Hypoxia Induces Up-Regulation Of P-Glycoprotein and Promotes Resistance To Carfilzomib In Multiple Myeloma

Multiple myeloma (MM) is a malignant neoplastic cancer of plasma cells that involves the bone marrow. Generally, patients will respond to treatment initially, but they later become resistant to therapy, and this is ultimately due to a change in the biology of the tumor. Multi-drug-resistance transporter proteins were shown to play a role in drug resistance in MM patients; P-glyco-protein (P-gp) is the most studied of the multi-drug resistance proteins, and it becomes up-regulated in response to many chemotheries. Hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. Tumor-hypoxia was shown todevelop many kinds of solid tumors and hematologic malignancies. Specifically, hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. In this study, we examinned the effect of hypoxia on the expression and activity of P-gp in MM and its contributing to drug resistance to therapies used in MM.

We tested the effect of hypoxia on the activity of P-gp in MM lines. We incubated MM cells under hypoxic and normoxic conditions, and we tested their ability to pump out Rhodamine (Rh) by measuring Rh content in the cells by fluorescent reader. First, we optimized the concentration of Rh and the time of incubation with the cells. We found that at all concentrations tested (0.1, 0.5, 1, 5 and 10 ug/ml) and at all incubation time of cells with Rh with MM cells (0.25, 0.5, 1, 2, 4, 6, 8 and 24hrs) , hypoxia increased the efflux of Rh. The most significant efflux was achieved when incubating the cells for 1hr with Rh 1ug/ml. We found that hypoxia increased the efflux of Rh in all MM cell lines tested. Incubation of RPMI cells under hypoxic for 24hrs and 48hrs decreased the Rh content of the cells by about 40% and 65%, respectively.

Carfilzomib was previously reported to be a substrate of P-gp, we tested the effect of carfilzomib on the efflux of Rd in the MM cells. Hypoxic and normoxic MM cells were treated for 5hrs with carfilzomib (5 nM) and then incubated for 1hrs with Rh (1ug/ml). We tested the Rh content of the cells by fluorescent reader and found that carfilzomib competed with Rh on the P-gp and decreased the efflux of Rh induced by hypoxic.

We tested the effect of carfilzomib on induction of P-gp in hypoxic and normoxic MM cells by treating RPMI cells with a low dose of carfilzomib (0.25nM) for 48hrs under hypoxic or normoxic conditions, and tested the cells ability to efflux Rh. We found that carfilzomib increased P-gp expression and induced efflux of about 30% of the Rh in non-treated normoxic cells.  Hypoxia induced efflux of about 65% of normoxic cells, but no effect was observed with the treatment of carfilzomib.

Furthermore, we tested the hypoxia-induced P-gp expression in MM on the sensitivity of MM cells to carfilzomib. We incubated MM cells for 24hrs in hypoxic and normoxic conditions, and cells were treated with carfilzomib (0 or 5nM) for additional 24hrs. We found that while carfilzomib induced the death of about 40% of the cells under normoxic condition, it had no significant effect on the survival of MM cell under hypoxic conditions.

Hypoxia induced a significant up-regulation of P-gp in MM cells, and increased MM drug resistance to carfilzomib. These results provide mechanistic evidence for drug resistance to carfilzomib in MM, and suggest hypoxia as a novel therapeutic to prevent upregulation of P-gp and drug resistance.

No relevant conflicts of interest to declare.