Elsevier

Leukemia Research

Volume 32, Issue 8, August 2008, Pages 1244-1258
Leukemia Research

Resistance of acute myeloid leukemic cells to the triterpenoid CDDO-Imidazolide is associated with low caspase-8 and FADD levels

https://doi.org/10.1016/j.leukres.2007.12.008Get rights and content

Abstract

The synthetic triterpenoid CDDO-Im-induced apoptosis of patient-derived AML blasts: 11/25 AMLs were highly sensitive, while the remaining were moderately sensitive to CDDO-Im. The addition of TRAIL significantly potentiated the cytotoxic effect of CDDO-Im, through mechanisms involving the induction of TRAIL-R1/TRAIL-R2 and downmodulation of TRAIL-R3/TRAIL-R4. Biochemical studies showed that CDDO-Im: induced a rapid and marked GSH depletion and antioxidants (GSH or NAC) completely inhibited its pro-apoptotic effect; sequentially activated caspase-8, -9 and -3; caspase inhibitors partially protected AML blasts from CDDO-Im-induced apoptosis; resistance of AML blasts to CDDO-Im-induced apoptosis correlated with low caspase-8/FADD and high Bcl-XL expression in leukemic blasts.

Introduction

Acute myelogenous leukemia (AML) is a heterogeneous group of malignant disorders characterized by uncontrolled proliferation of clonal leukemic hemopoietic precursor cells blocked at various stages of myeloid development, associated with a severely impaired normal hemopoiesis, thus leading to neutropenia, anemia and thrombocytopenia. The development of AML is associated with accumulation of acquired genetic alterations and epigenetic changes in hemopoietic progenitor cells which alter the normal mechanisms of cell growth, proliferation, cell death and differentiation. Approximately 50–75% of adult patients with AML achieve complete remission when treated with cytabarine and daunorubicin or idarubicin, or with topoisomerase II inhibitor, mitoxantrone [1], [2]. However, the majority of these patients relapse and develop chemoresistance [1], [2]. In part, this failure to respond to chemotherapy in first or in second line is due to defects in apoptosis pathways [3], [4]. Therefore, agents that overcome road-blocks to apoptosis could be very useful for the treatment of this disease.

Triterpenoids, biosynthesized in plants by the cyclization of squalene, are used for pharmaceutical aims in many Asian countries, and several studies have suggested that some of these compounds exhibit antitumor activity [5], [6]. However, the natural triterpenoids display only a relatively weak anticancer activity and therefore new analogous of these molecules have been synthesized in an attempt to identify potent antitumor agents [7], [8]. Synthetic oleanane triterpenoids have profound effects on inflammation and the redox state of the cells and tissues, as well as being anti-proliferative and pro-apoptotic agents (reviewed in [9]).

Recent studies on the synthetic triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester (CDDO-m) have revealed an ability of these chemical compound to induce apoptosis of malignant cells in vitro, at doses that are well tolerated in rodents [10], [11], [12], [13], [14]. The novel synthetic triterpenoid, CDDO-Imidazolide (CDDO-Im), was more potent than CDDO both in vitro and in vivo to inhibit tumor growth [15].

CDDO induces apoptosis of tumor cells through caspase-independent and caspase-dependent mechanisms, the latter ones involving activation of caspase-8 [11], [13], [14]. This conclusion is directly supported by studies showing that CDDO-induced apoptosis in AML and chronic lymphocytic leukemia cells is inhibited by caspase-8 inhibitors [11], [13], [14]. The mechanisms through which CDDO induces caspase-8 activation are unclear: the decrease in the levels of cFLIP, an antiapoptotic protein that operates as an endogenous antagonist of caspase-8, induced by CDDO could play a role in the mechanisms favouring caspase-8 activation [16]. In contrast, overexpression of Bcl-2 or Bcl-XL failed to protect from the apoptotic effects of CDDO [11], [13], [14].

Several studies have characterized the proapoptotic effects of CDDO and its derivatives CDDO-Me and CDDO-Im in leukemia cells [10], [12], [16]. The majority of these studies have been carried out on leukemic cell lines and few data are available on the effects of these compounds on primary AML leukemic blasts. In this context, Suh and workers reported the effects of in vitro of CDDO and CDDO-Me on 10 primary AMLs: 20 and 50% of these AMLs resulted to be responsive (i.e., induction of at least 25% cell death) to CDDO and CDDO-Me, respectively [15]. Subsequently, Konopleva et al. showed that 1 μM CDDO induced apoptosis in 6/16 AMLs; furthermore, it was shown that CDDO was active in the killing of leukemic progenitors [17]. Finally, Shishodia et al. reported the inhibitory effect of CDDO-Me on NF-κB activity in leukemic blasts derived from 13 AMLs [18]. Recent studies indicate that these triperpenoids affect also the differentiation of leukemic cells: thus, CDDO-Im induces monocytic differentiation through a mechanism involving increased CEBPbeta expression [19], while CDDO is a potent inducer of granulocytic differentiation of leukemic cell lines and patient-derived primary AML blasts by translationally enhancing the expression and function of CEBPA [20].

In the present study we have explored the proapoptotic effect of CDDO-Im on 25 primary AMLs. Furthermore, we explored whether CDDO could sensitize AML cells to the proapoptotic effects of the death ligand TRAIL which, as we previously showed, does not induce apoptosis of AML blasts when used alone [21]. On the basis of our results we identified a subset of AMLs particularly sensitive to the pro-apoptotic effects of CDDO-Im, that could represent a target for future clinical trials involving the use of these triterpenoids for the treatment of AMLs.

Section snippets

Cells

Fresh leukemic blasts from 25 patients with AML, obtained after informed consent, were isolated from either bone marrow or peripheral blood by Ficoll-Hypaque density gradient centrifugation and immediately processed. All patients were consecutively diagnosed at the Divisions of Hematology of the University “La Sapienza” and of the University “Tor Vergata”. Leukemias were classified by morphologic and cytochemical criteria according to the French–American–British (FAB) classification. All

Sensitivity of AML blasts to CDDO-Im

The aim of this study was to determine the sensitivity of AML cells to the synthetic triterpenoid CDDO-Im. In a first set of experiments we have evaluated the capacity of CDDO-Im to induce apoptosis of fresh AML blasts isolated from 25 AML patients, whose main clinical and biologic features are reported in Table 1A, Table 1B. To perform these experiments we have incubated leukemic blasts for 24 h either in the absence (C) or in the presence of either CDDO-Im or TRAIL or CDDO-Im + TRAIL.

Discussion

We have shown that a synthetic triterpenoid, such as CDDO-Imidazolide (which is a known potent antiproliferative and proapoptotic agent), is a potent inducer of apoptosis of AML cells. Particularly, CDDO-Im exerted a marked proapoptotic effect in 11/25 AMLs; furthermore, the proapoptotic effect of CDDO-Im was greatly potentiated by TRAIL, that, when added alone, did not induce apoptosis of AML blasts.

It was of interest to note that, in line also with previous studies carried out on few AML

Acknowledgments

This paper was supported by a grant from the Italian Health Ministry. We are grateful to Giuseppe Loreto for help in graph preparation.

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