Hypoxia-mediated 4-HPR Resistance In Childhood Acute Lymphoblastic Leukemia Cells.

N-(4-Hydroxyphenyl)-retinamide (4-HPR, Fenretinide) is a synthetic retinoid with cytotoxic activity in a wide variety of cancer cells both in vitro and in vivo including acute lymphoblastic leukemia (ALL) cell lines. The mechanisms of 4-HPR-mediated apoptosis have not fully elucidated. Previous reports have indicated that 4-HPR could induce apoptosis through RAR-dependent and/or -independent pathways. The activation of the c-Jun N terminal kinase, the activation of the mitochondrial pathway via generation of reactive oxygen species (ROS), and/or the induction of increased ceramide (a sphingoid-based lipid second messenger molecule) production have all been implicated in 4-HPR-medaited apoptosis. It was also reported that 4-HPR enhanced the effect of chemotherapy, and the mechanism is related to mitochondrial dysfunction, which plays an important role in 4-HPR-induced apoptosis.ALL is a malignant (clonal) disease of the bone marrow in which early lymphoid precursors proliferate and replace the normal hematopoietic cells of the marrow. A poor prognosis of ALL patients is related to the recurrence in the bone marrow, a hypoxic tissue compartment. It has been documented that hypoxic tissue may serve as a "sanctuary" site for tumor cells, resulting in resistance to many common chemotherapeutic agents due to hypoxia.Recently, a decrease in the cytotoxicity of 4-HPR by hypoxia was reported in Ewing's sarcoma and primitive neuroectodermal tumor cell lines. Therefore, we were interested in identifying whether hypoxia could exert inhibition effect on the sensitivity of ALL cells to 4-HPR.It has been demonstrated that a change in mitochondrial membrane0potential (A*Fm) plays a pivotal role in transducing a variety of proapoptotic stimuli. Opening of permeability transition pores in mitochondria induces the release of cell death-promoting factors, including cytochrome C and apoptosis-inducing factor (AIF). Cytochrome C has been demonstrated to be involved in the activation of a caspase cascade, while AIF has been shown to directly trigger apoptosis. It was reported that hypoxia modulated A*Fm and the expression of several proteins, such as ERK1/2 and XIAP, are involved in AYm stabilization and caspases activation. Thus, we hypothesized that hypoxia might inhibit 4-HPR activity via mitochondrial pathway.Together, there are two specific aims in the current study: (1) To determine the in vitro cytotoxicity of 4-HPR against ALL cell lines in hypoxia;and (2) To explore the mechanisms of hypoxia-mediated 4-HPR resistance in ALL cell lines.Aims: To determine the hypoxia-induced 4-HPR resistance and toinvestigate the mechanism of hypoxia-mediated 4-HPR resistance in Molt-4 cells.Methods: To evaluate the in vitro cytotoxicity of 4-HPR against Molt-4 cells in normoxia (20% O2) and hypoxia (2% O2) by MTT method;to detect the 4-HPR-induced apoptosis in normoxia and hypoxia by flow cytometry;JC1 stain was used to detect mitochondrial membrane potential (AYm) of Molt-4 cells treated with 4-HPR in two conditions;DCFDA stain was used to detect the ROS production in Molt-4 cells treated with 4-HPR under two conditions;Apoptosis-pathway-involved protein expressions of the Molt-4 cells treated with 4-HPR in normoxia and hypoxia were analyzed by western blotting.Results:1) The cytotoxicity of 4-HPR was measured by MTT method in normoxia and hypoxia. IC50 value of 4-HPR for Molt-4 cell line in 20% O2 were 2.6 ±0.1 nM and the IC50 values of 4-HPR in 2% O2 increased to 9.1 ± 0.2 \iM for Molt-4 cells. Hypoxia significantly (P < 0.01) inhibited, but not abrogated, the cell killing ability of 4-HPR.2) The Molt-4 cells treated with 10 ^M 4-HPR for 6, 12 and 24 hours were examined for apoptosis by propidium iodide staining and flow cytometry. Spontaneous apoptosis (1.1-1.4%) was observed in hypoxia and normoxia.In 20% O2,4-HPR induced apoptosis in 1.2%, 12.6%, and 76.3% at 6,12 and 24 hours with a time-dependent manne. In 2% O2, apoptosis induced by 4-HPR were declined to 1.4% (6 hours), 1.2% (12 hours), and 11.0% (24 hours) at each time point, respectively.3) To determine if hypoxia could effect 4-HPR-mediated ROS production, we measured ROS by flow cytometry using carboxy-DCFDA. Molt-4 cells were exposed to 10 ^M 4-HPR for 1 hour - 6 hours in normoxia and hypoxia prior to staining with carboxy-DCFDA and flow cytometric analysis. One hour treatment with 4-HPR increased the mean fluorescence 3.2-fold and 2.3-fold, respectively, over controls in 20% O2 and 2% O2. Afterward, ROS production showed a decreased trend at 2 hours - 6 hours after addition of 4-HPR. At 6 hours treatment point, 4-HPR-caused ROS production almost restored to the level of pre-treatment in 20% O2 and 2% O2. Compare to normoxia, 4-HPR still could elevate ROS level in hypoxia, but the efficiency somewhat depressed.4) To better explore the role of ROS in the cytotoxicity of 4-HPR in ALL cell lines, Molt-4 cells were pre-exposed to the thiol antioxidant NAC 50 fiM and 100 ^M for 3 hours, and then treated with or without 4-HPR 5 |xM in normoxia. ROS levels were assayed after 1-hour treatment with 5 |xM 4-HPR, and cell viability was measured after 72 hours. The 2.7-foldelevation of ROS level was observed in 4-HPR alone group, and a > 35% decline was caused by NAC-pre-treatment. Relative to controls, NAC treatment alone induced a > 1.2-fold (p < 0.05) elevation in cell viability. NAC 50 nM and 100 nM failed to significantly (p > 0.05) inhibit cytotoxicty of 4-HPR in Molt-4 cells. Thus, though partially suppressed 4-HPR-mediated ROS level increase, NAC exhibited no effect on 4-HPR activity.5) We determined A 6 - hour treatment) obviously decreased the expression of proCaspase3, ERK and XIAP in Molt-4 cells, and increased the cleavage of PARP in a time-dependent manner in 20% O2. Whereas in 2% O2,4-HPR only slightly decreased proCaspase3 and increased the cleavage of PARP at 24 hours. In addition, 4-HPR in 2% O2 failed to decrease the expression of ERK and enhanced the expression of XIAP at 24 hours.Conclusion: In conclusion, hypoxia induced 4-HPR resistance in Molt-4 cells via mitochondrial pathway. Its mechanisms might be related to the reduction of ROS production and the activation of ERK by maintaining level of XIAP, which can stabilize the ANPm and inhibit the activation of Caspase3.