Synergistic Effect of Panobinostat and Bortezomib on Chemoresistant Acute Myelogenous Leukemia Cells Via Akt/NF-κB Pathway,

The mechanism of MDR (multiple drug resistance) in acute myelogenous leukemia (AML) is complicated. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway plays a crucial role in cellular proliferation, survival and apoptosis in leukemia cells. Both histone deacetylase (HDAC) inhibitor and proteasome inhibitor have emerged as new kinds of therapeutic drugs against multiple myeloma (MM), their anti-tumor effects are later confirmed in other hematologic malignancies. Panobinostat, a novel HDAC inhibitor, can improve histone acetylation level and non-histone acetylase level associated with cell growth and apoptosis. Anticancer and drug-sensitization effect of Bortezomib have been investigated in lymphoma and leukemia through inhibiting NF-κB pathway. In this study, we evaluate the synergistic effect of panobinostat and bortezomib on proliferation, apoptosis and drug resistance in chemoresistant AML cells and its molecular mechanism.

HL60/ADR cell with high expression of MRP1 was maintained with adriamycin to ensure its drug resistance, which displayed almost 80-fold more resistance to adriamycin than the parental line. Bone marrow samples were obtain from five patients with refractory AML except M3. The synergistic effect of Panobinostat and Bortezomib in HL60/ADR cells and refractory acute myelogenous leukemia (AML) primary cells was determined by isobologram analysis based on cell viability in MTT assay. Apoptosis assessment and analysis of sensitivity to adriamycin were analyzed by fluorescence-activated cell sorting (FACS) and MTT. The change in protein level of HL60/ADR cell was analyzed by western blotting to further study its molecular mechanism.

Synergistic cytotoxicity was observed in the combined treatment with Panobinostat and Bortezomib in HL-60/ADM cells and primary patients AML cells. More powerful synergy was observed in 21nmol/L Panobinostat in combination with 12nmol/L Bortezomi, which CI value equal to 0.531 and 0.498. The accumulation of adriamycin in HL60/ADM cells was significantly increased by the combination (64.81±3.69%) compared with treatment with Panobinostat or Bortezomib alone (28.96±2.52% or 37.29±3.71%) (P <0.05). Simultaneously, MRP1 expression was down regulated in the combination (57.78±3.34%) (P <0.05), which was accompany with susceptibility to adriamycin. The synergistic cytotoxicity was partially depended on downregulation of X-linked inhibitor of apoptosis protein (XIAP), as well as the extrinsic apoptosis pathway consistent with initiator caspase-8 activation and downstream cleavage of caspase-3 and PARP. Our findings indicated that the combined treatment significantly induced apoptosis and intercellular adriamycin accumulation via p53-dependent Bcl-2 and MRP1 downregulation in HL60/ADR cells. Moreover, Panobinostat and Bortezomib individually attenuated phosphorylated form of AKT, which was further abrogated by the combination. The phosphorylation-dependent state of IKKα was significantly decreased by the combined treatment, and blockade of NF-κB nuclear translocation was more obvious in the cooperation of IKKα inactivation and proteasome inhibition.

Our results indicate that combination of Panobinostat and Bortezomib exerted a synergistic cytotoxicity in chemoresistant AML cells, and sensitivity to adriamycin was also increased in HL60/ADR cells. Cooperation of multiple apoptotic molecules and intracellular drug accumulation contributed to the synergistic interaction via inhibition of Akt/NF-κB pathway. These findings provided a rationale for clinic protocols using Panobinostat and Bortezomib to overcome drug resistance and improve therapeutic effect in refractory AML patients.

No relevant conflicts of interest to declare.