Elsevier

Immunology Letters

Volume 133, Issue 2, 30 October 2010, Pages 99-105
Immunology Letters

Direct inhibition of human acute myeloid leukemia cell growth by IL-12

https://doi.org/10.1016/j.imlet.2010.08.002Get rights and content

Abstract

Acute myeloid leukemia is a haematopoietic malignancy originating from the transformation of myeloid progenitors that proliferate and accumulate in the bone marrow. In AML patients the survival rate at 5 years is 40–50% highlighting the need for novel therapies. In this study we have asked whether IL-12, an immuno-modulatory cytokine with anti-tumor activity, may inhibit directly AML cell growth. We show that the human AML cell lines U937, K562 and THP-1 expressed both chains of the IL-12 receptor (R), i.e. IL-12Rβ1 and IL-12Rβ2. IL-12 inhibited the angiogenic potential of AML cells in vitro, but did not affect their survival or proliferation. In vivo experiments were performed using SCID–NOD mice injected intraperitoneally (i.p.) with the human U937 AML cell line and subsequently treated with human recombinant IL-12 or PBS i.p. Histological, immunohistochemical and flow cytometric analyses on explanted tumors revealed that IL-12 reduced new vessel formation, induced apoptosis and inhibited tumor cell proliferation. Studies on a panel of angiogenesis related genes in explanted tumors using PCR arrays showed significantly down-regulated expression of numerous pro-angiogenic genes including VEGF-C, IL-6, IL-8, CXCL1, CXCL6 and alanyl aminopeptidase in IL-12 vs PBS treated mice. This study shows for the first time that IL-12 targets directly AML cell growth and paves the way to further investigation of IL-12 as potential drug for AML treatment.

Introduction

Acute myeloid leukemia (AML) is a hematopoietic malignancy originating from the transformation of myeloid progenitors that proliferate and accumulate in the bone marrow [1], [2]. In many cases the leukemic cell infiltration in bone marrow is accompanied by anemia and thrombocytopenia. AML is a clinically and genetically heterogeneous disease that is often fatal [3]. Thus, AML patients need novel therapies. With this background, we investigated whether IL-12 may function as anti-tumor agent against AML cells.

IL-12 is a heterodimeric cytokine bridging innate and adaptive immunity [4], that is formed by the p35 and p40 subunits and binds to the IL-12R, composed of the β1 and the β2 chains [5]. IL-12Rβ2 represents the unique chain of the IL-12R necessary for IL-12 signal transduction and consequently for regulation of biological responses of target cells to IL-12 [5]. More importantly, IL-12 exerts potent anti-tumor activity through immuno-stimulatory and anti-angiogenic mechanisms and/or direct activity on tumor cells [6], [7], [8], [9]. We [10], [11] demonstrated that the IL-12Rβ2 chain functions as tumor suppressor in human chronic B-cell tumors and reintroduction of the IL-12Rβ2 gene in these cells enabled IL-12 to restrain their growth. Differently, multiple myeloma cells expressed constitutively IL-12Rβ2 and were responsive to IL-12 treatment, thus candidating IL-12 as a novel therapeutic agent against this malignancy [12].

Here, we have performed functional studies to assess the direct anti-tumor activity of IL-12 on AML cell lines in vitro and in vivo and to unravel the mechanisms involved.

Section snippets

Cell lines, antibodies, reagents and flow cytometry

The human U937, K562, THP-1 AML cell lines were provided by Interlab Cell Line Collection (Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy) that certifies their origin. Cells were cultured in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% FBS (Sigma).

The following antibodies (Abs) were used: phycoerythrin (PE)-conjugated anti-human IL-12Rβ1; goat IgG anti-human IL-12Rβ2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phytocyanine (FITC)-anti-human CD45 (Caltag,

Expression and function of IL-12R in AML cell lines

First, expression of IL-12Rβ1 and β2 chains on three human AML cell lines was investigated by flow cytometry. The U937, K562 and THP-1 cell lines expressed constitutively both chains of the IL-12R (IL-12Rβ1+ cells, mean ± SD: U937 78 ± 3%, K562 74 ± 5% and THP-1 78 ± 3%. IL-12Rβ2+ cells, mean ± SD: U937 85 ± 1%, K562 80 ± 2% and THP-1 79 ± 2%). Fig. 1A and B shows one representative histogram for IL-12Rβ1 and β2 expression, respectively, on U937 and K562 cells. In order to assess whether IL-12R was functional

Discussion

Here we show for the first time that AML cells expressed complete and functional IL-12R and that IL-12 treatment damped AML cell growth. The anti-tumor activity operated by IL-12 in vivo was related to significant inhibition of new vessel formation associated with inhibition of cell proliferation and induction of apoptosis. The severe vascular deficiency caused by IL-12 in vivo was related to down-modulation of different pro-angiogenic molecules including VEGF-C, alanyl aminopeptidase, IL-6,

Acknowledgments

This work was supported by grants from Associazione Italiana Ricerca sul Cancro (A.I.R.C.) Milano, Italy (Grant Number 4014 to I.A.), Fondazione Cassa di Risparmio della Provincia di Chieti (CariChieti), Italy to E.D.C., and A.I.R.C. to V.P.

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    These authors equally contributed as last authors.

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