Antibody-Targeted HRP Associated with Indole-3-Acetic Acid Induces in Vitro Apoptosis in Hematological Tumors.

Abstract 1270

Poster Board I-292

3-Indole-acetic acid (IAA) is a growth hormone found in plants that may be oxidized by horseradish peroxidase (HRP) generating cytotoxic molecules capable of inducing injury in mammal cells, including a variety of human tumors. The aim of this study was to establish an assay based on targeting HRP to hematopoietic tumor cells using antibodies and induce apoptosis by incubation with IAA. To set up the best experimental conditions, we used two human lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta 519, from Mantle Cell Lymphoma (MCL). The targeting of HRP was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with the enzyme. Eight groups of cells were incubated for 2, 8, 18, 24 and 48 hours: control, HRP targeted, HRP targeted with IAA at 1, 5 and 10 mM, cells not targeted with IAA at 1, 5 and 10 mM. Apoptosis was analyzed by flow cytometry, with anexin V-FITC and propidium iodide. The best experimental conditions for both NB4 and Granta 519 cells were achieved with incubation for 24 h with 10 mM IAA. For NB4, the group targeted with HRP and treated with 10 mM IAA for 24 hours presented 44.1% of apoptosis, whereas the control groups achieved 18.2 to 24.6% (P<0.05). In Granta 519, these results demonstrated higher values of cell death (64.1% versus 15.5 to 20.6% in control groups, P<0.05). Next, we tested cells from 12 patients with acute myeloid leukemia (AML) (six with APL and six with AML others than APL) and from 10 patients with chronic lymphocytic leukemia (CLL). In AML patients others than APL, cells targeted with HRP and treated for 24 h with 10 mM IAA presented 78.8% of apoptosis, while the results with control groups varied from 18.5 to 27.8% (P<0.05). These group of cells in APL patients presented 75.9% of apoptosis, while the controls varied from 18.1 to 44.8% (P<0.05). Interestingly, in APL patients, the cell group treated with 10 mM IAA and not targeted with HRP also had higher apoptosis (44.8%) when compared to the others controls (18.1 to 26.8%) (P<0.05), suggesting that the high concentrations of myeloperoxidase of these cells were capable of activating IAA, in the absence of HRP. In CLL, cells incubated for 24 hours, targeted with HRP and treated 10 mM IAA showed mean apoptosis value of 93.3%. In the control cells, these values varied from 35.7 to 53.7% (P<0.05). Interestingly, cells from patients with CLL presented higher apoptosis than AML cells (P<0.05). Our results demonstrated that the IAA/HRP association was capable of inducing apoptosis in hematopoietic tumors, which was dependant on the IAA dose, the time of IAA exposure and the lineage of the tumor. It is also suggested that the endogenous myeloperoxidase found in APL blasts was capable of activating HRP in the absence of IAA, leading to death of these cells.