Cell surface P-glycoprotein (ABCB1) is degraded by lysosomal/autophagic pathway

Wednesday, October 10, 2012 — Poster Session II
Noon – 2:00 p.m	Natcher Conference Center, Building 45	NCI	CELLBIO-5

Authors

• K. Kapoor
• K. Katayama
• S. Ohnuma
• S.V. Ambudkar

Abstract

Multidrug resistance mediated by P-glycoprotein (P-gp), is believed to be one of the major causes of failure of chemotherapy. Over-expression of P-gp confers resistance to a variety of structurally and functionally diverse anticancer drugs. The mechanism of internalization and degradation of cell surface P-gp is not well understood. The present study is aimed to understand the pathway of P-gp degradation in colon cancer cell line HCT-15 and doxorubicin resistant HCT-15 cell line (HCT-DOX) to compare the degradation pathways in drug-sensitive and resistant cells. We analyzed the disappearance of P-gp by biotinylating cell surface proteins at 4°C for 1 hr and then measuring the loss of biotinylated P-gp with time by flow cytometry and Western blotting. The half-life of cell surface P-gp was determined to be 25-27 hrs in HCT-15 cells. A lysosomal proton pump inhibitor bafilomycin A1 (BafA1, 1 nM) prolonged the half-life to 33-36 hrs. However, treatment with proteasomal inhibitor MG132 did not affect the half life. Immunodetection showed the co-localization of internalized P-gp with lysosomal marker LAMP1 demonstrating that P-gp is likely degraded by lysosomal/autophagic pathway. Similar studies are being carried out with HCT-DOX cell line to determine pathways involved in degradation of P-gp in drug selected cells.

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