Korean J Hematol.  2005 Dec;40(4):211-218. 10.5045/kjh.2005.40.4.211.

siRNA Targeting Vascular Endothelial Growth Factor and Recombinant Human Prothrombin Kringle 2 Inhibits Leukemia-induced Angiogenesis

• Kim BJ
• Kim SS

• KMID: 2083458
• DOI: http://doi.org/10.5045/kjh.2005.40.4.211

Abstract

BACKGROUND
Vascular endothelial growth factor (VEGF) plays a role in the development of cancer and the progression of liquid tumors such as chronic lymphatic leukemia, non-Hodgkin lymphomas, and multiple myeloma. VEGF also triggers endothelial cells to secrete hematopoietic growth factors such as interleukin-6 (IL-6); this in turn promotes further leukemia growth, thereby contributing to a paracrine loop between the leukemia and the endothelial cells.
METHODS
We transfected a small interfering RNA (siRNA) targeting VEGF into K562 cells in order to investigate the role of VEGF in the development of leukemic cancer. After the conditioned media (CM) of the K562 was cells added to the human umbilical endothelial cell (HUVEC) culture media, we compared the proliferation and tube formation of the HUVECs. Recombinant human prothrombin kringle2 (K2), which is a known angiogenic inhibitor, was also treated onto the HUVECs, and we then examined the level of IL-6 to determine the paracrine interaction between the leukemic and endothelial cells.
RESULTS
RT-PCR and western blot analysis demonstrated that the siRNA efficiently down regulated the expression of VEGF in the K562 cells. When the CM of the K562 cells was added to the HUVEC culture, the proliferation of the HUVECs was stimulated. The proliferation of the HUVEC induced by the CM from the siRNA-VEGF K562 cells was diminished, compared with that of the vector control K562 cells. K2 reduced not only the proliferation of the HUVECs, but also the secretion of IL-6 by the HUVEC.
CONCLUSION
The siRNA strategy is an alternative tool for inhibiting leukemia-induced angiogenesis. A combinated therapy with angiogenesis inhibitor K2 increases the efficiency. K2 modulates the production of IL-6, which may affect the paracrine interaction between leukemia and endothelial cells.

Keyword

Vascular endothelial growth factor; Leukemia; Angiogenesis; Interleukin-6; Kringle

MeSH Terms

Blotting, Western
Culture Media
Culture Media, Conditioned
Endothelial Cells
Humans*
Intercellular Signaling Peptides and Proteins
Interleukin-6
K562 Cells
Kringles*
Leukemia
Lymphoma, Non-Hodgkin
Multiple Myeloma
Prothrombin*
RNA, Small Interfering*
Vascular Endothelial Growth Factor A*
Culture Media
Culture Media, Conditioned
Intercellular Signaling Peptides and Proteins
Interleukin-6
Prothrombin
RNA, Small Interfering
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1. Vascular endothelial growth factor (VEGF) expression in K562 cells. Control or siRNA to VEGF was transfected into K562 cells. (A) Total RNA was extracted from the cells and VEGF transcripts were analyzed by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. (B) CM of K562 cells was analyzed by western blotting. The blot showed that VEGF secretion of K562 cell was significantly decreased by transfection of siRNA targeting VEGF.

• 

  Fig. 2. Endothelial cell proliferation. HUVECs were cultured in 96 well plates at 5103 cells per well with or without 50% (v/v) CM of K562 cells in the presence (□) or absence (■) of K2 for 72h. The proliferation of HUVECs was measured by MTT assay. Results represent the mean±SD (n=3).

• 

  Fig. 3. Tube formation assay. HUVECs were serum-starved for 24 h and cultured on growth factor-reduced Matrigel in the presence or absence of 50% (v/v) CM of K562 cells for 12h. Representative images were shown at ×40 magnification.

• 

  Fig. 4. Production of interleXkin-6 (IL-6) by HUVECs. HUVECs were cultured with 50% (v/v) CM of K562 cells in the presence (□) or absence (■) of 5 ug/mL K2 for 24h. The culture supernatant was centrifuged and IL-6 concentrations were determined by ELISA. Results represent the mean±SD (n=3).

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