Arsenic trioxide and ascorbic acid interfere with the BCL2 family genes in patients with myelodysplastic syndromes: an ex-vivo study

Abstract

Background: Arsenic Trioxide (ATO) is effective in about 20% of patients with myelodysplasia (MDS); its mechanisms of action have already been evaluated in vitro, but the in vivo activity is still not fully understood. Since ATO induces apoptosis in in vitro models, we compared the expression of 93 apoptotic genes in patients' bone marrow before and after ATO treatment. For this analysis, we selected 12 patients affected by MDS who received ATO in combination with Ascorbic Acid in the context of the Italian clinical trial NCT00803530, EudracT Number 2005-001321-28.

Methods: Real-time PCR quantitative assays for genes involved in apoptosis were performed using TaqMan® Assays in 384-Well Microfluidic Cards "TaqMan® Human Apoptosis Array".Quantitative RT-PCR for expression of EVI1 and WT1 genes was also performed. Gene expression values (Ct) were normalized to the median expression of 3 housekeeping genes present in the card (18S, ACTB and GAPDH).

Results: ATO treatment induced up-regulation of some pro-apoptotic genes, such as HRK, BAK1, CASPASE-5, BAD, TNFRSF1A, and BCL2L14 and down-regulation of ICEBERG. In the majority of cases with stable disease, apoptotic gene expression profile did not change, whereas in cases with advanced MDS more frequently pro-apoptotic genes were up-regulated. Two patients achieved a major response: in the patient with refractory anemia the treatment down-regulated 69% of the pro-apoptotic genes, whereas 91% of the pro-apoptotic genes were up-regulated in the patient affected by refractory anemia with excess of blasts-1. Responsive patients showed a higher induction of BAD than those with stable disease. Finally, WT1 gene expression was down-regulated by the treatment in responsive cases.

Conclusions: These results represent the basis for a possible association of ATO with other biological compounds able to modify the apoptotic pathways, such as inhibitors of the BCL2 family.

Figures

Figure 1

Unsupervised cluster analysis of apoptotic gene expression modification upon ATO and Ascorbic Acid treatment. Figure depicts -ΔΔCt of the “test patient” (lane #6) versus the remaining ones.

Figure 2

ATO and Ascorbic Acid induced gene expression modification of BAD in patients that reached stable disease or a response (major or minor).

Figure 3

Mean and standard deviation of gene expression before and after ATO and Ascorbic Acid treatment of ICEBERG (A), TNFRSF1A (B) and BCL2L14 (C) in the whole series of patients.

Figure 4

Relationships between differentially expressed genes have been explored in the pathway analysis. Up-regulated genes are reported in red, whereas down-regulated in green.