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.
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.


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 microM DOX for 72 h induced 8, 14 and 28% apoptosis, respectively. However, only 1 microM DOX was able to trigger about 8% apoptosis in MCF- 7/Adr-20 cells. Moreover, apoptosis triggered by 0.01 and 0.1 microM DOX in MCF-7 cells was mainly caspase-dependent, but at 1 microM about 70% of apoptosis was caspase-dependent. Western blot analysis revealed that caspase-7 was activated at 0.1 and 1 microM DOX treatment and caspases-6, -8, -9 and 10 were only activated at 1 microM 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 microM induced p53 and p21(WAF-1/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.
Park
Soo-Jung
SJ
Department of Pharmacology and Toxicology and Indiana University Cancer Center, 1044 West Walnut R4-119, Indianapolis, IN 46202, USA.
Wu
Ching-Haung
CH
Safa
Ahmad R
AR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D007527:Isoenzymes; D012333:RNA, Messenger; D004317:Doxorubicin; D020169:Caspases