Comparison of idarubicin and daunorubicin regarding intracellular uptake, induction of apoptosis, and resistance

doi:10.1016/S0304-3835(01)00824-2

Cancer Letters

Volume 178, Issue 2, 25 April 2002, Pages 141-149

https://doi.org/10.1016/S0304-3835(01)00824-2 Get rights and content

Abstract

Anthracycline antibiotics are widely used as anticancer agents. Idarubicin (4-demethoxydaunorubicin; Ida), a semisynthetic derivative of daunorubicin (Dnr) is more potent than the parent compound in vitro and in vivo. The equitoxic dose of Ida in patients is about one-fourth of that of Dnr. We compared these drugs regarding cytotoxicity, apoptosis induction, and resistance mechanisms in human leukaemic cell lines. Cytotoxicity was studied by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and drug-induced apoptosis by means of the Annexin V–fluorescein isothiocyanate method at similar intracellular concentrations (extracellular concentrations of 0.35 μM for Ida and 1 μM for Dnr). Ida was at least twice as potent as Dnr in MOLT-4, HL60, CEM, and K562 cell lines. It took 8 h for Ida to induce approximately 20% apoptosis, but at least 22 h for Dnr to reach 20% apoptosis at identical intracellular concentration. Ida induces a faster and higher apoptosis rate compared with Dnr. The human chronic myelogenous leukaemia cell line (K562) was selected for resistance to Dnr and Ida with and without verapamil (Ver). Continuous incubation with Dnr, but not with Ida, led to an increased mdr1 gene expression as assessed by real-time PCR. The development of mdr1 gene expression in Dnr-resistant cells could be reversed by the presence of Ver. Ver also reversed the cytotoxicity to Dnr, but not to Ida, in K562/Dnr cells. The results show that Ida is more effective than Dnr in inducing apoptosis and that there are differences in resistance mechanisms between the drugs.

Introduction

Anthracyclines are important components of many combination chemotherapy regimens [1]. They show activity against solid tumours as well as haematological malignancies. It has been reported that the cytotoxic action of anthracyclines is dependent on cellular uptake and interferences with DNA, enzymes like DNA topoisomerases, metal ions, molecular oxygen in the tumour cell, and with cell membranes [2]. The contribution of these mechanisms to tumour cell death remains to be clarified.

In the present study, two anthracyclines widely used in cancer therapy were compared in vitro: idarubicin (Ida) and daunorubicin (Dnr). It has been demonstrated that anthracycline permeation increases with increasing lipophilicity [3]. Ida lacks a methoxy group in position 4 of the Dnr chromophore ring system and is more lipophilic, judging from its higher octanol/buffer concentration ratio. Ida has been reported to be more effective both in vitro and in vivo (better remission rates and overall survival in acute myeloid leukaemia) probably due to higher intracellular uptake than the first-generation anthracycline Dnr [4], [5], [6].

Anthracyclines and several different classes of cytostatic drugs can induce the multidrug resistance (MDR) phenotype. MDR cells have reduced intracellular drug concentrations frequently associated with overexpression of an ATP-binding cassette (ABC) family of transporter proteins, P-glycoprotein (Pgp), which is encoded by the mdr1 gene [7] in human cells and is the most widely studied type of cellular drug resistance mechanism. Recent studies have shown that the Pgp-mediated efflux of different anthracycline-based drugs may not differ considerably. Therefore, if the diffusion rate of an anthracycline is high enough, as in the case of Ida due to its higher lipophilicity, the cytotoxicity in Pgp-positive cells could be enhanced [8], [9]. Chemosensitizers like verapamil (Ver) have shown promise in reversing Pgp, but responses have been transient, suggesting that tumour cells become resistant to the reversing effects of chemosensitizers or that other resistance mechanisms may develop. In an attempt to clarify the differences in Pgp-related resistance mechanisms between Ida and Dnr, we selected five cell lines for resistance to Ida or Dnr with and without Ver as a chemosensitizer with stepwise increasing concentrations of the drug in cultures of the human chronic myelogenous leukaemia cell line (K562/wt).

Many haematopoietic cell types, as well as leukaemia and lymphoma cell lines, have been found to undergo apoptosis in response to a range of stimuli, such as exposure to different antineoplastic drugs [10], [11]. Apoptosis can occur at therapeutic concentrations of anthracyclines [12], [13]. Therefore, the ability to induce apoptosis at identical intracellular concentrations of Ida and Dnr was compared, as well as drug cytotoxicity and the development of resistance.

Section snippets

Drugs, chemicals, and reagents

Dnr (Cerubidin^®) was purchased from Rhône-Poulenc Rorer (Bristol, UK) and Ida (Zavedos^®) from Pharmacia & Upjohn (Stockholm, Sweden). Ver was from NM Pharma (Stockholm, Sweden). RPMI 1640 medium, heat-inactivated foetal calf serum, l-glutamine and penicillin–streptomycin were all purchased from Gibco (Life Technologies, Paisley, UK). Other compounds were of analytical grade and purchased from Sigma Chemical Company (St. Louis, MO).

