Enhanced Cytotoxic Effects of Arenite in Combination with Active Bufadienolide Compounds against Human Glioblastoma Cell Line U-87

The cytotoxicity of a trivalent arsenic derivative (arsenite, As(III)) combined with arenobufagin or gamabufotalin was evaluated in human U-87 glioblastoma cells. Synergistic cytotoxicity with upregulated intracellular arsenic levels was observed, when treated with As(III) combined with arenobufagin instead of gamabufotalin. Apoptosis and the activation of caspase-9/-8/-3 were induced by As(III) and further strengthened by arenobufagin. The magnitude of increase in the activities of caspase-9/-3 was much greater than that of caspase-8, suggesting that the intrinsic pathway played a much more important role in the apoptosis. An increase in the number of necrotic cells, enhanced LDH leakage, and intensified G(2)/M phase arrest were observed. A remarkable increase in the expression level of γH2AX, a DNA damage marker, was induced by As(III)+arenobufagin. Concomitantly, the activation of autophagy was observed, suggesting that autophagic cell death associated with DNA damage was partially attributed to the cytotoxicity of As(III)+arenobufagin. Suppression of Notch signaling was confirmed in the combined regimen-treated cells, suggesting that inactivation of Jagged1/Notch signaling would probably contribute to the synergistic cytotoxic effect of As(III)+arenobufagin. Given that both As(III) and arenobufagin are capable of penetrating into the blood–brain barrier, our findings may provide fundamental insight into the clinical application of the combined regimen for glioblastoma.