Abstract
We have previously demonstrated (
The siRNA molecules were transfected into the APL cells by electroporation and resulted in almost complete (>95%) knock-down of VEGF expression. The VEGF knock-down resulted in significant apoptosis (>50%) within 96 hours. To verify the specificity of the VEGF siRNA, we used a siRNA molecule with a single nucleotide mismatch mutation, as well as a scrambled siRNA molecule. These control siRNA molecules did not result in VEGF knock-down, nor did they cause apoptosis, confirming the specificity of the VEGF siRNA and its effect.
In contrast to the striking induction of apoptosis by VEGF knock-down, external blocking of VEGF and VEGF receptors by addition of neutralizing antibodies did not cause apoptosis. In addition, the concentration of VEGF within the cells was over 100-fold higher than the external VEGF concentration. These results suggest that VEGF mediates its actions through an internal autocrine mechanism. Intriguingly, immunfluorescence studies showed that VEGF is strongly expressed in the nucleus, suggesting a novel nuclear role for VEGF.
To study the possible role of the VEGF receptors in internal loops we knocked-down the VEGF receptors using siRNA molecules. VEGF receptor knock-down did not affect survival of the APL cells. To summarize, we demonstrate that VEGF is a survival factor for APL cells, and acts independent of the VEGF receptors through an internal autocrine mechanism. These results indicate that targeting internal VEGF may be an effective therapeutic strategy for APL.
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