A P-glycoprotein- and MRP1-independent Doxorubicin-resistant Variant of the MCF-7 Breast Cancer Cell Line with Defects in Caspase-6, -7, -8, -9 and -10 Activation Pathways

Soo Jung Park, Ching Haung Wu, Ahmad Safa

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Background: Several mechanisms are known to cause resistance to chemotherapy in cancer cells, but the mechanisms of drug resistance due to a lack of apoptosis are not well elucidated. Materials and Methods: To understand the mechanisms of resistance to apoptosis induced by doxorubicin (DOX), we developed a DOX-resistant variant of MCF-7 referred to as MCF-7/Adr-20, measured growth inhibition by methylene blue cell survival assay, quantitated apoptosis by annexin V binding assay and detected activation of caspases-6, -7, -8, -9 and -10 in these cells. Results: The resistant cells expressed 20-fold resistance to apoptosis induced by DOX compared to MCF-7 cells. MCF-7/Adr-20 cells did not express MDR1 mRNA or its product P-glycoprotein and they did not overexpress MRP-1. Treating MCF-7 cells with 0.01, 0.1 and 1 μM DOX for 72 h induced 8, 14 and 28% apoptosis, respectively. However, only 1 μM DOX was able to trigger about 8% apoptosis in MCF-7/Adr-20 cells. Moreover, apoptosis triggered by 0.01 and 0.1 μM DOX in MCF-7 cells was mainly caspase-dependent, but at 1 μM about 70% of apoptosis was caspase-dependent. Western blot analysis revealed that caspase-7 was activated at 0.1 and 1 μM DOX treatment and caspases-6, -8, -9 and 10 were only activated at 1 μM DOX treatment in MCF-7 cells, but none of the caspases checked were activated in MCF-7/Adr-20 cells. Moreover, DOX at 0.01 and 0.1 μM induced p53 and p21 WAF1/CIP-1 to the same extent in both MCF-7 and MCF-7/Adr-20 cells. Therefore, while DOX triggers growth arrest and induces p53 and p21 WAF-1/CIP-1 in these cells, defects in activation of the initiator and executioner caspases play a major role in resistance to apoptosis triggered by DOX.

Original languageEnglish
Pages (from-to)123-131
Number of pages9
JournalAnticancer Research
Volume24
Issue number1
StatePublished - Jan 2004

Fingerprint

Caspase 6
Caspase 7
P-Glycoprotein
Doxorubicin
Breast Neoplasms
Cell Line
Apoptosis
MCF-7 Cells
Caspases
Caspase 10
Initiator Caspases
Caspase 8
Annexin A5
Methylene Blue
Growth
Drug Resistance
Cell Survival

Keywords

  • Apoptosis
  • Caspases
  • Cytochrome c
  • Death receptors
  • Leukemia
  • Mitochondria
  • Taxol

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

@article{4759b5c7b0d14ee7a0af3e43c7b490cd,
title = "A P-glycoprotein- and MRP1-independent Doxorubicin-resistant Variant of the MCF-7 Breast Cancer Cell Line with Defects in Caspase-6, -7, -8, -9 and -10 Activation Pathways",
abstract = "Background: Several mechanisms are known to cause resistance to chemotherapy in cancer cells, but the mechanisms of drug resistance due to a lack of apoptosis are not well elucidated. Materials and Methods: To understand the mechanisms of resistance to apoptosis induced by doxorubicin (DOX), we developed a DOX-resistant variant of MCF-7 referred to as MCF-7/Adr-20, measured growth inhibition by methylene blue cell survival assay, quantitated apoptosis by annexin V binding assay and detected activation of caspases-6, -7, -8, -9 and -10 in these cells. Results: The resistant cells expressed 20-fold resistance to apoptosis induced by DOX compared to MCF-7 cells. MCF-7/Adr-20 cells did not express MDR1 mRNA or its product P-glycoprotein and they did not overexpress MRP-1. Treating MCF-7 cells with 0.01, 0.1 and 1 μM DOX for 72 h induced 8, 14 and 28{\%} apoptosis, respectively. However, only 1 μM DOX was able to trigger about 8{\%} apoptosis in MCF-7/Adr-20 cells. Moreover, apoptosis triggered by 0.01 and 0.1 μM DOX in MCF-7 cells was mainly caspase-dependent, but at 1 μM about 70{\%} of apoptosis was caspase-dependent. Western blot analysis revealed that caspase-7 was activated at 0.1 and 1 μM DOX treatment and caspases-6, -8, -9 and 10 were only activated at 1 μM DOX treatment in MCF-7 cells, but none of the caspases checked were activated in MCF-7/Adr-20 cells. Moreover, DOX at 0.01 and 0.1 μM induced p53 and p21 WAF1/CIP-1 to the same extent in both MCF-7 and MCF-7/Adr-20 cells. Therefore, while DOX triggers growth arrest and induces p53 and p21 WAF-1/CIP-1 in these cells, defects in activation of the initiator and executioner caspases play a major role in resistance to apoptosis triggered by DOX.",
keywords = "Apoptosis, Caspases, Cytochrome c, Death receptors, Leukemia, Mitochondria, Taxol",
author = "Park, {Soo Jung} and Wu, {Ching Haung} and Ahmad Safa",
year = "2004",
month = "1",
language = "English",
volume = "24",
pages = "123--131",
journal = "Anticancer Research",
issn = "0250-7005",
publisher = "International Institute of Anticancer Research",
number = "1",

