetal calf serum and 10% human serum until subconfluency and confluency, in separated cultures, was reached. Isolated subconfluent and confluent cultures of HT1080 cells were washed twice with serum-free DMEM medium and the experiment was begun by the addition to each well of 0.5 ml DMEM medium, supplemented with 0.5% of heat-inactivated charcoal-stripped fetal calf serum and 0.25% bovine serum albumin. Cells were incubated overnight in low-serum conditions before stimulation. Blackseed oil was dissolved in DMSO at a concentration of 10-200 mg and stored at -80[degrees]C. The appropriate concentration was added to the medium in a volume corresponding to 0.1% of the culture medium. The control medium contained an equal amount of excipient.

Preparation of conditioned medium

Separated subconfluent and confluent cultures were washed gently twice with 0.5 ml of serum-free growth medium pre-warmed to 37[degrees]C, followed by incubation for 24 h at 37[degrees]C with various concentrations of oil in serum-free growth medium (0.6 ml final volume in each well). The aliquots were removed after 24 h incubation and were immediately frozen at -80[degrees]C for later measurement of t-PA, u-PA and PAI-1 (see below). At the end of each experiment, the cells were treated with trypsin/EDTA for 2-3 min at room temperature, and the cells counted in an electronic counter (Coulter). Each concentration was tested in triplicate and triplicate control wells containing only serum-free growth medium were included in each experiment. Viability of cells was confirmed at 24 h in each experiment by visual inspection.

Assay for tissue type PA (t-PA) antigen

t-PA antigen in HT1080 cell-conditioned medium was determined by two-site, 'sandwich' specific commercially available enzyme-linked immunosorbent assays (ELISA) (Technoclone, Austria) according to manufacturer's instructions. The test range for this assay is 0.2-2.5 ng/ml. The t-PA ELISA detects free t-PA and t-PA complexed with PAI-1. The t-PA secretion observed with each oil concentration was expressed as nanogram (ng) per 10,000 for subconfluent cultures and 100,000 cells for confluent cultures to correct for any differences in cellular proliferation induced by the various agents used.

Assay for urokinase type PA (u-PA) antigen

u-PA antigen in conditioned medium was determined by an ELISA as described by Wojta et al. (1989).

Assay for plasminogen activator inhibitor type-1 (PAI-1) antigen

PAI-1 antigen in conditioned medium was measured by a two-site 'sandwich' ELISA. A monoclonal anti-PAI-1 antibody (5PAI12) that recognizes active PAI-1, latent PAI-1 and PAI-1 in complex with t-PA immobilized to a micro-ELISA plate was used to bind the PAI-1 contained in the samples. A second peroxidase-labeled monoclonal anti-PAI-1 antibody (3PAI15) that recognizes active and latent PAI-1 as well as PAI-1 in complex with t-PA was used to quantify the amount of bound PAI-1. Purified melanoma PAI-1 (Wagner and Binder, 1986) was used as a calibration standard (Resh et al., 1989). The PAI-1 levels in conditioned medium were expressed as nanogram (ng) per 10,000 and 100,000 cells for subconfluent and confluent cultures, respectively, to correct for variations in cellular proliferation induced by the various agents used.

Statistical analysis

The significance of differences in t-PA, u-PA and PAI-1 secretion in response to various oil concentrations was assessed using the Statgraph Program student's two-tailed t-test, with p<0.05 considered significant. The figures have been plotted using Sigma Plot Scientific Graphing Software Program, Version 2.01.

Results

Effects of blackseed oil on the release of t-PA, u-PA and PAI-1 antigen

Subconfluent cultures: As shown in Table 1 and Figs. 1a-c, increasing concentrations of oil caused significant, dose-dependent decreases in t-PA, u-PA and PAI-1 production by HT1080 fibrosarcoma cells in conditioned medium, as compared with control. Cultures treated with 200 [micro]g/ml oil released t-PA related antigen (0.08 [+ or -] 0.02 ng/[10.sup.4] cells/24 h vs. control 0.35 [+ or -] 0.03 ng/[10.sup.4] cells/24 h, n = 9, p < 0.001), u-PA related antigen (0.7 [+ or -] 0.26 ng/[10.sup.4] cells/24 h vs. control 3.2 [+ or -] 0.22 ng/[10.sup.4] cells/24 h, n = 9, p < 0.001) and PAI-1 related antigen (0.7 [+ or -] 0.2 ng/[10.sup.4] cells/24 h vs. control 51 [+ or -] 3.2 ng/[10.sup.4] cells/24 h, n = 9, p < 0.001).

Confluent cultures: The levels of t-PA, u-PA and PAI-1-related antigen secretions were dramatically decreased by the oil, at concentrations between 0.0 and 112.5 [micro]g/ml oil, in HT1080 fibrosarcoma cells (p<0.001) (Table 2, Figs. 2a-c).

