The Para-Isomer of Nitric Oxide Donating Acetylsalicylic Acid (p-NO-ASA) Induces Apoptosis in Chronic Lymphocytic Leukemia (CLL) in Vitro and In Vivo without Gross Systemic Toxicities.

Abstract 3783

Poster Board III-719

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, non functional B cells. WNT/β-catenin(CTNNB1)/TCF/Lef-1 signalling appears to be constitutively and aberrantly activated in these cells. Furthermore, several compounds related to the non-steroidal anti-inflammatory drugs (NSAIDs) are reported to inhibit β-catenin stability and/or function in WNT active cancers in vitro. 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; hence, the required high concentrations limit their clinical use. Recently, nitric oxide-donating acetylsalicylic acid (NO-ASA) has been shown to achieve high plasma levels in doses not leading to any relevant side effects in humans. In addition, NO-ASA could disrupt complexation of β-catenin and TCF-4 in vitro. Because the general structure of NO-ASA enables more variants, the aim of our study was to evaluate the effect of the para- (p-NO-ASA) and meta-isomer (m-NO-ASA) in CLL in vitro and in vivo.

Primary CLL cells as well as healthy peripheral blood monocytes (PBMCs) and healthy B cells were treated with varying concentrations of p- and m-NO-ASA. Cytotoxicity was assessed by microscopic cell viability testing and measurement of the reduction of the ATP content. Induction of apoptosis was investigated by Annexin V-FITC/propidiumiodide staining and immunoblotting of the caspases-9, -3 as well as PARP. Further, the β-catenin protein amount and the expression of WNT effector proteins like cyclin D1 (CCND1), C-MYC and LEF-1 was evaluated by immunoblot analysis. In vivo activity of NO-ASA was evaluated by treating irradiated CD1 nu/nu female mice, containing a JVM-3 cell line xenograft, with 100 mg/kg/day of p- and m-NO-ASA or vehicle control p.o. for 3 weeks continuously.

We found a significant concentration dependent reduction of the ATP content in CLL cells after treatment with p-NO-ASA, whereas the meta-isomer showed no effect on CLL cells. While healthy B cells and healthy PBMCs were not significantly affected by any of the isomers the mean lethal concentration (LC₅₀) was 4.64 μM in CLL cells. Annexin V-FITC/PI staining revealed that reduced cell survival occurs in a time and concentration dependent manner and is mediated by apoptosis. Treatment with 10 μM of p-NO-ASA for 24 hours reduced survival to 46.3 ± 10.1%. This effect was achieved as early as 6 hours after treatment. Immunoblot analysis showed that only p-NO-ASA but not m-NO-ASA activates caspases-9, -3 and cleaves PARP at the same concentrations, which lead to induction of cell death. β-catenin protein levels and WNT pathway target genes are down regulated between 1 to 10 μM also only by the para-isomer. In vivo results revealed that exclusively p-NO-ASA show a strong antitumor efficacy with an IR_max value of 83.1%. After 9 days of treatment p-NO-ASA lead to a significantly reduced tumor volume compared to vehicle control (126.4 ± 22.3 mm³ for p-NO-ASA vs. 290.0 ± 65.9 mm³ for the vehicle control, p=0.0303). Tumor volume of vehicle treated controls increased up to 775.4 ± 219.6 mm³ whereas the tumor volume of p-NO-ASA treated group remained stable at 128.7 ± 27.6 mm³ (p=0.0091) over a treatment period of 21 days. The meta-isomer exhibited no significant antineoplastic effect. Our findings show that the para- but not the meta-isomer of NO-ASA selectively induces caspase mediated apoptosis in CLL cells. The mechanism of action might include inhibition of β-catenin/Lef-1 signaling since we observed downregulation of specific target gene expression. Due to our promising in vivo results, discovering a strong inhibition of tumor growth without producing gross side effects, p-NO-ASA might be a valuable compound for the treatment of CLL. More investigations of the mechanism of action and the specific difference between the positional isomers are needed.