Proteomics reveals protein profile changes in doxorubicin – treated MCF-7 human breast cancer cells

doi:10.1016/S0304-3835(02)00025-3

Cancer Letters

Volume 181, Issue 1, 8 July 2002, Pages 95-107

https://doi.org/10.1016/S0304-3835(02)00025-3 Get rights and content

Abstract

MCF-7 cells are extensively used as a cell model to investigate human breast tumors and the cellular mechanism of antitumor drugs such as doxorubicin (DOX), an anthracycline antitumor drug widely used in clinical chemotherapy. To understand the effects of DOX on the protein expression, we perform a comprehensive proteomics to survey global changes in proteins after DOX treatment in MCF-7 cells. Exposure of MCF-7 cells to 0.1 μM DOX for 2 days induced a differentiation-like phenotype with prominent perinuclear autocatalytic vacuoles, abundant filamentous material, and irregular microvilli at the cell surface. In this study, we also present a proteome reference map of MCF-7 cells with 21 identified protein spots via analysis of N-terminal sequencing, mass spectrometry, immunoblot and/or computer matching with protein database. Based on the proteome map, we found that DOX causes a markedly decrease in the levels of three isoforms of heat shock protein 27 (HSP27) whereas the levels of other stress associated proteins including HSP60, calreticulin, and protein disulfide isomerase were not significantly altered in DOX-treated MCF-7 cells. Taken together, we suggest that that action of DOX on breast tumor cells may be partly related to dysregulation of HSP27 expression. Modulation of HSP27 levels may be a clinically useful potential target for design of antitumor drugs and controlling breast tumor growth.

Introduction

Doxorubicin (DOX) remains an important part of chemotherapy regimens in the clinic and is considered to be the most effective agent in treatment of advanced breast cancer [1]. Understanding the cellular mechanism of DOX is a key step in changing its cytotoxicity and in developing new strategies for breast cancer treatment. Previous studies indicated the effect of DOX on tumor cells was mediated through multiple mechanisms [2]. These include intercalation between DNA bases [3], interference with DNA unwinding and topoisomerase II [4], generation of reactive oxygen species [5], and alteration of membrane structure [6].

DOX had also been reported to induce distinct cell fates including growth arrest, differentiation, and apoptosis in various tumor cell lines [7], [8]. Although screening of DOX-induced gene expression has been described by using DNA microarray technique [9], [10], little is known about the associated effects on the expression of protein molecules. In order to tackle this question we carried out a comprehensive proteome analysis, this allowing us to compare to simultaneously visualize more than seven hundred proteins present in MCF-7 cells and to detect the alterations of two-dimensional (2D) protein patterns caused by DOX exposure in MCF-7 cells. Twenty-one protein spots were identified using N-terminal sequencing, mass spectrometry, immunoblot and/or computer matching with protein database. Three of the 21 protein spots were downregulated by DOX treatment and were identified by immunoblotting as corresponding to isoforms of heat shock protein 27 (HSP27). By contrast, other stress associated proteins, namely HSP60, calreticulin, and protein disulfide isomerase remained unaffected in DOX-treated MCF-7 cells. These results imply that the mechanism of the action of DOX on tumor cells may be partly related to dysregulation of HSP27 expression.

Section snippets

Materials

MCF-7 cells (CRL 1721) were bought from the American Type Culture Collection (Rockville, MD, USA). Dulbecco's modified Eagle's medium (DMEM), and horse serum were the products of GIBCO (Grand Island, NY, USA).

Amido Black (Naphthol Blue Black, C.I. 20470), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), dithiothreitol (DTT), and doxorubicin were from Sigma (St. Louis, MO, USA), Antisera were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were

Effect of DOX on MCF-7 cell morphology

As shown in Fig. 1, incubation of DOX at concentrations from 0.01 to 10 μM for 2 days causes an anti-proliferative effect with IC₅₀ from 0.9 to 0.1 μM in MCF-7 cells cultured DMEM supplemented with 1% of bovine fetal serum. MCF-7 cells treated with 0.1 μM DOX for 2 days showed a significant cell aggregation and formed short processes (Fig. 2, 1A). As ultrastructural analysis via transmission electron microscopy, DOX-treated MCF-7 cells exhibited prominent perinuclear autocatalytic vacuoles,

Discussion

In agreement with previous findings [21], continuous exposure to a sublethal concentration of DOX induces MCF-7 cell morphological alterations consistent with the induction of differentiation. However, in contrast to some earlier results that illustrated DOX induces apoptosis in many tumor cells [10], [31], [32]. After DOX (concentrations from 0.01 to 10 μM) treatment throughout the culture period for 4 days, no apoptotic MCF-7 cells have been found by observing electron microscopy (Fig. 2) and

Acknowledgements

This study was supported by a grant from the National Science Council, Taiwan. We would like to thank Ms Pei-Wei Chu and Ms Ya-Hui Huang for their excellent skills in carrying out the 2D PAGE.

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Proteomics reveals protein profile changes in doxorubicin – treated MCF-7 human breast cancer cells

Abstract

Introduction

Section snippets

Materials

Effect of DOX on MCF-7 cell morphology

Discussion

Acknowledgements

Eur. J. Cancer

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cancer Lett.

Anal. Biochem.

Mol. Brain Res.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

Blood

Semin. Cancer Biol.

Biochem. Pharmacol.

FEBS Lett.

Chem. Biol.

Cell Biol. Int.

J. Biol. Chem.

Cancer Lett.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Evolving concepts in the systemic adjuvant treatment of breast cancer

Cancer Res.

Resistance to adriamycin: relationship of cytotoxicity to drug uptake and DNA single- and double-strand breakage in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia

Cancer Res.

Induction of differentiation and growth arrest associated with nascent (non-oligosomal) DNA fragmentation and reduced c-myc expression in MCF-7 human breast tumor cells after continuous exposure to a sublethal concentration of doxorubicin

Cell Growth Differ.

Prostaglandin E2 enhances human endometrial stromal cell differentiation

Endocrinology

Monitoring the expression profiles of doxorubicin-induced and doxorubicin-resistant cancer cells by cDNA microarray

Cancer Res.