Abstract: In this study, we found that zinc exerted a dual inhibition effect on plasminogen activator/plasmin system by inhibiting plasminogen activator activation and down-regulating plasmin activity. Zinc demonstrated significant inhibition effects on plasminogen activator and plasmin induced cell morphology change and movement. These results explain the anti-tumor and metastasis blocking effects of zinc observed in clinical studies. In addition, the strong down-regulation effects of zinc on plasminogen activator/plasmin system also suggest the potential role of zinc in other fibrinolysis associated diseases including neurodegenerative diseases.
Introduction
Zinc, an essential trace element for growth, is required for the normal functioning of a large number of enzymes and is a structural component of many proteins. Zinc deficiency has a profound adverse effect on cellular biochemistry including the impairment of memory, growth retardation, frequent infections, delayed wound healing and depressed immune function (Shankar and Prasad, 1998). Zinc has also been reported to inhibit the growth of prostate (Shankar and Prasad, 1998; Huang et al., 2001; Uzzo et al., 2002; Liang et al., 1999), lymphoblastoid (Prasad et al., 2001) and colon (Jaiswal and Narayan, 2004; Park et al., 2002) tumors both in vitro and in vivo. For example, the malignant prostate tissue is characterized by a dramatic decrease in zinc level (~85%) as compared to extremely high zinc levels in normal and hyperplastic glands and zinc can inhibit growth and invasive capabilities of malignant prostate (Zowczak et al., 2001). Now it is clearly that intracellular zinc depletion results from down-regulated zinc uptake transporter (ZIP1) expression on prostate tumor cells and zinc inhibits the proliferation of malignant cells predominantly through suppression of N F-κB signaling (Jaiswal et al., 2004; Costello and Frankin, 2006). However, the possible mechanism for zinc blocked metastasis in prostate cancer is nevertheless not fully understood. On the other hand, it has been found that the activity of tissue-type plasminogen activator or plasmin correlates well with a poor prognosis in several cancers and inhibitors to plasminogen activators or plasmin can restrain tumor growth and improve the prognosis of cancer treatment (Baker and Leaper, 2003; Gonzalez-Gronow et al., 2005). Effects of zinc on plasminogen activator and plasminogen/plasmin system have not been investigated yet. The objective of this study is to test the effects of zinc on plasminogen activation and examine if zinc blocked tumor metastasis comes from inhibited plasminogen activation or plasmin activity.
Materials and Methods
Materials
Human lys-plasminogen, plasmin, α-casein, H-D-Val-Leu-Lys p-nitroanilide
(S-2251), H-D-Ile-Arg p-nitroanilide (S-2288) and Coomassie blue R-250 were
purchased from Sigma (St. Louis, MO, USA). Two-chain t-PA was obtained from
Genentech Inc. (South San Francisco, CA, USA).
Amidolytic Activity of t-PA
Amidolytic activity of t-PA was determined in wells of microtitration plates
by using S-2288 as the chromogenic substrate. S-2288 was prepared in TBS buffer
(10 mM Tris-HCl, 0.15 M NaCl, pH 7.4) containing 0.01% Tween 80. Unless otherwise
stated, the final chromogenic substrate concentration in the assay mixture was
1.2 mM. To examine the effects of zinc on tPA activity, ZnCl2 and
t-PA were mixed in wells and then S-2288 was added. The initial rates of substrate
hydrolysis at 25°C were determined by measuring the absorbance at 405 nm
(A405nm) at various time intervals by using a microplate reader (BioRad
Laboratories, Richmond, CA, USA) and they were expressed as A405nm/min.
