Abstract
BackgroundA major problem with the use of current chemotherapy regimens for several cancers, including breast cancer, is development of intrinsic or acquired drug resistance, which results in disease recurrence and metastasis. However, the mechanisms underlying this drug resistance are unknown. To study the molecular mechanisms underlying the invasive and metastatic activities of drug-resistant cancer cells, we generated a doxorubicin-resistant MCF-7 breast cancer cell line (MCF-7/DOX).MethodsWe used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, DNA fragmentation assays, Western blot analysis, cell invasion assays, small interfering RNA (siRNA) transfection, reverse transcription-polymerase chain reaction, experimental lung metastasis models, and gelatin and fibrinogen/plasminogen zymography to study the molecular mechanism of metastatic activities in MCF-7/DOX cells.ResultsWe found that MCF-7/DOX acquired invasive activities. In addition, Western blot analysis showed increased expression of epidermal growth factor receptor (EGFR) and Cox-2 in MCF-7/DOX cells. Inhibition of Cox-2, phosphoinositide 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase (MAPK) pathways effectively inhibited the invasive activities of MCF-7/DOX cells. Gelatin and fibrinogen/plasminogen zymography analysis showed that the enzymatic activities of matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator were markedly higher in MCF-7/DOX cells than in the MCF-7 cells. In vitro invasion assays and mouse models of lung metastasis demonstrated that MCF-7/DOX cells acquired invasive abilities. Using siRNAs and agonists specific for prostaglandin E (EP) receptors, we found that EP1 and EP3 played important roles in the invasiveness of MCF-7/DOX cells.ConclusionsWe found that the invasive activity of MCF-7/DOX cells is mediated by Cox-2, which is induced by the EGFR-activated PI3K/Akt and MAPK pathways. In addition, EP1 and EP3 are important in the Cox-2-induced invasion of MCF-7/DOX cells. Therefore, not only Cox-2 but also EP1 and EP3 could be important targets for chemosensitization and inhibition of metastasis in breast cancers that are resistant to chemotherapy.
Highlights
A major problem with the use of current chemotherapy regimens for several cancers, including breast cancer, is development of intrinsic or acquired drug resistance, which results in disease recurrence and metastasis
To confirm the role of the phosphoinositide 3-kinase (PI3K)/ Akt and mitogen-activated protein kinase (MAPK) pathways in Cox-2 expression, we studied the effect of the PI3K/Akt inhibitor LY294002 and the MAPK inhibitor U0126 on Epidermal growth factor (EGF)-induced expression of pAkt and Cox-2 in MCF-7/DOX cells
We demonstrated that invasiveness of MCF-7/DOX cells resulted from Cox-2 activation, which was induced by either the epidermal growth factor receptor (EGFR)-activated PI3K/Akt or MAPK pathway
Summary
A major problem with the use of current chemotherapy regimens for several cancers, including breast cancer, is development of intrinsic or acquired drug resistance, which results in disease recurrence and metastasis. To study the molecular mechanisms underlying the invasive and metastatic activities of drug-resistant cancer cells, we generated a doxorubicin-resistant MCF-7 breast cancer cell line (MCF-7/DOX). A major problem with the use of chemotherapy to treat many cancers (including breast cancer) is intrinsic or acquired drug resistance, which results in disease recurrence and metastasis. Recent results from several laboratories have investigated the mechanism by which breast cancer cells become resistant to doxorubicin, as well as the molecular profile of breast cancer cells that are resistant to doxorubicin [3,4]. Recent evidence has suggested that the ability of tumor cells to acquire an aggressive phenotype may result from accumulation of genetic alterations conferred by extended survival [7,8]
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