Abstract:

Objective To evaluate the effect of low-molecular weight heparin （LMWH） on biological behavior of a human cutaneous squamous cell carcinoma cell line A431. Methods To optimize the concentration of LMWH, A431 cells were treated with different concentrations （12.5, 25, 50, 100 and 200 IU/ml） of LMWH for 24, 48 and 72 hours followed by CCK-8 assay for the detection of cell viability. Then, A431 cells were cultured with or without the presence of LMWH at 200 IU/ml for 24, 48 and 72 hours. Subsequently, flow cytometry was performed to assess cell cycle, real time quantitative PCR （RT-qPCR） and Western blot to quantify the expression of vascular endothelial growth factor （VEGF） mRNA and protein respectively, double-antibody sandwich enzyme linked immunosorbent assay （ELISA） to determine the expression level of VEGF protein in the supernatant of A431 cells, wound-healing assay, Transwell assay, and adhesion assay to observe the migration and adhesivity of A431 cells. Analysis of variance and t test were carried out for statistical analysis. Results The optimal concentration of LMWH was determined as 200 IU/ml according to the CCK-8 assay, and used in the following experiment. The LMWH of 200 IU/ml resulted in a decrease in cell viability, cell cycle arrest, an increase in cell percentage in G1 phase, and a reduction in cell percentage in S phase. The proliferation index was 23.41 ± 5.51 and 11.76 ± 5.13 respectively in A431 cells at 48 and 72 hours after treatment with LMWH of 200 IU/ml, significantly lower than that in untreated A431 cells （48.62 ± 4.50, t = 6.14, P < 0.05; 46.86 ± 3.51, t = 9.78, P < 0.05）. A significant decrease was observed in LMWH-treated A431 cells at 48 and 72 hours compared with the untreated A431 cells in the expression level of VEGF mRNA （10.16 ± 0.07 vs. 18.77 ± 0.11, 4.11 ± 0.01 vs. 17.39 ± 0.05, t =114.38, 451.10, both P < 0.05）, VEGF protein （0.16 ± 0.01 vs. 0.20 ± 0.01, 0.12 ± 0.01 vs. 0.21 ± 0.01, t = 4.90, 11.02, both P < 0.05）, and in the supernatant level of VEGF protein （（67.17 ± 3.34） ng/L vs. （122.63 ± 23.17） ng/L, （28.14 ± 3.14） ng/L vs. （86.76 ± 1.18） ng/L, t = 4.10, 30.27, both P < 0.05）. The percentage of adherent cells was 29.7% ± 1.92% and 17.5% ± 0.79% in LMWH-treated A431 cells at 48 and 72 hours, respectively, significantly lower than that in untreated A431 cells （36.9% ± 0.35%, 34.6% ± 0.96%, respectively, both P < 0.05）. The migration of A431 cells was also obviously inhibited by the treatment with LMWH for 24, 48 and 72 hours. Conclusion LMWH may suppress the proliferation, migration and adhesion of A431 cells via downregulating cellular viability and VEGF expression.

Key words: VEGF