Deglycosylated bleomycin induces apoptosis in lymphoma cell via c-jun NH2-terminal kinase but not reactive oxygen species
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.
References (49)
- et al.
Clinical trials with bleomycin in lymphomas and in solid tumors
Eur J Cancer
(1972) Bleomycin-induced pneumonitis
Chest
(2001)- et al.
Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs
J Biol Chem
(1999) Apoptotic pathways: paper wraps stone blunts scissors
Cell
(2000)Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins
Methods Enzymol
(2000)- et al.
Rescue of early-stage myelodysplastic syndrome-deriving erythroid precursors by the ectopic expression of a dominant-negative form of FADD
Blood
(2005) - et al.
Influence of the nitric oxide donor glyceryl trinitrate on apoptotic pathways in human colon cancer cells
Gastroenterology
(2002) - et al.
Characterization of N-acetylcysteine and ambroxol in anti-oxidant therapy
Respir Med
(1998) - et al.
New antibiotics, bleomycin A and B
J Antibiot (Tokyo)
(1966) - et al.
Bleomycins: towards better therapeutics
Nat Rev Cancer
(2005)
Minimal bone marrow damage in mice given bleomycin
Cancer Res
The effects of bleomycin on immunocompetence in man
Cancer Res
Bleomycin in non-Hodgkin's lymphoma
Semin Oncol
Histological effects and predictive biomarkers of TPP induction chemotherapy for oral carcinoma
J Oral Pathol Med
The importance of bleomycin in combination chemotherapy for good-prognosis germ cell carcinoma
J Clin Oncol
Bleomycin pulmonary toxicity has a negative impact on the outcome of patients with Hodgkin's lymphoma
J Clin Oncol
Effect of chelating agents and metal ions on the degradation of DNA by bleomycin
Biochemistry
Single-strand scission and repair of DNA in mammalian cells by bleomycin
Cancer Res
Ultrarapid recovery from lethal effects of bleomycin and gamma-radiation in stationary-phase human diploid fibroblasts
Cancer Res
The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway
Br J Cancer
Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway
Am J Physiol Lung Cell Mol Physiol
Bleomycin initiates apoptosis of lung epithelial cells by ROS but not by Fas/FasL pathway
Am J Physiol Lung Cell Mol Physiol
MEK inhibition enhances bleomycin A5-induced apoptosis in an oral cancer cell line: signaling mechanisms and therapeutic opportunities
J Oral Pathol Med
Induction of apoptosis by bleomycin in p53-null HL-60 leukemia cells
Int J Oncol
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These authors contributed equally to this work.