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Mechanisms of acquired resistance to 2-(4-Amino-3-methylphenyl)benzothiazole in breast cancer cell lines

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Abstract

Compounds within the 2-(4-aminophenyl)benzothiazole class represent extremely potent and selective experimental antitumour agents. The lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is undergoing phase I clinical evaluation. Extensive studies to elucidate mechanisms underlying the stark selectivity demonstrated potent cytosolic AhR ligand binding and cytochrome P450 1A1-catalysed bioactivation. Two human derived breast cell lines, initially exquisitely sensitive to this class of agent (GI50 < 5 nM) have been derived displaying acquired resistance to 2-(4-amino-3-methylphenyl)benzothiazole (DF 203; GI50 > 50 μM). Cross resistance to 2-(4-amino-3-iodophenyl)benzothiazole and 2-(4-amino-3-cyanophenyl)benzothiazole is observed (GI50 > 30 μM) as is > 100-fold reduced sensitivity of the two variant lines to 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). In contrast, cell lines possessing acquired resistance to DF 203 (203R) retain sensitivity to benzo[a]pyrene and doxorubicin. Examination of DF 203-treated cells by confocal microscopy and HPLC analyses of nutrient media concur revealing diminished depletion of DF 203 from medium and impaired intracellular DF 203 retention. In contrast to cytosolic arylhydrocarbon (AhR) receptors of wild type cells, AhR appears constitutively localised within nuclei of 203R cells; consequently, DF 203 fails to drive transcription of cyp1a1. DF 203- and 5F 203-derived DNA adducts fall significantly in 203R cells. Reduced number and intensity of γH2AX foci report protection against DF 203-evoked DNA double strand breaks. In conclusion, aberrant AhR signalling underlies at least in part acquired resistance to DF 203. Intriguingly, comparisons of gene transcription profiles between sensitive and resistant paired lines reveal > 5-fold up-regulation of cyp1b1 expression, a protein implicated in resistance to therapeutic agents.

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Abbreviations

Ab:

Antibody

ADPRT:

ADP ribosyl transferase

AhR:

Arylhydrocarbon receptor

B[a]P:

Benzo[a]pyrene

cy:

Cyanine

CYP1A1:

Cytochrome P450 1A1

CYP1B1:

Cytochrome P450 1B1

DF 129:

2-(4-amino-3-iodophenyl)benzothiazole

DF 203:

2-(4-amino-3-methylphenyl)benzothiazole

DMSO:

Dimethylsulphoxide

DSB:

Double strand break

ER:

Oestrogen receptor

5F 203:

2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole

GI50 :

Agent concentration which inhibits growth by 50%

γH2AX:

Serine 139 phosphorylation of histone H2AX

HPLC:

High performance liquid chromatography

IH 191:

2-(4-amino-3-cyanophenyl)benzothiazole

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS:

Phosphate-buffered saline

PD:

Pharmacodynamic

203R:

DF 203-resistance

Rt:

Retention time

TCDD:

2,3,7,8-tetrachlorodibenzo-p-dioxin

TK:

Thymidine kinase

wt:

Wild type

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Acknowledgement

Authors would like to thanks Professor Paul Workman for collaboration.

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Authors and Affiliations

  1. Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, UK

    Tracey D. Bradshaw, Erica L. Stone, Valentina Trapani, Chee-Onn Leong, Charles S. Matthews & Malcolm F. G. Stevens

  2. Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, UK

    Robert te Poele

Authors
  1. Tracey D. Bradshaw
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  2. Erica L. Stone
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  3. Valentina Trapani
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  4. Chee-Onn Leong
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  5. Charles S. Matthews
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  6. Robert te Poele
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  7. Malcolm F. G. Stevens
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Correspondence to Tracey D. Bradshaw.

Additional information

Part 28 in the “Antitumour Benzothiazoles” series.

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Bradshaw, T.D., Stone, E.L., Trapani, V. et al. Mechanisms of acquired resistance to 2-(4-Amino-3-methylphenyl)benzothiazole in breast cancer cell lines. Breast Cancer Res Treat 110, 57–68 (2008). https://doi.org/10.1007/s10549-007-9690-9

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  • DOI: https://doi.org/10.1007/s10549-007-9690-9

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