Cathepsin G induces cell aggregation of human breast cancer MCF-7 cells via a 2-step mechanism: catalytic site-independent binding to the cell surface and enzymatic activity-dependent induction of the cell aggregation

Abstract

Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG) is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering) CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.

Figure 1

MCF-7 cell aggregation-inducing activities of cathepsin G (CG) and chymotrypsin. (a) MCF-7 cell aggregation assay using CG and chymotrypsin. MCF-7 cells (1 × 10⁴ cells/well) were seeded in 96-well plates in RPMI 1640 medium containing 5% fatal bovine serum (FBS). The cells were cultured overnight and then incubated overnight with the serine proteases in RPMI 1640 medium containing 1% BSA. After washing, the residual cells were stained with crystal violet, and the aggregation index was calculated as described in Section 2. The results are expressed as mean ± SD (n = 3). When the bars are not shown, they are smaller than the size of the symbols. (b) Images of MCF-7 cells at 24 h after incubation with the serine proteases. Scale bar = 50 μm.

Figure 2

The MCF-7 cell aggregation-inducing activity of CG is inhibited by serine protease inhibitors. CG was simultaneously added to the medium with the serine protease inhibitor chymostatin (16.5 μM) (a) or Suc-Val-Pro-Phe^P-(OPh)₂ (10 μM) (b). PMSF-treated CG was added to MCF-7 cells (c). The aggregation index is shown in the left panels of Figures 2(a), 2(b), and 2(c). The results are shown as mean ± SD (n = 3). When the bars are not shown, they are smaller than the size of the symbols. The inhibitory effect of the serine protease inhibitors on the enzymatic activity of CG is also shown (right panels). The enzymatic activity of CG was analyzed by measuring the release rate of 4-nitroanilide following the addition of CG (667 nM, right panels of (a) and (b)) and the inhibitors (16.5 μM chymostatin, right panel of (a); 10 μM Suc-Val-Pro-Phe^P-(OPh)₂, right panel of (b)) to N-succinyl Ala-Ala-Pro-Phe p-nitroanilide (1.1 mg/mL) in 0.1 M HEPES buffer (pH 7.5) containing 0.5 M NaCl and 10% dimethyl sulfoxide at 25°C. The released p-nitroanilide was detected by measuring the absorbance at 405 nm. In the right panel of (c), the effect of 417 nM intact or PMSF-treated CG was measured. The data of the enzymatic activity are indicated as single-point values.

Figure 3

MCF-7 cell aggregation is induced by wild-type (WT) CG, but not the enzymatically inactive S195G mutant. (a) MCF-7 cell aggregation assay using CG-overexpressing cell lysates. MCF-7 cells were cultured overnight in medium containing 5% FBS and incubated with lysates serially diluted in serum-free medium containing 1% BSA. After washing, the residual cells were quantified by crystal violet staining. The results are shown as mean ± SD (n = 3). When the bars are not shown, they are smaller than the size of the symbols. (b) Immunoreactivities of CG and β-actin in the lysates assayed in Figure 3(a). A 10-μL aliquot of the whole cell lysate was analyzed by western blotting using anti-CG or β-actin antibody. (c) Images of cells that were analyzed in Figure 3(a) at 24 h after incubation with the diluted lysates. Scale bar = 50 μm.

Figure 4

CG binds to the surface of MCF-7 cells. (a) Detection of CG purified from human neutrophils in the MCF-7 cell surface fraction. MCF-7 cells were seeded in dishes containing RPMI 1640 medium supplemented with 5% FBS. The cells were cultured overnight and subsequently incubated with CG (209 nM) and chymostatin (33 μM), AT (4.5 or 11.4 μM), or ACT (75 or 188 nM) in serum-free RPMI 1640 medium for 90 min on ice. After washing, cell surface proteins were biotinylated and collected using avidin-conjugated agarose beads. The cell surface protein fractions were analyzed by western blot analysis using anti-CG antibody. (b) Immunohistochemical detection of CG on MCF-7 cells. MCF-7 cells that were treated with CG (209 nM) and a protease inhibitor (chymostatin, 16.5 μM; ACT, 18.8 nM) were immunostained with anti-CG antibody in the absence of permeabilization. Scale bar = 20 μm.

Figure 5

Binding of CG to the surface of MCF-7 cells is independent of its catalytic site. (a) The S195G CG mutant binds to MCF-7 cells. The cells were incubated with CG-overexpressing RBL-2H3 cell lysate or purified human CG from neutrophils (final concentration, 209 nM) for 90 min on ice. The CG bound to the cell surface was detected by immunostaining with anti-CG antibody in the absence of permeabilization. Scale bar = 10 μm. (b) Western blot analysis of CG in the lysates used in Figure 5(a) using anti-CG antibody. A 10-μL aliquot of the lysate was loaded in each lane.

Figure 6

Characterization of the binding of ¹²⁵I-labeled CG to the MCF-7 cell surface. (a) Dose dependency of ¹²⁵I-CG binding to MCF-7 cells. The cells were incubated with ¹²⁵I-labeled CG in RPMI 1640 medium containing 1% BSA for 60 min on ice. After washing, the cells were disrupted by the addition of 0.1 M NaOH, and the radioactivity of the lysate was determined using a γ-counter. In the cold inhibition experiment, a 20-fold excess of unlabeled CG was simultaneously added to the medium for competitive binding. Specific binding was determined by subtracting the value obtained for nonspecific binding (cold inhibition) from the total binding. The data are expressed as single-point values. (b) Hill plot analysis of ¹²⁵I-CG binding to MCF-7 cells. The slope of the Hill plot is the Hill coefficient (n _H), which indicates cooperativity. (c) Time course of ¹²⁵I-CG binding to MCF-7 cells. ¹²⁵I-CG was added at a final concentration of 834 nM. (d) Binding activities of ¹²⁵I-trypsin and ¹²⁵I-chymotrypsin. (e) Suc-Val-Pro-Phe^P-(OPh)₂ and chymostatin have no effect on CG binding to MCF-7 cells. MCF-7 cells were incubated with ¹²⁵I-CG (83.4 nM) that was pretreated with serine protease inhibitors (Suc-Val-Pro-Phe^P-(OPh)₂, 100 μM; chymostatin, 82.5 nM). The bound ¹²⁵I-CG is expressed as relative binding comparing the radioactivity of bound intact ¹²⁵I-CG with that of serine protease-treated ¹²⁵I-CG. Unless otherwise indicated, similar results were obtained from 2 independent experiments, each with duplicates. The results are shown as mean ± SD. When the bars are not shown, they are smaller than the size of the symbols.