Chemosensitizing Effect of Fenretinide-Induced NF-κb Inhibition in AML Therapy

INTRODUCTION: Acute myeloid leukemia (AML) represents a genetically heterogeneous hematological malignancy and is among top 10 common cancers in China. Though most cases achieve complete remission with current therapy, relapses eventually occur and subsequent therapies fail to eliminate the leukemic cells again and sustain long-term remission. Acquired resistance might be the real instigator of treatment failure. Nuclear factor kappa B (NF-κB) signaling pathway activation, a hallmark of primary AML cells, especially of leukemic stem cells (LCSs) and in vitro cell lines, and associated with multi-layered roles in AML pathogenesis, i.e., pre-leukemia myelodysplastic syndrome (MDS), LSCs, drug response/toxicity, relapse, and leukemic maintenance. Thus, NF-κB might be an attractive strategy for better treatment response and survival but less toxicity in AML therapy.

METHODS: Luciferase reporter assays were performed to define the effects of fenretinide on transcriptional signaling pathways. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the NF-κB down-stream genes expression, and immunoblotting was performed to confirm the role of fenretinide on NF-κB inihition and apoptosis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assay were performed to test the drug response and chemosensitizing effect of fenretinide on AML cell lines and primary AML samples.

RESULTS:

Initially, using series of specific reporter assay kits we experimentally evaluated the effects of fenretinide on cell signaling in HEK293T cells and ten leukemic cell lines. Among all tested signaling, we found that fenretinide-treated could significantly suppress the NF-κB activation induced by TNF-α in HEK293T cells and daunorubin (DNR)-induced NF-κB activationin AML cell lines. To molecularly confirm NF-κB suppression, we found that anti-apoptotic gene BCL2 was decreased and pro-apoptotic genes cIAP, XIAP, BID were increased. Also, immunoblotting showed the decreased protein level of p65 NF-κB in accompanied by increased level of cleaved-PARP, and BID, while no alteration of JNK, ERK proteins. Next, we went on testing the fenretinide-induced NF-κB inhibition in AML chemosensitizing using series of MTT and flow cytometric apoptotic assays. In this regard, we found that even low dose of fenretinide could chemosensitize AML cells to DNR treatment. Mechanistic studies showed that while ROS maintenance successfully rendered AML cells sensitive to DNR treatment, abolishing ROS production using N-Acetyl Cysteine (NAC) could not reversed the response of AML cells to DNR. In the meanwhile, NF-κB inhibition was the main cause of fenretinide-induced AML chemosensitizing. Finally, we made use of nine primary AML samples and then treated with fenretinide and/or DNR. In general, fenretinide could suppress NF-κB signaling but the inhibition effects varied between patients. Acutally, adding fenretinide indeed substantially potentiated the effect of DNR on AML cells. Also, the chemosensitizing effect was correlated with the level of fenretinide-induced NF-κB inhibition and the MDR1 gene down-regulation.

CONCLUSIONS: The significance in our study is to identify the role of fenretinide-induced NF-κB inhibition in AML chemosensitizing through systemic in vitro experiments. Using in vitro AML cell lines and primary samples, we found that fenretinide could suppress NF-κB signaling pathway and sensitize AML cells to DNR treatment via reducing the pro-apoptotic/apoptotic genes expression, affecting proliferation associated genes expression and down regulating MDR1 expression. Low dose DNR in combination with low dose fenretinide produced similar effect comparable to that of high dose DNR treatment. Further, we identified that NF-κB inhibition but not ROS induction was the main reason for fenretinide-induced chemosensitizing on AML cells. These entire results highlight that fenretinide is a very promising chemosensitizing agent that would be of help in AML therapy.