Identification of a Compound Overcoming Drug Resistance of Myeloma Cells at Hypoxic Condition

Introduction

Hypoxia plays a crucial role in the development of drug resistance in various cancer cells. Therefore, many attempts targeting hypoxia are underway to overcome the drug resistance mediated by hypoxia. This strategy is useful for multiple myeloma (MM) cells, as MM cells reside within the bone marrow, where oxygen concentrations are quite low. To elucidate influence of oxygen concentration to drug sensitivity of MM cells, we evaluated sensitivity of MM cells to lenalidomide and melphalan under hypoxia (oxygen concentration: 1%). We found that significant elevation of LD₅₀ for MM cell lines, KMS12PE and U266, were observed (Fig. 1), indicating drug resistances to these reagents are indeed induced under hypoxia.

Materials and Methods

MM cell lines, KMS12PE and U266, were utilized. Natural compound library was obtained from Institute of Natural Medicine, Toyama University (Toyama, Japan). Cell viability was analyzed either by MTT assay or trypan blue dye eclusion analysis. DNA double strand break response was analyzed by western blot detetcting gamma-H2AX. Melphalan was utilized as an conventional anti-MM reagent causing DNA double strand break.

Results and Discussion

We screened natural compound library to identify compounds exerting cytotoxicity in MM cells under hypoxic condition. Bufalin, a component of a chinese medicine (Chan Su), exhibited marked cytotoxicity to MM cells under both hypoxic and normoxic conditions at nano molar levels (compound#5 in Fig. 2). Importantly, no significant toxicity by bufalin was observed for PBMC obtained from normal donors.

At normoxic condition, bufalin induced significant DNA double strand break (DSB) response, ROS induction and caspase activation within 24 hours which is a more rapid response compared with melphalan. Interestingly, the bufalin-induced DSB response was not impaired by low oxygen concentrations while DSB response by melphalan was reduced (Fig. 3). Moreover, treatment with bufalin abolished HIF-1α expression under hypoxia (Fig. 4), suggesting bufalin exerts cytotoxicity under hypoxia by regulating HIF-1α.

These results suggest that bufalin induces apoptosis to MM cells through DSB under hypoxic condition by inhibiting HIF-1α, suggesting bufalin could be useful for eradication of drug resistant MM cells in the hypoxic microenvironment.

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