Oridonin Upregulates All-Trans Retinoic Acid Receptor Alpha and Induces Differentiation Of NB4 Cells Though NF-Kb Pathway

Nuclear retinoic acid receptors (RARs) function as ligand-dependent transcriptional regulators, often heterodimerized with retinoid X receptors. They play critical roles in a variety of biological processes, including development, differentiation and homeostasis. However, it is still not very clear about the mechanism of RARα catabolism. Almost all cases of acute promyelocytic leukemia (APL) express oncogenic fusion protein involving the RARα due to chromosome translocations. Despite the great success of ATRA therapy, it is still important to search for a variety of promising compounds to enhance the pharmacologic activity of ATRA and alleviate its adverse effects. And some of these compounds may be useful as a probe to investigate the mechanism of RARα catabolism.

Our previous studies showed that oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, could have an ability to accumulate RARα, enhance ATRA-induced differentiation in ATRA-sensitive as well as -resistant APL cells, and induce apoptosis which is initiated by reactive oxygen species (ROS) production. Based on those discoveries, we supposed that inhibition of RARα degradation may be responsible for these serial actions. Herein, we used Ori as a probe to explore how Ori stabilized RARα and its potent mechanisms. Firstly, our results showed that Ori upregulated RARa in a dose- and time- dependent manner in NB4 cells while anti-oxidant agent N-acetyl-L-cysteine (NAC) addition completely blocked this effect, indicating that RARa was stabilized via Ori-induced intracellular ROS formation. Secondly, we found H₂O₂, which could increase ROS level also, upregulated RARa in a dose- and time- dependent manner. More of interest, we found that both Ori and H₂O₂ upregulated RARa through NF-kB-signaling, proved by the accumulate evidence that either NF-κB pathway inhibitor or knockdown of P65 or inhibition IkBa phosphorylation could block the RARa stabilization. Whereas, P65 overexpression or knockdown of IkBa or some NF-κB pathway activating agents like tumor necrosis factor alpha (TNFa) and lipopolysaccharide ( LPS) could increase the RARa level.

Collectively, these findings suggested that Ori is an extraordinary tool to enhance the pathophysiological and potential therapeutic roles of RARα expression though NF-kB pathway. Moreover, an approach combining ATRA with Ori warrants further investigation as a therapeutic strategy for APL patients.