Chemically modified heparin inhibits the in vitro adhesion of nonsmall cell lung cancer cells to P-selectin

Abstract

Purpose: Several independent studies have indicated that tumor metastasis can be inhibited by chemically modified heparin with low anticoagulant activity in the different tumor models. The mechanism of inhibition by the heparin derivatives in part accounts for the interference of tumor cell-platelet interaction mediated by P-selectin.

Methods: In the present study, we demonstrated that both heparin and chemically modified heparins inhibited the adhesion of nonsmall cell lung cancer (NSCLC) cells to P-selectin under static or flow conditions in vitro.

Results: Flow cytometric analysis with the heparan sulfate-specific monoclonal antibody revealed that both NSCLC cells express heparan sulfate-like proteoglycans. Furthermore, heparinase treatment impaired P-selectin binding, indicating that heparan sulfate-like proteoglycans on the tumor cell surface are implicated in the adhesion of NSCLC cells to P-selectin.

Conclusions: These findings suggest that some chemically modified heparins with low anticoagulant activity may deserve further testing in the experimental NSCLC treatment protocols.

Fig. 1

The binding of P-selectin to NCI H460 and SPC-A-1 cells. Interaction of P-selectin chimeras with NCI H460 (left) and SPC-A-1 (right) cells was analyzed by flow cytometry in the presence of P-selectin blocking mAb (9E1) or nonblocking mAb (AC1.2). An isotype-matched human IgG was used as negative control. The results are representative of three independent experiments

Fig. 2

a, b Effect of heparin and modified heparins on P-selectin binding to NCI H460 and SPC-A-1 cells. NCI H460 cells (a) and SPC-A-1 cells (b) were incubated with P-selcetin chimeras preincubated with heparin or modified heparins at indicated concentration, then followed by a FITC-labeled goat antihuman IgG. The binding events were measured by flow cytometry. The percentage of positive control that tumor cells bound to P-selcetin in the absence of heparin and modified heparins was 73.7 (NCI H460) and 80.5 (SPC-A-1), respectively. The results represent those obtained from three independent experiments

Fig. 3

a, b Inhibition of NSCLC cells to CHO-P by mAbs, heparin and modified heparins under flow conditions. After 30 min of preincubation with mAb (9E1 or AC1.2), heparin and modified heparins, CHO-P monolayers were washed and equipped into the flow chamber, and then tumor cells were perfused on the monolayers. Parent CHO cells were used as negative control. Adhesion of tumor cells to the CHO monolayers were measured by videomicroscopy in 10–20 fields of view by using a ×10 objective lens. Data were the mean ± SE of three to five experiments and were normalized to the adhesion in the absence of mAbs, heparin and modified heparins. Inhibition by heparin or modified heparins was statistically significant corresponding to that in the presence of P-selectin nonblocking mAb (AC1.2). NCI H460, P<0.01; SPC-A-1, P<0.01

Fig. 4

a–c Inhibition of NSCLC cells adhesion to surface-anchored platelets by mAbs, heparin and modified heparins under flow conditions. The adhesion assay was performed in a flow chamber at 0.3 dyn/cm² as described in Materials and methods. The results were the mean ± SE from three to five independent experiments and represented the percentage adhesion with respect to that in the absence of mAbs, heparin and modified heparins. Inhibition by heparin and modified heparins was statistically significant corresponding to that in the presence of P-selectin nonblocking mAb (AC1.2). NCI H460, P<0.01; SPC-A-1, P<0.01

Fig. 5

a–c The detection of heparan sulfate proteoglycan and other carbohydrate determinants involved in P-selectin binding on the tumor cell surface. Flow cytometric analysis of NCI H460 (left) and SPC-A-1 (right) cells was performed as described in Materials and methods. a Tumor cells exhibit deficiencies in sLe^x and PSGL-1 expression, but express heparan sulfate proteoglycans. Isotype-matched mAbs were used as negative control. KPL-1, a mAb to PSGL-1; 10E4, a mAb to heparan sulfate proteoglycan; CSLEX-1, a mAb to sLe^x. b The effect of tumor cells treated with heparinase I, II and III (Hep) on P-selectin binding. The proportion of positive cells compared with negative control was as follows: P-Fc, 87.6; Hep/P-Fc, 72.4 (NCI H460), P-Fc, 93.5; Hep/P-Fc, 76.4 (SPC-A-1). c The detection of carbohydrate determinants involved P-selectin binding. After digestion with neuraminidase (Neu) or trypsin (Try), tumor cells were analyzed by flow cytometry. The proportion of positive cells compared with negative control was as follows: P-Fc, 81.6; Neu/P-Fc, 80.9; Try/P-Fc, 13.2 (NCI H460), P-Fc, 90.1; Neu/P-Fc, 88.7; Try/P-Fc, 9.8 (SPC-A-1)