Nitric Oxide Sensitizes B-NHL Cells to TRAIL-Mediated Apoptosis through Induction of RKIP, Inhibition of YY1 and Upregulation of DR5.
Authors: Baritaki Stavroula , Katsman Alina , Huerta-Yepez Sara , Yeung Kam C. , Berenson James , Bonavida Benjamin 
Published: April 2020

Abstract
A better knowledge of the mechanisms of survival and escape from apoptosis of B-cell lymphoma cells has led to the proposal of new drugs that selectively interfere at the different steps of these cascades. Among the novel agents that have recently emerged for the sensitization of several resistant tumor cells to chemo- or immuno-mediated cell death are agents that induce nitric oxide (NO) (Bonavida B et al., Drug Resistance Update, 2006). The molecular mechanisms which are implicated in the NO-induced sensitization of tumor cells to apoptosis are not clearly elucidated. Recent findings in our laboratory have reported that treatment of various cancer cell lines with the NO donor, DETA/NONOate, sensitizes tumor cells to FasL- and TRAIL-mediated apoptosis. Sensitization is mediated through inhibition of NF-κB and YY1 activities and upregulation of death receptors. The objective of the present study was to examine whether DETA/NONOate is also able to sensitize B-NHL cells to TRAIL-mediated apoptosis and to identify additional molecular targets which could be involved in the NO-mediated chemo- and immuno-sensitization of tumor cells. Contrary to YY1, RKIP (Raf kinase inhibitor protein) has been shown to play a pivotal role in suppression of mitogenesis through inhibition of Raf/MEK/ERK and NF-κB survival signaling pathways (Yeung et al., Molecular Cellular Biology; 21:7207, 2001). Since both NO and RKIP inhibit NF-κB, we hypothesized that NO may induce RKIP expression and may participate in NO-mediated effects. The TRAIL-resistant B-NHL cell line, Ramos, was treated with DETA/NONOate and examined for changes in YY1 and RKIP protein and m-RNA levels, as well as for sensitization to TRAIL-mediated apoptosis. The findings demonstrate that treatment of Ramos cells with DETA/NONOate results in slight induction of RKIP m-RNA levels followed by significantly elevated RKIP protein expression. In contrast, both YY1 transcript and protein levels were found remarkably reduced after DETA/NONOate treatment. Since YY1 has been recently shown to negatively regulate DR5 transcription, NO-induced suppression of YY1 was able to sensitize Ramos cells to TRAIL-mediated apoptosis through significant DR5 upregulation, as assessed by flow cytometry. However, DR4 surface protein expression remained unchanged. Noteworthy, the rituximab resistant Ramos clone (RR1) was not sensitized by NO, while no changes were observed in both DR4 and DR5 levels, suggesting different regulation of cell sensitivity to TRAIL. The above findings support the role of NO as an immuno-sensitizing agent via inhibition of YY1 and suggest, for the first time, the pivotal role of RKIP as one potential NO target for apoptosis induction in tumor cells. Further experimental studies will clarify the interlink between RKIP and YY1 involved in NO-based B-cell lymphoma sensitization to apoptosis and validation with patient derived tissues.