Carnosol-induced apoptosis and downregulation of Bcl-2 in B-lineage leukemia cells
Introduction
B-lineage acute lymphoblastic leukemia (ALL) is a disease that is prevalent in infants and in early childhood [1]. The chromosomal translocation t(4;11)(q21;q23) is frequently involved in childhood ALL and is present in greater than 60% of infants, 2% of children, and 3–6% of adults with ALL [1]. The t(4;11) ALLs, often classified as pro-B cells, display a mixed-lineage phenotype with both B and myeloid cell surface markers, suggesting that these leukemias may be generated from pluripotent hematopoietic progenitors that can potentially differentiate into lymphoid or myeloid cells. This high-risk subgroup of ALL is highly resistant to conventional chemotherapeutics and has an exceedingly poor prognosis.
Carnosol, a phenolic antioxidant extracted from the herb rosemary, exhibits anti-cancer activity in animal models for both breast and skin tumorigenesis [2], [3]. In this report, we determined whether carnosol was cytotoxic to several B-lineage ALL-derived cell lines, including three t(4;11) lines established from patients with ALL carrying the t(4;11)(q21;q23) chromosomal translocation. We show that carnosol induced apoptosis accompanied by a disruption of the mitochondrial membrane potential. Further analysis revealed that carnosol treatment downregulated the anti-apoptotic protein, Bcl-2, in each of the leukemia lines and this reduction of Bcl-2 may contribute to the apoptotic effects of carnosol.
Section snippets
Cell culture and reagents
SEM, RS4;11, and MV4;11 lines were established from patients diagnosed with ALL containing the chromosomal translocation t(4;11)(q21;q23) [4], [5], [6]. The three t(4;11) ALL lines and the pre-B acute leukemia lines, REH and Nalm-6, were maintained at 37°C, 5% CO2 in RPMI 1640 medium (Life Technologies, Inc., Karlsruhe, Germany) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 1 mM sodium pyruvate, and 2 mM L-glutamine (Life
Carnosol induces apoptotic cell death in the acute leukemia lines
Dose response analyses were performed with the three t(4;11) ALL lines, SEM, RS4;11, and MV4;11, and the two pre-B leukemia lines, REH and Nalm-6, to determine the cytotoxicity of carnosol. The cells were treated once with DMSO (untreated) or 3, 6 and 9 μg/ml carnosol (approximately 9, 18 and 27 μM, respectively). PBMCs were isolated from healthy volunteers and also treated with the above doses of carnosol. The fraction of non-viable cells (percentage cell death) was assessed after a period of
Discussion
The Bcl-2 family consists of anti-apoptotic and pro-apoptotic proteins that interact with each other to regulate the survival or death of the cell [15]. The Bcl-2 gene was originally cloned from the breakpoint of a t(14;18) translocation in B cell lymphomas [15]. This translocation placed the Bcl-2 gene under the control of the immunoglobulin enhancer, resulting in increased Bcl-2 expression and protection against cell death. Bcl-2 is found in the membranes of mitochondria, the endoplasmic
Acknowledgements
This work was supported by grants from the Wilhelm Sander-Stiftung and the Madeleine Schickedanz Kinderkrebs-Stiftung.
References (18)
- et al.
Clinical significance of cytogenetic abnormalities in adult acute lymphoblastic leukemia
Blood
(1998) - et al.
Inhibition by rosemary and carnosol of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumorigenesis and in vivo DMBA–DNA adduct formation
Cancer Lett.
(1996) - et al.
Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics
Blood
(1985) - et al.
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry
J. Immunol. Methods
(1991) - et al.
Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry
Biochem. Biophys. Acta
(1992) - et al.
A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V
J. Immunol. Methods
(1995) - et al.
Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis
J. Biol. Chem.
(1999) - et al.
Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level
Exp. Cell Res.
(1994) - et al.
Clinical relevance of Bcl-2 overexpression in childhood acute lymphoblastic leukemia
Blood
(1996)