Silencing of hypoxia inducible factor-1α by RNA interference attenuates human glioma cell growth in vivo

Purpose: Higher-grade gliomas are distinguished by increased vascular endothelial cell proliferation and peritumoral edema. These are thought to be instigated by vascular endothelial growth factor, which, in turn, is regulated by cellular oxygen tension. Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a main responder to intracellular hypoxia and is overexpressed in many human cancers, including gliomas. Experimental Design: We investigated the role of HIF-1 alpha. in glioma growth in vivo using RNA interference (RNAi) in U251, U87, and U373 glioma cells. Results: We found that RNAi can be used to significantly attenuate glioma growth by reducing HIF-1 alpha levels constitutively using short hairpin RNAs and transiently using small interfering RNAs (siRNA). HIF-1 alpha levels on average were reduced 55% in normoxia and 71% in hypoxia. Vascular endothelial growth factor and GLUT-1 levels were reduced 81% and 71%, respectively, in the stable HIF-1 alpha- reduced clones. These clones showed significant growth attenuation (up to 73%) compared with negative controls when grown in vivo in mouse flanks. Cellular proliferation was also reduced significantly, as determined by MIB-1 staining. Treating gliomas grown in mouse flank transiently with siRNA against HIF-1 alpha by intratumoral injection resulted in a significant reduction of HIF-1 alpha activity. This activity was followed using a hypoxia-responsive luciferase construct that enabled hypoxia imaging in vivo. Tumor volume in these si RNA injection experiments was reduced by 50% over the negative controls. Conclusions: These results indicate that transient RNAi directed against HIF-1 alpha can effectively curb glioma growth in vivo.