Plasminogen Conversion Activity of t-PA
The t-PA mediated plasminogen activation was determined using chromogenic
substrate 2251 as described previously. Briefly, plasminogen, t-PA and ZnCl2
were all prepared in TBS buffer (10 mM Tris-HCl, 0.15 M NaCl, pH 7.4). Zinc
solutions of various concentrations were mixed with t-PA in wells. After 10
min incubation at room temperature, S-2251 and plasminogen were added. The final
concentrations of S-2251, plasminogen and t-PA were 0.8 mM, 0.05 U mL-1
and 10 μg mL-1, respectively. The initial rates of substrate
hydrolysis at room temperature were determined by measuring the absorbance at
405 nm using a microplate reader (Biorad, Richmond, CA, USA) at various time
intervals. The results were expressed as ΔA405 min-2.
Conversion of plasminogen to plasmin was further confirmed by 10% SDS-PAGE.
Amidolytic Activity Assay for Plasmin
Plasmin, ZnCl2 were mixed in wells first and S-2251 were added.
The final concentrations of plasmin and S-2251 were 5 μg mL-1
and 0.8 mM. And the Zn 2+ concentrations were 0, 25, 50, 100, 200 μM, respectively.
The initial rates of substrate hydrolysis at room temperature were expressed
as ΔA405/min. Amidolytic activity of plasmin was also measured
by a pretylatic assay using α-Casein as substrate. In that experiment,
α-Casein, plasmin and ZnCl2 were mixed in eppendorf tubes and
incubated at 37°C for 2 h. After that, reaction mixtures were analyzed using
10% SDS-PAGE.
Cell Scatter Assay
HT-29 cells (4x104 cm-2) were seeded in 24-well plates
and cultured in complete medium for 24 h. After that, cells were fed with serum-free
medium. After 24 h culture, cells were treated with t-PA/plasminogen or plasmin
in the presence or absence of Zn2+ for another 16 h. Cell morphology
changes were then examined under microscopy and average cell cluster sizes were
calculated.
Results and Discussion
Effects of Zn2+ on t-PA/plasminogen System
The overall effect of zinc on plasminogen/plasmin system was first examined
in vitro using a small substrate system containing t-PA, plasminogen
and small substrate S-2251. The reaction conditions had been optimized in our
previous studies (Liang et al., 2000).
| Fig. 1: | Effect of Zn2+ on the tPA-mediated plasminogen activation. Amidolytic activities of plasmin produced by the reaction of tPA with plasminogen were measured using S-2251 as the substrate in the presence of various concentrations of zinc |
Zinc concentrations (0 ~ 400 μM) in these experiments was determined by the zinc concentrations used in most clinical studies. Zn2+ exerted a profound inhibitory effect on S-2251 conversion even at very low concentration (25 μM) (Fig. 1). As t-PA concentration was increased from 2.0 to 10 μg mL-1, the IC50 value of zinc was increased from 16 μM to about 45 μM correspondingly. Therefore, the possibility of inhibited amidolytic activity of t-PA/plasminogen system from plasminogen/zinc interaction could be excluded. Although S-2251 is a substrate of plasmin, the inhibited S-2251 conversion can also come from reduced plasmin production as a result of zinc inhibited tPA activity.
The possible interaction between t-PA and zinc was studied using a t-PA specific substrate, S-2288. Zinc clearly inhibited t-PA mediated S-2288 substrate conversation and dose-dependent inhibition curves could be obtained. However, inhibition curves from t-PA activity (S-2251) and amidolytic activity assay (S-2251) were different. Increasing zinc concentration above 100 FM would not result in further decrease in t-PA activity (Fig. 2A). This result was confirmed in followed plasmin production assays using SDS-PAGE (Fig. 2B). High concentration of zinc (higher than 200 μM) hardly produced any additional effects on t-PA mediated plasmin production, as justified from unchanged plasmin band intensity on the gel. Since reducing SDS-PAGE was used, two plasmin chains, which were linked by two disulfide bonds under physiological condition, were visualized as two close bands on the gel.
