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Tumor hypoxia is a potential therapeutic problem because it is closely associated with resistance to anti-cancer therapies and with the phenomenon of malignant progression. Therefore, although hypoxic tumor cells account for a very limited area in a solid tumor, conquering tumor hypoxia is crucial for treatment of malignant tumors. To target tumor hypoxia, we have recently developed a fusion protein, TOP3, which is designed to specifically stabilize and efficiently induce apoptosis in hypoxic tumor cells. To overcome poor delivery efficiency to the hypoxic region of solid tumors, TOP3 possesses a protein transduction domain (PTD) derived from HIV-1 tat protein. The PTD has been shown to efficiently pass through the cell membrane, and therefore in vivo delivery of the fusion protein to cells throughout the body is possible. TOP3 also contains oxygen-dependent degradation (ODD) sequences of the human hypoxia-inducible factor-1α (HIF-1α) ODD domain, which tightly regulate the fusion protein stability in a manner that depends on the surrounding oxygen concentration. The molecular mechanism of oxygen-dependent regulation of HIF-1α protein stability has been extensively studied, and the identical mechanism regulates TOP3 stability. The third domain of TOP3 is procaspase-3, a dormant form of caspase-3. TOP3 did not affect well-oxygenized cells but efficiently increased caspase-3 activity and induced cell death to hypoxic cells in vitro. This cytotoxicity hardly influenced the radiation effect and TOP3 consequently showed additive effects on radiosensitivity in colony formation assay. However, intraperitoneal TOP3 injection combined with x-ray irradiation significantly delayed tumor growth, and tumor growth doubling time of the combination was markedly prolonged in comparison with the one of x-ray irradiation alone (p<0.01). The growth speed of xenografts treated with TOP3 alone was reduced by half in comparison with untreated xenografts. To investigate whether the growth delay is attributable to the specific targeting of tumor hypoxia by TOP3, we monitored the hypoxic cells in xenografts of HeLa/5HRE-Luc cells, whose luciferase activity under hypoxic conditions was more than 60 times higher than under aerobic conditions, via an in vivo real-time imaging system during TOP3 treatment. The monitored optical bioluminescence signals from hypoxic cells clearly revealed that the increase of hypoxic regions was continuously suppressed during the sequential administration of TOP3. Furthermore immunohistochemical analysis of the TOP3-treated xenografts revealed that hypoxic tumor cells underwent apoptosis and were significantly and time-dependently reduced within 48 h after TOP3 treatment. Here we demonstrate that TOP3 is a useful tool to specifically target hypoxic tumor cells in vivo and provide direct evidence that hypoxic tumor cells play a crucial role in the tumor growth.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]