Discussion

Blackseed oil and its preparations have demonstrated significant in vitro and in vivo antineoplastic activity (Salomi et al., 1992; Hailat et al., 1995; Worthen et al., 1998; Badary and Gamal el-Din, 2001; Gunduz et al., 2002). It is generally accepted that the fibrinolytic potential of tumor cells correlates with their respective malignancy and might play an important role in tumor invasion and metastasis (Binder, 1990). Therefore, the regulation of t-PA, u-PA and PAI-1 synthesis may be helpful in developing drugs to counteract insufficient endogenous t-PA, u-PA and PAI-1 by decreasing their production. For metastatic activity, Pourreau-Schneider et al. (1989) reported that PA production by metastatic breast cancer cells could be modulated through interactions with basement membrane components. In this study, NSO was shown to have a significant inhibitory effect on fibrinolytic enzymes in the fibrosarcoma cell line, HT1080, indicating a stong antitumor activity. This result suggests that the oil contains some ingredient(s), which may directly counteract PA proteolytic enzymes or indirectly induce other factors downstream in the signaling cascade with the respective promoter of t-PA, u-PA and PAI-1. This speculation gains further strength with recent reports (Swamy and Tan, 2000; Khan et al., 2003). Such coordinated regulation leads to modulation of the fibrinolytic potential of the HT1080 cell line which may contribute to the mechanisms by which oil has been observed to affect the behavior of transformed cells (Salomi et al., 1992; Hailat et al., 1995; Worthen et al., 1998; Badary et al., 1999; Swamy et al., 2000) and might be one of the explanations for the beneficial effects seen with local blackseed extract treatment of skin carcinogenesis in mice (Salomi et al., 1991). It has been reported that the antitumor effect of Nigella can be attributed to its content of certain long-chain fattty acids (Salomi et al., 1992; Mabrouk et al., 2002). The existence of such fatty acids in NSO may explain its antiumor effect in affecting the fibrinolytic cascade in this study. Among the mechanisms associating cancer prevention with using such nutrients are inhibition of cylooxygenase-2 and prostaglandins (Sharma et al., 2001), inhibition of inducible nitric oxide synthase (Hur et al., 2001), arresting the cell cycle at S/G2 transition and apoptosis (Joe et al., 2001).

[FIGURE 1 OMITTED]

These data indicate that NSO are exerting inhibitory actions on the secretion of fibrinolytic enzymes of HT1080 cells in vitro. This modulation of fibrinolytic potential is due to a decrease in the amount of free, active t-PA and a concomitant reduction of fibrinolytic activity by inhibited complex formation between PAI-1 and t-PA. In line with this, it has been shown that azeliac acid (AZA) decreases the fibrinolytic potential of the melanoma cell lines in vitro (Wagner et al., 1985, 1986; Addo-Boadu et al., 1996) suggesting several mechanisms of action to explain the beneficial effects of AZA seen in vivo. These include inhibition of tyrosinase activity (Nazzaro-Porro et al., 1979), inhibition of mitochondrial enzymes (Passi et al., 1984; Robins et al., 1985), the migratory behavior of melanoma cells and inhibition of RNA and DNA synthesis (Leibl et al., 1985; Galhaup, 1989). Also, the synthetic glucocorticoid dexamethasone (DEX) has a negative effect on u-PA expression by reducing the transcription rate in HT1080 cell line (Bulens et al., 1997).

There is a positive correlation between PAI-1 expression and pulmonary metastatic potential in human fibrosarcoma cells, HT1080 (Matsuda et al., 1993), in vitro or by facilitating tumor cell lodgement in vessels in vivo (Tsuchiya et al., 1995). This study showed that blackseed oil reduced PAI-1 antigen significantly in the HT1080 cell line in a concentration-dependent manner. High expression of both u-PA and its inhibitor PAI-1 in tumor extracts suggests a tightly linked regulation of these genes (De Vries et al., 1996a), which may explain the concomitant changes of u-PA and PAI-1 antigens in HT1080 oil-treated cells. Evidence for a linkage of expression of the genes of the plasminogen activation system is also present at the molecular level. Promoters of the t-PA and PAI-1 genes, for instance, share a great deal of homology (Luskutoff, 1991). Both the u-PAR (Lund et al., 1991) and the PAI-1 gene promoters contain TGF-[beta]1-responsive elements (Riccio et al., 1992). The decrease of t-PA, u-PA and PAI-1 induced by NSO might result from the repression of the transcription from the promotors of the human u-PA, t-PA and PAI-1 gene (Kunz et al., 1995), involving interaction of some oil constituents or another factor downstream in the signaling cascade with the respective promoter of t-PA, u-PA and PAI-1. Such coordinated regulation leading to a decrease in the production of fibrinolytic proteins would result in a net decrease in the fibrinolytic activity and allow for a lesser potential for fibrinolysis in the cells, leading to impaired proteolysis and inhibition of metastasis. Therefore, evaluation of the effect of blackseed oil in HT1080 cells might be revealed as a complex differential regulation of the fibrinolytic components t-PA, u-PA and PAI-1.

