Bufalin induces apoptosis in myeloma cells through DNA damage at hypoxic conditions

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Introduction: Although novel chemotherapeutic agents have become available for multiple myeloma (MM), recurrence of the disease has yet been challenging. The recurrence of the disease has been partially explained by the acquisition of drug resistance. Resistance of tumor stem cells under hypoxic conditions within bone marrow for conventional anti-cancer agents has been thought as a source of drug-resistant cells. Therefore, novel drugs targeting MM cells under hypoxic conditions are urgently required. Here we describe a compound, called bufalin, induces cytotoxicity to MM cells at hypoxic condition. Methods and Materials: Human MM cell lines were cultured either under normoxic or hypoxic (1% oxygen) conditions with compounds from a natural compound library (provided from Toyama University). Caspase-3, H2A.X, phosphorylated H2A.X and PARP analyses were conducted with Western blotting. Results and Discussion: Melphalan and lenalidomide induced apoptosis in MM cells under normoxic conditions, whereas they were less effective (approximately 2 to 5 folds) under hypoxic conditions. In contrast, a chemical compound, bufalin, induced apoptosis in MM cells under both normoxic and hypoxic conditions. Exposure of MM cells to bufalin at 8 nM for 6 hours induced significant cell death, suggesting its high potential as an anti-myeloma reagent. Interestingly, the cleaved form of PARP, H2AX and its phosphorylated form, which are indicators of DNA double-strand break (DSB), were significantly induced by treatment with bufalin under both hypoxic and normoxic conditions. On the other hand, bufalin induced no significant toxicity to lymphocytes obtained from normal donors. Taken these findings together, bufalin induces cell death through DNA DSB even at hypoxic condition. Cytotoxicity introduced by bufalin may be effective for tumor stem cells at hypoxic conditions thus contribute to overcome drug resistance.