Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway

Kazuhiro Katayama, Khyati Kapoor, Shinobu Ohnuma, Atish Patel, William Swaim, Indu S. Ambudkar, Suresh V. Ambudkar

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

Original languageEnglish
Pages (from-to)2361-2370
Number of pages10
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1853
Issue number10
DOIs
Publication statusPublished - 2015 Oct 1
Externally publishedYes

Fingerprint

P-Glycoprotein
Half-Life
Pharmaceutical Preparations
Vacuolar Proton-Translocating ATPases
Biotinylation
Rhodamine 123
Antibodies
Fluorescent Antibody Technique
Colorectal Neoplasms
Flow Cytometry
Membrane Proteins
Therapeutics
Western Blotting

Keywords

  • Degradation
  • Endosome
  • Half-life
  • Lysosome
  • P-glycoprotein
  • Proteasome

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Revealing the fate of cell surface human P-glycoprotein (ABCB1) : The lysosomal degradation pathway. / Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S.; Ambudkar, Suresh V.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1853, No. 10, 01.10.2015, p. 2361-2370.

Research output: Contribution to journalArticle

Katayama, Kazuhiro ; Kapoor, Khyati ; Ohnuma, Shinobu ; Patel, Atish ; Swaim, William ; Ambudkar, Indu S. ; Ambudkar, Suresh V. / Revealing the fate of cell surface human P-glycoprotein (ABCB1) : The lysosomal degradation pathway. In: Biochimica et Biophysica Acta - Molecular Cell Research. 2015 ; Vol. 1853, No. 10. pp. 2361-2370.
@article{bb7e4816a31b4ca3984b5914a7045650,
title = "Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway",
abstract = "P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.",
keywords = "Degradation, Endosome, Half-life, Lysosome, P-glycoprotein, Proteasome",
author = "Kazuhiro Katayama and Khyati Kapoor and Shinobu Ohnuma and Atish Patel and William Swaim and Ambudkar, {Indu S.} and Ambudkar, {Suresh V.}",
year = "2015",
month = "10",
day = "1",
doi = "10.1016/j.bbamcr.2015.06.001",
language = "English",
volume = "1853",
pages = "2361--2370",
journal = "Biochimica et Biophysica Acta - Molecular Cell Research",
issn = "0167-4889",
publisher = "Elsevier",
number = "10",

}

TY - JOUR

T1 - Revealing the fate of cell surface human P-glycoprotein (ABCB1)

T2 - The lysosomal degradation pathway

AU - Katayama, Kazuhiro

AU - Kapoor, Khyati

AU - Ohnuma, Shinobu

AU - Patel, Atish

AU - Swaim, William

AU - Ambudkar, Indu S.

AU - Ambudkar, Suresh V.

PY - 2015/10/1

Y1 - 2015/10/1

N2 - P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

AB - P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

KW - Degradation

KW - Endosome

KW - Half-life

KW - Lysosome

KW - P-glycoprotein

KW - Proteasome

UR - http://www.scopus.com/inward/record.url?scp=84938125626&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84938125626&partnerID=8YFLogxK

U2 - 10.1016/j.bbamcr.2015.06.001

DO - 10.1016/j.bbamcr.2015.06.001

M3 - Article

C2 - 26057472

AN - SCOPUS:84938125626

VL - 1853

SP - 2361

EP - 2370

JO - Biochimica et Biophysica Acta - Molecular Cell Research

JF - Biochimica et Biophysica Acta - Molecular Cell Research

SN - 0167-4889

IS - 10

ER -