The interaction of plasmin and zinc was significant. Zinc demonstrated profound inhibitory effects on plasmin’s activity in tested concentration ranges (Fig. 3). In the amidolytic activity assay using protein substrate, α-casein, α-casein degradation by plasmin could be totally abolished (99%) in the presence of 400 μM zinc (Fig. 3B). Obviously, zinc can down-regulate plasminogen activator/plasmin system through its interaction with both t-PA and plasmin. However, the inhibitory effect of zinc to plasmin is more profound and effective.
Effects of Zn2+ on Cell Morphology and Movement
Plasmin, through the direct degradation of proteins of the basement membrane
and the extracellular matrix o r the activation of zymogen forms of ECM-degrading
metalloproteases such as interstitial procollagenase (matrix metalloproteinase-1)
and prostromelysin (matrix metalloproteinase-3), plays a crucial part in tissue
remodeling (Tsirka, 2002; Seeds et al., 1999; Indyk et al.,
2003; Kim et al., 1999), angiogenesis and tumor metastasis (Baker and
Leaper, 2003; Gonzalez-Gronow et al., 2005).
| Fig. 2: | Effect of Zn2+ on t-PA activity. The activity of t-PA was measured using small substrate 2288 (A) and by examining t-PA mediated plasminogen conversion into plasmin using reducing SDS-PAGE (B) in the presence of various concentrations of zinc |
| Fig. 3: | Effect of Zn2+ on plasmin activity. The midolytic activity of plasmin was measured using small substrate 2251(A) and by examining the α-casein degradation using reducing SDS-PAGE (B) in the presence of various concentrations of zinc |
| Fig. 4: | Plasmin or t-PA/plasminogen induced cell morphology changes. Cells were incubated with blank medium (A, B), culture medium with plasmin (C, D) and culture medium with t-PA/plasminogen (E, F) in the absence (A, C, E) or presence (B, D, F) of 100 μM zinc for 16 h. Cell morphology was observed under microscopy |
Effects of zinc on cell morphology and cell mobility were tested on cultured human colon carcinoma cells in the presence of plasmin or t-PA/plasminogen. In agreement with previous experiments, zinc did induce measurable and observable morphology changes, suggesting that zinc concentrations used in this experiment are safe to cells (Fig. 4A and 4B). In the presence of plasmin or plasminogen/t-PA, cells demonstrated a significant morphology change, from plat (Fig. 4A) to round (Fig. 4C and E) shapes. Such cell morphology changes were associated with significantly increased cell motility/scatting, as reflected by decreased cell numbers in fixed area (Fig. 5). Addition of zinc reversed plasmin or plasminogen/t-PA caused cell morphology change, from round to plat (Fig. 4D and F) shapes. In agree with this result, no significant cell cluster size changes could be observed if zinc was added with plasmin or plasminogen/t-PA, suggesting that plasmin or plasminogen/t-PA increased cell mobility was blocked by zinc (Fig. 5).
Taken together, the dual inhibition effect of zinc on plasminogen activator/plasmin system, inhibiting plasminogen activator activation and blocking plasmin activity, make zinc become a strong and effective inhibitor to plasminogen activator/plasminogen system. Present finding can explain the anti-tumor and metastasis blocking effects of zinc observed in clinical studies. In addition, the strong down-regulation effects of zinc on plasminogen activator/plasmin system suggest the potential roles of zinc in fibrinolysis and associated diseases including neurodegenerative diseases (Kim et al., 1999; Liang et al., 2005).
| Fig. 5: | Inhibition effect of Zn2+ on plasmin or t-PA/plasminogen induced cell mobility. Cells were incubated with blank medium, culture medium with plasmin and culture medium with t-PA/plasminogen in the absence (light grey) or presence (black) of 100 μM zinc. Cell clusters were counted and average cluster sizes were calculated after 16 h incubation. * p<0.05 compared to the control group without zinc; ** p<0.05 compared to the same treated group without zinc |
Acknowledgment
This research was partially supported by research grants from Ruth Estrin Goldberg Memorial for Cancer Research and Bristol-Myers Squibb.