Daunorubicin and vincristine binding to plasma membrane vesicles from daunorubicin-resistant and wild type Ehrlich ascites tumor cells.
Tumor cell resistance to anthracyclines, epipodophyllotoxins and vinca alkaloids, called multi-drug resistance (MDR) is intimately linked to changes in the plasma membrane which facilitate an increased energy dependent drug extrusion in the resistant cell compared to the wild type cell. Isolated plasma membrane vesicles from wild type Ehrlich ascites tumor cells (EHR2) and the daunorubicin (DNR) resistant subline EHR2/DNR+ were utilised to study binding and possible transport of DNR and vincristine (VCR). A significant ATP enhanced increase in VCR binding to vesicles from EHR2/DNR+ compared to EHR2 was demonstrated. Furthermore, an increase in ATP enhanced VCR binding in proportion to content of the MDR associated P-glycoprotein was seen in plasma membrane vesicles prepared from various benign human endocrine tumors. VCR binding to EHR2/DNR+ vesicles was inhibited by other vinca alkaloids greater than actinomycin D greater than colchicine greater than anthracyclines, with 35-75 microM concentrations of anthracyclines needed for 50% inhibition. VCR binding to EHR2/DNR+ vesicles was pH and temperature dependent with an activation energy of -30 kJ/mol and was decreased by replacement of Na+ with K+ and by addition of Ca2+. Preincubation of vesicles with monoclonal antibody against the C terminal of P-glycoprotein had no effect on VCR binding and osmolality tests failed to show genuine transmembranal transport of VCR. DNR binding was similar in plasma membrane vesicles from both cell lines, and showed none of the characteristics mentioned for VCR. Furthermore, a radiolabeled N-hydroxysuccinimide ester derivative of doxorubicin, which inhibited VCR binding to EHR2/DNR+ membranes to an even greater extent than doxorubicin, labeled plasma membrane proteins from EHR2 and EHR2/DNR+ identically and did not demonstrate any binding to P-glycoprotein. Therefore, even though the study confirms the close link between vinca alkaloid binding and P-glycoprotein, it could not detect a similar association between anthracyclines and P-glycoprotein thus attesting to the complexity of the MDR phenotype.
Sehested
M
M
Department of Pathology, Herlev University Hospital, Denmark.
Bindslev
N
N
Demant
E J
EJ
Skovsgaard
T
T
Jensen
P B
PB
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003630:Daunorubicin / Q000378:metabolism*; D004351:Drug Resistance; D005260:Female; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008811:Mice, Inbred DBA; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014750:Vincristine; D003630:Daunorubicin