Hypoxia-mediated fenretinide (4-HPR) resistance in childhood acute lymphoblastic leukemia cells

N-(4-Hydroxyphenyl)-retinamide (4-HPR, Fenretinide) is a synthetic retinoid with cytotoxicity in acute lymphoblastic leukemia (ALL) cell lines. Since ALL is a disease of the bone marrow, a hypoxic tissue compartment, and it has been reported that there is an antagonistic effect of hypoxia on many chemotherapeutic agents, our purpose was to observe whether hypoxia is able to inhibit the effect of 4-HPR for ALL cell lines and to investigate its mechanisms of antagonism to 4-HPR.

Cytotoxicity was measured by MTT method, and apoptosis was measured by flow cytometry. Mitochondrial membrane potential (DeltaPsim) was detected by JC1 staining and flow cytometry. Protein expression was analyzed by western blotting.

Hypoxia (2% O2) induced 4-HPR resistance in the tested two ALL cell lines (Molt-4 and Molt-3), with at least a 2.8-fold increase in IC50 values (P<0.01) compared with the IC50 values in normoxia (20% O2). Apoptotic detection showed that 2% O2 significantly suppressed 4-HPR-induced apoptosis and the percentages of 4-HPR-induced apoptotic cells at 12 and 24 h were 1.2 and 11.0%, respectively, compared with 12.6 and 76.3% in 20% O2. In addition, in 20% O2, but not in 2% O2, 4-HPR obviously downregulated the protein expression of procaspase-3, ERK1/2 and XIAP, and increased the cleavage of PARP. Also, a significant DeltaPsim loss in response to 4-HPR was observed in normoxia, but not in hypoxia.

Hypoxia is able to induce 4-HPR resistance in Molt-4 cells and the mechanism may be involved in the inhibition of 4HPR-induced DeltaPsim depolarization and regulation of mitochondrial pathway-related proteins associated in signaling apoptosis.