Cepharanthin enhanced irradiation sensitivity through the inhibition of NF-kappaB induced anti-apoptotic agents in human oral cancer cells.

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[Background] The nuclear factor κB (NF-κB) plays an important role in the development and progression of cancers. We had already reported that NF-κB activity in head and neck 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 that cepharanthin enhances radiosensitivity in B88 cells.

[Methods and Results] Cepharanthin (donated by Kaken Pharmaceutical Inc., Osaka, Japan) is a biscoclaurine alkaloid extracted from the roots of Stephania ceharantha hayata, and is widely used in Japan for the treatment of patients with leucopenia and venomous snake bites. 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 Cepharanthin (0.1 mg/kg/day, three times a week for 4 weeks) or irradiation (IR) (1.5 Gy/day, three times a week for 4 weeks) alone, or combination of Cepharanthin with IR. Suppression of tumor growth was significantly augmented by the combined treatment with IR and Cepharanthin as compared to IR or Cepharanthin alone. To investigate the mechanism involved in the enhancement of radiosensitization by Cepharanthin, expression of NF-κB-regulated antiapoptotic proteins, including cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2, XIAP, Bcl-2 and Bcl-x, was examined. When B88 cells reached subconfluence in culture, they were exposed to IR (15 Gy) with or without Cepharanthin (5 μg/ml). Twenty-four hours later, total RNA was extracted and examined the expression of anti-apoptotic proteins by quantitative real-time RT-PCR. Although IR alone enhanced the expression of both cIAP-1 and cIAP-2 mRNAs in B88 cells, treatment of cells with Cepharanthin and IR caused the suppression of these mRNA expression. However, expression levels of XIAP and Bcl-2 mRNAs were not enhanced by IR, significant suppression was found when the cells were treated with Cepharanthin and IR. Next, we examined the cleavage of PARP protein to determine whether treatment with Cepharanthin and IR enhances the apoptotic cell death in B88 cells. This analysis showed that the treatment with the combination of Cepharanthin and IR led to the enhanced cleavage of PARP protein with a molecular weight of 29 kDa, indicating the augmentation of apoptotic cell death in B88 cells.

[Conclusion] These findings indicate that Cepharanthin enhances the apoptosis in oral cancer cells by inhibiting the irradiation-induced production of antiapoptotic proteins through the inhibition of NF-κB activation.