Gefitinib inhibition of drug resistance to doxorubicin by inactivating ABCG2 in thyroid cancer cell lines.
OBJECTIVE
To investigate the regulation of the breast cancer resistance protein ABCG2/BRCP1 drug transporter by epidermal growth factor receptor (EGFR) kinase activity, and to determine whether gefitinib, an EGFR small molecule inhibitor, will modulate the effects of doxorubicin hydrochloride by inhibiting its extrusion from thyroid cancer cells.


METHODS
Extrusion assays using flow cytometry analysis were used to determine the ability of thyroid cancer cells to extrude the chemotherapy drug, doxorubicin, via the ABCG2 drug transporter in the presence or absence of gefitinib. Immunofluorescence was employed to determine the cellular expression of ABCG2. The ABCG2 expression in ARO and WRO cell lines was analyzed by Western blot analysis. Inactivation of EGFR kinase by gefitinib was analyzed by Western blot analysis and immunofluorescence. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay was performed to demonstrate ABCG2-mediated apoptosis in the presence of doxorubicin. Colony formation assays were performed to determine the effect of gefitinib on thyroid cancer cell survival in response to gefitinib, doxorubicin, or the combination of both drugs.


RESULTS
Inhibition of EGFR kinase activity by gefitinib causes the translocation of the ABCG2 drug transporter away from the plasma membrane, resulting in a concomitant decrease in doxorubicin extrusion in thyroid cancer cell lines. Both ARO and WRO demonstrated differential ABCG2 expression, whereas both were sensitized to doxorubicin-induced apoptosis on ABCG2 knockdown with short interfering RNA. The addition of gefitinib increases doxorubicin-induced cell death in thyroid cancer cells as measured by colony formation assay.


CONCLUSIONS
Epidermal growth factor receptor regulates the function of the drug transporter ABCG2/BCRP1 and correlates with ABCG2 protein expression levels. Inactivation of the EGFR kinase by gefitinib potentiates the cytotoxic effect of doxorubicin in thyroid cancer, most likely by decreasing the ability of the cell to extrude doxorubicin. The expression of ABCG2 may explain in part the ineffectiveness of doxorubicin as a single modality treatment for anaplastic thyroid cancer or for treatment of metastatic follicular thyroid cancer. Use of this combination treatment of gefitinib and doxorubicin may be a promising therapy for anaplastic thyroid and metastatic follicular thyroid cancer and needs to be investigated further.
Lopez
Jay Patrick
JP
Department of Surgery, VA San Diego Healthcare System, and Department of Radiology, University of California, San Diego, Biomedical Sciences Bldg, Room 1202, Mail Code 0612, 9500 Gilman Dr, La Jolla, CA 92093-0612, USA.
Wang-Rodriguez
Jessica
J
Chang
Catherine
C
Chen
Jocelyn S
JS
Pardo
Francisco S
FS
Aguilera
Joseph
J
Ongkeko
Weg M
WM
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000970:Antineoplastic Agents / Q000627:therapeutic use; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003470:Culture Media; D004317:Doxorubicin / Q000008:administration & dosage; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020287:In Situ Nick-End Labeling; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011799:Quinazolines / Q000008:administration & dosage; D012334:RNA, Neoplasm / Q000235:genetics*; D013964:Thyroid Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D003470:Culture Media; D009363:Neoplasm Proteins; D011799:Quinazolines; D012334:RNA, Neoplasm; D004317:Doxorubicin; D066246:ErbB Receptors; D000077156:Gefitinib