| Recent evidence indicates that tumor microvascular apoptosis, mediated by acid sphingomyelinase (ASMase), plays a prominent role in the tumor response to ionizing radiation (IR). Here, we investigate the mechanism by which ASMase initiates apoptotic signaling in endothelium. These studies show that IR initiates the rapid activation and translocation of ASMase to the exoplasmic leaflet of the plasma membrane. Ceramide generation ensues, driving the formation of ceramide-rich platforms (CRPs), macromolecular structures that regulate signal transduction. CRPs are required for initiation of endothelial apoptosis in vitro and in vivo. Pharmacologic inhibition of CRP formation inhibits IR-induced apoptosis in endothelium, suggesting that platforms are obligate for IR-induced endothelial apoptosis both in vitro and in vivo. We then hypothesized that enhancement of CRP formation might enhance platform-mediated apoptosis, radiosensitizing endothelium in vitro and in vivo. To this end, we developed Ad5HEPPE-3x(ASM), an adenovirus designed to upregulate ASMase expression specifically within tumor microvasculature. In vitro transduction of AdSHEPPE-3x(ASM) resulted in endothelial-specific ASMase overexpression, conferring radiation hypersensitivity to endothelial cells via increased CRP generation and providing proof-of-concept that radiosensitization can be achieved by genetic upregulation of ASMase. Finally, we demonstrate that Ad5HEPPE-3x(ASM)-mediated ASMase overexpression impacts tumor growth in vivo. Ad5HEPPE-3x(ASM) infection restored radiation sensitivity to MCA/129 fibrosarcoma implanted into radiation-resistant asmase mice. Additionally, overexpression of ASMase in tumor-bearing wild-type mice resulted in an enhanced tumor response to radiation, leading to an increase in tumor cure in a dose-dependent manner, while not impacting endothelium within other radiation-sensitive organs such as the gastrointestinal (01) tract. Taken together, these data demonstrate the potential of Ad5HEPPE-3x(ASM) to increase the effectiveness of radiotherapy without incurring unwanted normal tissue toxicity. |
