Abstract
Low-molecular-weight heparin (LMWH) has been used in cancer patients with venous thromboembolic complications, resulting in a higher survival rate and an inhibitory action on experimental metastasis. In the present study, human umbilical vein endothelial cells (HUVECs) were treated with LMWH for 24 h. We found that the resulting HUVECs could significantly inhibit the highly metastatic human prostate cancer cell line (PC-3M) in terms of its adhesion to the endothelium and migration across the endothelium, according to scanning electron microscopy. We also determined the elevated levels of endothelial intercellular Ca2+ concentration after the adhesion of PC-3M cells to HUVECs was greatly reduced by incubation with LMWH. Using proteomics, we surveyed the global protein changes in HUVECs after LMWH treatment and identified four down-regulated proteins that were possible isoforms of cytoskeletal vimentin intermediate filaments, cartilage-derived C-type lectin, and serine/threonine protein phosphatase 1β (PP-1B). LMWH affected the morphology of vimentin and the expression levels of αv integrin and PP-1B in HUVECs bound to PC-3M cells. Vimentin assists in the adhesion of PC-3M cells, which was confirmed by short interfering RNA experiments. Furthermore, the direct binding of purified vimentin protein with LMWH was detected with surface plasmon resonance methods. However, when we used fluorescence-labeled heparin for 24 h to identify whether this binding occurred within cells, heparin was distributed principally around endothelial cells. Taken together, these findings suggest that the monoincubation of LMWH with HUVECs could inhibit PC-3M cell adhesion to, and migration through, endothelium. LMWH's regulation of vimentin plays a role in the antimetastatic action.
Footnotes
This work was supported by the National Natural Science Foundation of China [Grants 81020108031, 30572202, 30973558, 30772571, 30901803, 30901815]; the Major Specialized Research Fund from the Ministry of Science and Technology in China [Grant 2009ZX09103-144]; the Research Fund from Ministry of Education of China (111 Projects) [Grant B07001]; and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of China.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.182055.
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ABBREVIATIONS:
- LMWH
- low-molecular-weight heparin
- CFDA
- carboxyfluorescein diacetate
- DTT
- dithiothreitol
- EC
- endothelial cell
- HUVEC
- human umbilical vein endothelial cell
- IEF
- isoelectric focusing
- IPG
- immobilized pH gradient
- MALDI-TOF-MS
- matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- PBS
- phosphate-buffered saline
- RU
- response units
- PAGE
- polyacrylamide gel electrophoresis
- SEM
- scanning electron microscopy
- siRNA
- short interfering RNA
- SPR
- surface plasmon resonance
- TGF-β
- transforming growth factor-β
- PP-1B
- protein phosphatase 1β
- 2D
- two-dimensional
- FITC
- fluorescein isothiocyanate
- RT-PCR
- reverse transcription-polymerase chain reaction; [Ca2+]i intracellular calcium concentration
- ns-siRNA
- nonsilencing siRNA
- siVimentin
- vimentin-targeted siRNA.
- Received March 28, 2011.
- Accepted June 30, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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