Cancer Letters

Cancer Letters

Volume 170, Issue 1, 10 September 2001, Pages 33-39
Cancer Letters

Carnosol-induced apoptosis and downregulation of Bcl-2 in B-lineage leukemia cells

https://doi.org/10.1016/S0304-3835(01)00549-3Get rights and content

Abstract

Carnosol, a phenolic compound extracted from the herb rosemary has been reported to have anti-cancer activity. We investigated whether carnosol was cytotoxic against several pro-B and pre-B acute lymphoblastic leukemia (ALL) lines. In all ALL lines tested, carnosol induced apoptotic cell death distinguished by loss of nuclear DNA, externalization of cell membrane phosphatidylserine, and depolarization of mitochondrial membranes. Flow cytometric measurement of Bcl-2 protein levels revealed that carnosol induced a 34–53% decrease in Bcl-2 in the cell population exhibiting a viable phenotype prior to detectable apoptotic changes in morphology. These results suggest that carnosol may be useful as a novel chemotherapeutic agent against B-lineage leukemias, and possibly other types of cancers that express high levels of the protective protein, Bcl-2.

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.

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