The Para-Isomer of Nitric Oxide-Donating Acetylic Salicylic Acid (p-NO-ASA) Effectively Induces Cell Death in B-Cell Chronic Lymphocytic Leukemia (CLL) Cells at Low Micromolar Concentrations.

Introduction: B-cell chronic lymphocytic leukemia CLL is characterized by an accumulation of mature, non functional B cells. We and others have shown that WNT/β-catenin (CTNNB1) signaling appears to be constitutively activated in these cells. Furthermore, it is already long known that several compounds related to the non-steroidal antiinflamnmatory drugs (NSAID) can inhibit CTNNB1 stability and/or function. However, so far clinical studies with such substances generated disappointing results which is likely to the fact that therapeutic plasma concentrations could not be reached without producing significant toxicities. Recently, nitric oxide donating derivatives of acetylic salicylic acid (NO-ASA) have been shown to be well tolerated in humans. In addition NO-ASA could disrupt complexation of CTNNB1 and TCF-4 in vitro, whereas the latter belongs to the transcription factors which posses a central function in mediating WNT signaling. The aim of our study was to evaluate whether the para- and meta-isomers of NO-ASA selectively induce apoptosis in CLL cells.

Methods: Primary CLL cells as well as healthy peripheral blood monocytes (PBMC) were treated with varying concentrations of p- and m-NO-ASA for different time periods. Cytotoxicity was assessed by microscopic cell viability testing and an ATP assay. Induction of apoptosis was investigated by immunoblotting of PARP, caspases 3 and 9. Further, CTNNB1 protein amount was measured by immunoblotting and expression of WNT effector proteins like cyclin D1 (CCND1), C-MYC and LEF-1 was evaluated with immunoblot analysis as well.

Results: The meta-isoform of NO-ASA did not have any effect on CLL cells whereas the para-isomer showed a selective cytotoxic effect. Mean lethal concentration (LC50) values were 4.83 μM and 4.64 μM in CLL cells, respectively. LC50 values for healthy controls were more than 25-fold higher. Immunoblot analysis revealed that p-NO-ASA cleaves PARP; caspase 3 and caspase 9, decreases CTNNB1 protein levels and downregulates WNT pathway target genes in a concentration dependent manner.

Conclusion: Our findings show that the para- but not the meta-isomer of NO-ASA induces caspase-mediated apoptosis by inhibition of WNT signaling in CLL cells. NO-ASA, consisting of a traditional molecule of ASA where the NO moiety is covalently bound via a spacer, has been shown to exhibit a lower gastric toxicity than traditional NSAIDs. Therefore p-NO-ASA might be a valuable compound for the treatment of CLL. More investigations of the exact mechanism of action and the specific difference between the positional isomers are indicated.