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

Abstract

The cytotoxicity of a trivalent arsenic derivative (arsenite, AsIII) 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 AsIII combined with arenobufagin instead of gamabufotalin. Apoptosis and the activation of caspase-9/-8/-3 were induced by AsIII 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 G2/M phase arrest were observed. A remarkable increase in the expression level of γH2AX, a DNA damage marker, was induced by AsIII+arenobufagin. Concomitantly, the activation of autophagy was observed, suggesting that autophagic cell death associated with DNA damage was partially attributed to the cytotoxicity of AsIII+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 AsIII+arenobufagin. Given that both AsIII 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.

Keywords

• arsenite
• arenobufagin
• gamabufotalin
• glioblastoma
• DNA damage
• cell death