[FIGURE 2 OMITTED]

In this study, the production of the respective fibrinolytic components in cell line HT1080 was normalized for cell numbers so the observed changes are not likely to be caused by antiproliferative effects of the oil. It has to be pointed out, however, that the actual fibrinolytic activity is also influenced by factors not determined in this study, such as u-PA receptors, PAI-2 and [[alpha].sub.2]-antiplasmin. Blackseed oil might therefore offer a therapeutic approach in ameliorating or treating fibrosarcoma, as in the other tumors, which have been mentioned previously. This may be appropriate because its always much better, from a drug development perspective, to inhibit or to interfere with an enzymatic activity than to interfere with ligand binding to a receptor, especially when the threshold for activating signaling in tumor cells is not known.

Conclusion

In conclusion, Nigella sativa oil (NSO) possesses significant antitumor activity in vitro via depletion of the plasminogen activation system. To our knowledge, this is the first report of a possible role for blackseed oil in modulating the plasminogen activation system in a tumor cell line and of a possible therapeutic role for Nigella sativa oil modulation of the fibrinolytic potential of fibrosarcoma. Further in vivo and clinical investigations will be necessary prior to the recommendation of its use as a safe and effective antifibrinolytic remedy for cancer patients.
Table 1. Effects of NSO on the fibrinolytic parameters in subconfluent
cultured HT1080 conditioned medium (CM)

Oil ([micro]g/ t-PA antigen u-PA antigen
ml) (ng/ml/[10.sup.4] cells) (ng/ml/[10.sup.4] cells)

Control 0.35[+ or -]0.03 3.2[+ or -]0.22
 25 0.26[+ or -]0.02** 2.6[+ or -]0.26*
 50 0.23[+ or -]0.02*** 1.9[+ or -]0.19***
100 0.1[+ or -]0.03*** 1.4[+ or -]0.24***
200 0.08[+ or -]0.02*** 0.7[+ or -]0.26***

Oil ([micro]g/ PAI-1 antigen
ml) (ng/ml/[10.sup.4] cells)

Control 51[+ or -]3.2
 25 33[+ or -]3.1***
 50 18[+ or -]2.8***
100 1.1[+ or -]0.4***
200 0.7[+ or -]0.2***

HT1080 cells were treated with the indicated concentrations of oil.
Samples of the conditioned medium were taken after 24 h. Data are
expressed relative to control and represent the mean [+ or -] SD (n = 9)
values of three experiments, each performed in triplicate. *p < 0.05;
**p<0.01; ***p<0.001.

Table 2. Effects of NSO on the fibrinolytic parameters in confluent
cultured HT1080 conditioned medium (CM)

Oil ([micro]g/ t-PA antigen u-PA antigen
ml) (ng/ml/[10.sup.5] cells) (ng/ml/[10.sup.5] cells)

Control 1.7[+ or -]0.18 8.7[+ or -]0.23
14.06 1.4[+ or -]0.18* 7.8[+ or -]0.32***
28.125 1.35[+ or -]0.21* 7.0[+ or -]0.22***
56.25 1.07[+ or -]0.12*** 5.9[+ or -]0.26***
112.5 0.78[+ or -]0.15*** 4.2[+ or -]0.21***

Oil ([micro]g/ PAI-1 antigen
ml) (ng/ml/[10.sup.5] cells)

Control 534[+ or -]9.0
14.06 503[+ or -]11***
28.125 455[+ or -]16***
56.25 295[+ or -]13***
112.5 130[+ or -]7.0***

HT1080 cells were treated with the indicated concentrations of oil.
Samples of the conditioned medium were taken after 24 h. Data are
expressed relative to control and represent the mean[+ or -]SD (n = 9)
values of three experiments, each performed in triplicate. *p < 0.05;
**p < 0.01; ***p < 0.001.


Acknowledgements

The author wishes to acknowledge the kindness and scientific guidance of Professor Bernd R. Binder, Director of the Institute of Vascular Biology and Thrombosis Research, Faculty of Medicine, Vienna University, Austria, who introduced me to the field of fibrinolysis and provided both laboratory facilities and kind continous advice.

Received 22 July 2003; accepted 3 September 2003

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E.M. Awad*

Department of Zoology, Faculty of Science, University of Minia, Minia, Egypt

*Corresponding author. Otto-Meyerhof-Zentrum, Abt. Innere Med. I, Labor Prof. Nawroth, Raum: 00.031, Im Neuenheimer Feld 350, Heidelberg (HD) 69120, Germany. Tel.: +49-06221/56-4750; fax: +49-06221/56-4754.

E-mail address: ezzat_awad@med.uni-heidelberg.de (E.M. Awad).
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Author:Awad, E.M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Jan 1, 2005
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