Elsevier

Biochemical Pharmacology

Volume 74, Issue 10, 15 November 2007, Pages 1445-1455
Biochemical Pharmacology

Deglycosylated bleomycin induces apoptosis in lymphoma cell via c-jun NH2-terminal kinase but not reactive oxygen species

https://doi.org/10.1016/j.bcp.2007.07.036Get rights and content

Abstract

Bleomycin (BLM) has demonstrated potent activity in treating malignant lymphomas but its therapeutic efficacy is hampered by induction of lung fibrosis. This side effect is related to the ability of the drug to generate reactive oxygen species in lung cells. In the present study, we evaluated the consequences of deglycosylation of BLM in term of cytotoxic activity and generation of reactive oxygen species. When tested on U937 human lymphoma cells, both compounds generated a typical apoptotic phenotype. Cell death induction was associated with Bax oligomerization, dissipation of the mitochondrial membrane potential, release of cytochrome c, caspase activation, chromatin condensation and internucleosomal degradation. Whereas both reactive oxygen species and c-jun NH2-terminal kinase (JNK) inhibitors prevented BLM-induced U937 cell death, only JNK inhibition prevented deglycosylated BLM-mediated cell death. Both compounds induced clustering of TRAIL receptors (DR4 and DR5) and Fas at the cell surface but neither a chimeric soluble DR5 receptor that inhibits TRAIL-induced cell death nor a dominant negative version of the adaptor molecule Fas-associated death domain prevented BLM-induced cytotoxicity. These observations indicate that deglycosylation of BLM does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway but prevents induction of reactive oxygen species. This observation suggests that deglycosylated BLM could exhibit less toxic side effects and could warrant its use in clinic.

Introduction

Bleomycins (BLM) are a family of glycopeptides isolated from Streptomyces verticullis in 1966 [1] which exhibit antibiotic properties (for review [2]). They are attractive therapeutic drugs hardly induce myelosuppression [3] or immunosuppression [4]. They are commonly included in chemotherapy regimens used to treat patients with Hodgkin's or non Hodgkin's malignant lymphoma [5], [6], squamous-cell carcinoma [7] or germ-cell tumor [8]. Their therapeutic use is limited by a dose-dependent lung toxicity that eventually leads to fibrosis [9], [10].

The clinically administrated form of BLM, Bleonoxane (Bristol-Meyers Squibb), is essentially composed of two molecules, BLM-A2 (∼60%) and BLM-B2 (∼40%), which differ in their positively charged tail. The anti-neoplastic properties of BLM were attributed to their ability to link metals including iron and to form a complex that reduces molecular oxygen to superoxide and hydroxyl radicals. This causes single- and double-stranded DNA breaks and ultimately leads to cell death [11], [12], the extent of which depends on drug concentration and incubation [12], [13]. At low doses, BLM arrests cells in the G2/M phase of the cell cycle, which possibly ends with mitotic catastrophe. Extensive DNA double stranded-breaks induced by BLM at higher doses can trigger apoptosis [14], as observed in alveolar epithelial cells [15], [16] as well as in limited number of tumor cells [17], [18].

Deglycosylation of BLM was proposed as a potential mean to reduce the toxicity of the molecule [19] but whether deletion of the carbohydrate moieties of BLM affected the ability of the compound to trigger apoptosis remained obscure.

A series of experimental evidences indicates that while most anti-tumor drugs activate the intrinsic death pathway [20], [21], [22], [23], death receptor extrinsic pathway could contribute to the cytotoxic activity of a limited number of specific anticancer drugs [24], [25], [26]. The intrinsic pathway, also called mitochondria-dependent pathway, requires sentinel, such as BH3-only proteins of the Bcl-2 family that bind to and inhibit anti-apoptotic members of this family in order to activate multi-domain pro-apoptotic members such as Bax and Bak. The outer mitochondrial membrane (OMM) then becomes permeable to cytochrome c that, in the cytosol, enables the assembly of the apoptosome in which caspase-9 is activated, leading to subsequent activation of a caspase cascade and cell demise [27]. The death receptor-mediated pathway, also known as the extrinsic pathway, is activated upon engagement of death receptors such as Fas and TNF-related apoptosis inducing ligand (TRAIL) receptors DR4 and DR5 at the cell surface. Interaction of these receptors to their cognate ligands results in the recruitment of the adaptor molecule Fas-associated death domain protein (FADD) that, in turn, recruits the initiator caspase-8 within the Death inducing Signaling Complex (DISC) in which the protease is activated. Depending on the cell type, caspase-8 either directly activates downstream effector caspases such as caspase-3 or cleaves the sentinel BH3-only protein Bid to connect the extrinsic to the intrinsic pathway to death [28].

The present study was undertaken to determine the influence of deglycosylation on the ability of BLM to trigger cell death by apoptosis. We show that both parental drug and its derivative activate the intrinsic pathway through the activation of the JNK pathway. Death receptors, although redistributed at the cell surface upon drug exposure are, however, unlikely to be involved in the cytotoxic effect. Whereas BLM also induces the formation of ROS, the deglycosylated derivative does not, which might indicate a less toxic profile.

Section snippets

Drugs and reagents

Lyophilized BLM (kindly provided by Nippon Kayaku (Tokyo, Japan)) was dissolved in sterile water and stored at −20 °C. Nonglycosylated form of BLM (D-BLM) was obtained by β-elimination under mild alkaline conditions, and by solvolysis with hydrogen fluoride as previously described [19]. The recombinant soluble FasL was collected from the supernatant of FasL-transfected Neuro2A cells (Dr. Fontana, Lausanne, Switzerland). A unique pool of sFasL supernatant was used throughout the study. The Ig

BLM and D-BLM-induced apoptosis in U937 human lymphoma cells is mediated by caspases

Exposure of U937 human lymphoma cells to BLM and D-BLM decreased both cell viability (Fig. 1A and B) and clonogenic survival (Fig. 1C and D) in a concentration- and time-dependent manner. This cytotoxic effect was observed at low concentrations, e.g. exposure of U937 cells to 1 μM BLM or D-BLM induced a 60 and 40% decrease in their clonogenicity, respectively (Fig. 1C and D). The cytotoxic effect of BLM and its deglycosylated form appeared to be apoptotic as demonstrated by the condensation of

Discussion

BLM-containing regiments remain commonly used to treat malignant lymphomas [36], but the use of BLM exposes patients to lung fibrosis [37], [38], side effects related to the ability of the molecule to generate free radicals such as ROS [16], [39]. Here, we show that deglycosylation of the molecule generates a compound that remains able to trigger apoptosis in a lymphoma cell line but does not generate ROS.

When studied in a lymphoma cell line, this pathway appears to involve Bax, which is in

Acknowledgments

We are grateful for Professor Eric Solary, and Arlette Hammann (Inserm U866, IFR100, Dijon, France) for help in manuscript revision, and flow cytometry data analysis, respectively. Our group is supported by grants from Inserm, France and the DGRSRT, Tunisia. SB is the recipient of the Education Ministry of Tunisia. LP is the recipient of INSERM and région de Bourgogne fellowship.

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