Proteasome inhibitor enhances radiosensitization in human oral squamous carcinoma cells via suppressing the radiation-induced expression of antiapoptotic proteins and cytokines

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[Background] The nuclear factor κB(NF-κB) plays an important role in the development and progression of cancers, and the promotion of cell survival. We had already reported that NF-κB activity in oral carcinoma cells was significantly higher than that in normal oral epithelial cells due to the enhanced phosphorylation and degradation of IκBα protein, and that inhibition of NF-κB activity by introducing a super-repressor form of IκBα cDNA into oral cancer (B88) cells led to a drastic decrease in tumorigenicity in nude mice and an acquisition of enhanced radiosensitization.
 [Purpose] In this study, we examined a possibility whether proteasome inhibitor, bortezomib (VELCADE^®, Millennium Pharmaceutical, Inc.), would inhibit the tumor growth, production of cytokines, and expression of anti-apoptotic proteins, and enhance the radiosensitivity in oral carcinoma cells.
 [Methods and Results] We investigated the effects of bortezomib alone, and the combination of bortezomib with RT on in vitro cell growth and in vivo tumor growth. B88 cells (5×10⁶) were subcutaneously inoculated into the backs of nude mice. When the tumor reached 0.5 cm in diameter, tumor-bearing nude mice received either the intravenous administration of bortezomib (0.1 mg/kg/day, three times a week) or radiation (RT) (1.5 Gy/day, three times a week) alone, or combination of bortezomib with RT. Suppression of the cell growth in vitro and tumor growth in vivo was significantly augmented by the combined treatment with RT and bortezomib as compared to RT or bortezomib alone. In order to investigate the mechanism involved in the enhancement of radiosensitization by bortezomib, the expression of NF-κB-regulated antiapoptotic proteins, including cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2, XIAP, and FLICE inhibitory protein (FLIP), and VEGF, IL-6, and IL-8 proteins was examined in vivo. Although the expression of anti-apoptotic proteins was induced by RT, bortezomib inhibited the expression of these anti-apoptotic proteins. Bortezomib significantly inhibited the RT-induced production of VEGF, IL-6, and IL-8 proteins. Vascularization in the tissues of tumor-bearing nude mice after treatment with RT, or bortezomib alone, and combination of both, was assessed by the immunohistochemistry for Factor VIII. The Factor VIII positive vessels showed a significant decrease in the bortezomib-treated tumors as compared to untreated controls.
 [Conclusion] These findings suggest that bortezomib and combination with RT would be effective in the treatment of oral cancer through the inhibition of RT-induced expression of VEGF, IL-6, IL-8, cIAP-1, cIAP-2, XIAP, and FLIP through the inhibition of NF-κB activation.