[Inhibitory effect of low molecular weight heparin on the secretion of vascular endothelial growth factor by tumor cells in vitro].
OBJECTIVE
To investigate whether low molecular weight heparin (LMWH) may suppress the expression and secretion of vascular endothelial growth factor (VEGF) from tumor cells in vitro and inhibit the VEGF-induced proliferation of human tumor vascular endothelial cells.


METHODS
Human lung cancer cell line A549, human liver cancer cell line HepG2, human colon carcinoma cell lines HCT116 and HCT8 were used in this study. The expression levels of VEGF and TNF-alpha (tumor necrosis factor-alpha) in the tumor cells with or without pretreatment of LMWH/heparin were measured by standard sandwich ELISA technique. The VEGF mRNA level of HepG2 cells cultured with or without LMWH/heparin was determined by RT-PCR and real time PCR. Human umbilical vein endothelial cells (HUVEC) were cultured in tissue culture medium (TCM) with or without LMWH/heparin for 3 days. Then non-radioactive cell proliferation assay (MTS) kit and cell cycle assay by flow cytometry were performed to measure the proliferation of HUVEC.


RESULTS
The VEGF levels in the control, LMWH, and heparin groups of the pulmonary adenocarcinoma cell line A549 were (1045.89 +/- 165.30) pg/ml, (782.45 +/- 67.17) pg/ml and (916.54 +/- 71.25) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups of the colon adenocarcinoma cell line HCT116 were (955.76 +/- 51.14) pg/ml, (822.89 +/- 142.39) pg/ml and (951.77 +/- 188.22) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the colon adenocarcinoma cell line HCT8 were (1290.62 +/- 41.23) pg/ml, (1063.34 +/- 63.82) pg/ml and (1257.14 +/- 11.40) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the liver cancer cell line HepG2 were (1083.00 +/- 134.35) pg/ml, (758.00 +/- 84.85) pg/ml and (874.00 +/- 22.62) pg/ml, respectively. The VEGF expression levels in the above mentioned cell lines cultured in TCM were significantly reduced in the LMWH-treated groups compared with that of the control group (P < 0.05). But the level of TNF-alpha in TCM-cultured cells was unaffected by LMWH. The VEGF mRNA was reduced in the LMWH-treated HepG2 cell line. Moreover, TCM exhibited stimulating effect on proliferation of HUVEC and the effect was significantly impaired by LMWH treatment. Flow cytometric analysis revealed that LMWH treatment arrested HUVECs at the G1 phase of cell cycle.


CONCLUSIONS
LMWH can suppress the expression and secretion of VEGF by tumor cell lines and therefore have a potential inhibiting effect on angiogenesis induced by VEGF.
Department of Oncology, Peking Union Medical College Hospital, Beijing 100730, China.
Zhao
Z
Sun
Zong-lan
ZL
Hu
Xiao-hong
XH
Ning
Jian-feng
JF
Zhou
Ya-juan
YJ
Shao
Jin-hong
JH
Duan
Xian-da
XD
Yang
Chun-mei
CM
Bai
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D042783:Endothelial Cells / Q000166:cytology*; D045325:HCT116 Cells; D056945:Hep G2 Cells; D006493:Heparin / Q000494:pharmacology; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D017077:Culture Media, Conditioned; D006495:Heparin, Low-Molecular-Weight; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D006493:Heparin