Abstract A23: Expression profile of genes altered by ester-protected hydroxybenzyl phosphates (EHBP) in T-leukemia cells
Authors: Nath Niharika , Kodela Ravinder , Boring Daniel , Crowell James , Kashfi Khosrow 
Published: May 2012

A23

Introduction

Acute lymphoblastic leukemia (ALL) is the primary cause of cancer-related mortality in children. Novel compounds and different therapeutic approaches are required for more effective treatments. Nitric oxide-releasing aspirin (NO-ASA), comprised of an ASA molecule covalently bound to an NO liberating moiety through an aromatic spacer was reported to inhibit Jurkat T cell growth and other cancer cell lines of colon, pancreatic and breast origin, including experimentally-induced tumors in various animal models. Recently, we and others showed that the mechanism of action of NO-ASA did not involve released NO or aspirin but that the spacer molecule joining these two entities was the biologically active component. The role of the NO-releasing moiety was simply that of a leaving group in the production of a reactive quinone methide (QM) intermediate. The ASA component apparently had a very limited or no biological contribution. On this basis, we synthesized two compounds as models in which the NO-releasing group was replaced with a substituted phosphate (Agent 1: No NO, NO-ASA); and one in which the aspirin component was replaced with an acetyl group and the -ONO2 leaving group was replaced by a substituted phosphate group (Agent 2: No NO, No-aspirin, NO-ASA). In this study we investigated whether these three compounds may alter the gene expression of biological signaling pathways that are potential targets in carcinogenesis, and to elucidate the role of the NO-releasing moiety and the aspirin component of NO-ASA in the differential expression of these genes.

Methods

Agents: NO-ASA (2-(acetyloxy)-4-[(nitrooxy)methyl] phenyl ester, Agent 2: (2-(acetyloxy)-4-[(diethylphosphate)methyl] phenyl ester, Agent 3: (4-acetyloxybenzyl diethylphosphate) were synthesized, purified and NMR verified by us. Cell line: Jurkat T-acute lymphoblastic leukemia; the IC50 for cell growth inhibition for the three compounds was determined by an MTT assay. Following treatment of the Jurkat cells with NO-ASA, agent 2 or agent 3, at their respective IC50s, mRNA was isolated and labeled according to standard protocols and kits. The oligo arrays set from SuperArray were used. The blots were hybridized and the ratios of the expression levels quantified and calculated. Potential candidate genes that were differentially expressed were validated by real time RT-PCR.

Results

All three drugs were potent growth inhibitors of human leukemia Jurkat T cells. The genes that were prominently induced by all three drugs included FMO4, HSPA1A, HSPA6, HSPH1, FOS, CYP24 A1, DDIT3; this induction occurred in a concentration dependent manner. These may provide mechanistic insights into the growth inhibition observed with these agents. Other moderately altered genes included HYOU1, SOD1, SOD2. Interestingly, all 3 agents had the same effect in terms of fold-changes in gene expression for many of these candidate genes. This strongly suggests that the NO-releasing group and the aspirin component of NO-ASA have a negligible role in modulating these gene expressions. We propose that the growth inhibitory properties of all three agents are associated with the spacer component of the molecules which generates a quinone methide intermediate. The significance of the genes induced or repressed and the relationship of their gene expression with the cell growth inhibition will be discussed.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):A23.