Cell culture and treatments

Human acute T lymphoblastic leukaemia (CEM), promyelocytic

In vitro drug chemosensitivity

Human leukaemia cell lines growing in suspension cultures were incubated with different concentrations of Ida or Dnr. The antiproliferative activities of Ida and Dnr toward MOLT-4 (Fig. 1), CEM, K562, and HL60 cell lines were analyzed by MTT assays and described as IC₅₀ values (Table 1). In all lines, Ida was 2–3-fold more potent than Dnr. The antiproliferative activity of five resistant cell lines to Ida or Dnr was studied with and without Ver (Table 2). Ver increased the cytotoxic activity of

Discussion

In view of increasing evidence that anthracyclines can trigger apoptosis and the discrepancy between drug accumulation and the cellular toxicity of anthracyclines in multidrug-resistant cells [18], we decided to compare the occurrence, duration, and frequency of apoptosis induced by Ida and Dnr. We also developed five cell lines resistant to Ida or Dnr with and without Ver to study differences in resistance mechanisms. Here, we show that continuous incubation with Dnr led to an increased

Acknowledgements

This study was supported by grants from the Swedish Children Cancer Foundation and the Swedish Cancer Society.

References (44)

P.H. Wiernik et al.
Cytarabine plus idarubicin or daunorubicin as induction and consolidation therapy for previously untreated adult patients with acute myeloid leukemia
Blood
(1992)
C. Marbeuf-Gueye et al.
Correlation between the kinetics of anthracycline uptake and the resistance factor in cancer cells expressing the multidrug resistance protein or the P-glycoprotein
Biochim. Biophys. Acta
(1999)
Y.A. Hannun
Apoptosis and the dilemma of cancer chemotherapy
Blood
(1997)
P.W. Mesner et al.
Chemotherapy-induced apoptosis
Adv. Pharmacol.
(1997)
A. Skladanowski et al.
Adriamycin and daunomycin induce programmed cell death (apoptosis) in tumour cells
Biochem. Pharmacol.
(1993)
G. Koopman et al.
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis
Blood
(1994)
I. Vermes 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)
X.F. Hu et al.
Altered multidrug resistance phenotype caused by anthracycline analogues and cytosine arabinoside in myeloid leukemia
Blood
(1999)
M. Nakayama et al.
Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias
Blood
(1998)
K. Kohno et al.
The direct activation of human multidrug resistance gene (MDR1) by anticancer agents
Biochem. Biophys. Res. Commun.
(1989)

D.J. Roovers et al.

Idarubicin overcomes P-glycoprotein-related multidrug resistance: comparison with doxorubicin and daunorubicin in human multiple myeloma cell lines

Leuk. Res.

(1999)

K.M. Carlson et al.

Characterization of drug-resistant cell lines by comparative genomic hybridization

Cancer Genet. Cytogenet.

(1999)

J.P. Vial et al.

Study of the apoptosis induced in vitro by antitumoral drugs on leukaemic cells (See comments)

Leuk. Res.

(1997)

E. Berman et al.

Comparative cellular pharmacology of daunorubicin and idarubicin in human multidrug-resistant leukemia cells

Blood

(1992)

J.M. Boiron et al.

Flow cytometric study of idarubicin and daunorubicin accumulation and the effect of verapamil in leukemic cell lines and fresh cells from patients with acute non-lymphoblastic leukemia

Leuk. Res.

(1994)

R.B. Weiss et al.

Anthracycline analogs: the past, present, and future

Cancer Chemother. Pharmacol.

(1986)

J.W. Lown

Anthracycline and anthraquinone anticancer agents: current status and recent developments

Pharmacol. Ther.

(1993)

P.A. Speth et al.

Idarubicin v daunorubicin: preclinical and clinical pharmacokinetic studies

Semin. Oncol.

(1989)

A systematic collaborative overview of randomized trials comparing idarubicin with daunorubicin (or other anthracyclines) as induction therapy for acute myeloid leukaemia

Br. J. Haematol.

(1998)

W.R. Vogler et al.

A phase III trial comparing idarubicin and daunorubicin in combination with cytarabine in acute myelogenous leukemia: a Southeastern Cancer Study Group Study

J. Clin. Oncol.

(1992)

C.F. Higgins

ABC transporters from microorganisms to man

Annu. Rev. Cell Biol.

(1992)

U. Tidefelt et al.

Comparison of idarubicin and daunorubicin and their main metabolites regarding intracellular uptake and effect on sensitive and multidrug-resistant HL60 cells

Cancer Chemother. Pharmacol.

(1996)

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Comparison of idarubicin and daunorubicin regarding intracellular uptake, induction of apoptosis, and resistance

Abstract

Introduction

Section snippets

Drugs, chemicals, and reagents

Cell culture and treatments

In vitro drug chemosensitivity

Discussion

Acknowledgements

Blood

Biochim. Biophys. Acta

Blood

Adv. Pharmacol.

Biochem. Pharmacol.

Blood

J. Immunol. Methods

Blood

Blood

Biochem. Biophys. Res. Commun.

Leuk. Res.

Cancer Genet. Cytogenet.

Leuk. Res.

Blood

Leuk. Res.

Anthracycline analogs: the past, present, and future

Cancer Chemother. Pharmacol.

Anthracycline and anthraquinone anticancer agents: current status and recent developments

Pharmacol. Ther.

Idarubicin v daunorubicin: preclinical and clinical pharmacokinetic studies

Semin. Oncol.

A systematic collaborative overview of randomized trials comparing idarubicin with daunorubicin (or other anthracyclines) as induction therapy for acute myeloid leukaemia

Br. J. Haematol.

A phase III trial comparing idarubicin and daunorubicin in combination with cytarabine in acute myelogenous leukemia: a Southeastern Cancer Study Group Study

J. Clin. Oncol.

ABC transporters from microorganisms to man

Annu. Rev. Cell Biol.

Comparison of idarubicin and daunorubicin and their main metabolites regarding intracellular uptake and effect on sensitive and multidrug-resistant HL60 cells

Cancer Chemother. Pharmacol.