}

TY - JOUR

T1 - A P-glycoprotein- and MRP1-independent Doxorubicin-resistant Variant of the MCF-7 Breast Cancer Cell Line with Defects in Caspase-6, -7, -8, -9 and -10 Activation Pathways

AU - Park, Soo Jung

AU - Wu, Ching Haung

AU - Safa, Ahmad

PY - 2004/1

Y1 - 2004/1

N2 - Background: Several mechanisms are known to cause resistance to chemotherapy in cancer cells, but the mechanisms of drug resistance due to a lack of apoptosis are not well elucidated. Materials and Methods: To understand the mechanisms of resistance to apoptosis induced by doxorubicin (DOX), we developed a DOX-resistant variant of MCF-7 referred to as MCF-7/Adr-20, measured growth inhibition by methylene blue cell survival assay, quantitated apoptosis by annexin V binding assay and detected activation of caspases-6, -7, -8, -9 and -10 in these cells. Results: The resistant cells expressed 20-fold resistance to apoptosis induced by DOX compared to MCF-7 cells. MCF-7/Adr-20 cells did not express MDR1 mRNA or its product P-glycoprotein and they did not overexpress MRP-1. Treating MCF-7 cells with 0.01, 0.1 and 1 μM DOX for 72 h induced 8, 14 and 28% apoptosis, respectively. However, only 1 μM DOX was able to trigger about 8% apoptosis in MCF-7/Adr-20 cells. Moreover, apoptosis triggered by 0.01 and 0.1 μM DOX in MCF-7 cells was mainly caspase-dependent, but at 1 μM about 70% of apoptosis was caspase-dependent. Western blot analysis revealed that caspase-7 was activated at 0.1 and 1 μM DOX treatment and caspases-6, -8, -9 and 10 were only activated at 1 μM DOX treatment in MCF-7 cells, but none of the caspases checked were activated in MCF-7/Adr-20 cells. Moreover, DOX at 0.01 and 0.1 μM induced p53 and p21 WAF1/CIP-1 to the same extent in both MCF-7 and MCF-7/Adr-20 cells. Therefore, while DOX triggers growth arrest and induces p53 and p21 WAF-1/CIP-1 in these cells, defects in activation of the initiator and executioner caspases play a major role in resistance to apoptosis triggered by DOX.

AB - Background: Several mechanisms are known to cause resistance to chemotherapy in cancer cells, but the mechanisms of drug resistance due to a lack of apoptosis are not well elucidated. Materials and Methods: To understand the mechanisms of resistance to apoptosis induced by doxorubicin (DOX), we developed a DOX-resistant variant of MCF-7 referred to as MCF-7/Adr-20, measured growth inhibition by methylene blue cell survival assay, quantitated apoptosis by annexin V binding assay and detected activation of caspases-6, -7, -8, -9 and -10 in these cells. Results: The resistant cells expressed 20-fold resistance to apoptosis induced by DOX compared to MCF-7 cells. MCF-7/Adr-20 cells did not express MDR1 mRNA or its product P-glycoprotein and they did not overexpress MRP-1. Treating MCF-7 cells with 0.01, 0.1 and 1 μM DOX for 72 h induced 8, 14 and 28% apoptosis, respectively. However, only 1 μM DOX was able to trigger about 8% apoptosis in MCF-7/Adr-20 cells. Moreover, apoptosis triggered by 0.01 and 0.1 μM DOX in MCF-7 cells was mainly caspase-dependent, but at 1 μM about 70% of apoptosis was caspase-dependent. Western blot analysis revealed that caspase-7 was activated at 0.1 and 1 μM DOX treatment and caspases-6, -8, -9 and 10 were only activated at 1 μM DOX treatment in MCF-7 cells, but none of the caspases checked were activated in MCF-7/Adr-20 cells. Moreover, DOX at 0.01 and 0.1 μM induced p53 and p21 WAF1/CIP-1 to the same extent in both MCF-7 and MCF-7/Adr-20 cells. Therefore, while DOX triggers growth arrest and induces p53 and p21 WAF-1/CIP-1 in these cells, defects in activation of the initiator and executioner caspases play a major role in resistance to apoptosis triggered by DOX.

KW - Apoptosis

KW - Caspases

KW - Cytochrome c

KW - Death receptors

KW - Leukemia

KW - Mitochondria

KW - Taxol

UR - http://www.scopus.com/inward/record.url?scp=1442326136&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1442326136&partnerID=8YFLogxK

M3 - Article

VL - 24

SP - 123

EP - 131

JO - Anticancer Research

JF - Anticancer Research

SN - 0250-7005

IS - 1

ER -