Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein.
An established gastric-carcinoma cell line, EPG85-257P, is extremely sensitive to mitoxantrone (IC50, 0.12 ng/ml). Stepwise selection with mitoxantrone for 3 years resulted in a cell line (EPG85-257RN) that is 7,056-fold resistant to mitoxantrone (IC50, 846 ng/ml) and displays cross-resistance to the topoisomerase(topo)-II poisons ametantrone (411x), etoposide (112x) and teniposide (60x) as well as the topo-I poisons 7-ethyl-10-hydroxycamptothecin (331x) and topotecan (58x). We now show that this resistance is multifactorial. Western blotting revealed a 5-fold decrease in topo-IIalpha polypeptide in the mitoxantrone-resistant cells. Immunohistochemistry and Western blotting failed to demonstrate P-glycoprotein overexpression. Formation of trapped topo-II-DNA complexes in the resistant cells required higher mitoxantrone concentrations than in parental cells, even though nuclei isolated from the EPG85-257RN cells formed cleavage complexes normally. In agreement with these observations, which suggest the possibility of a defect in mitoxantrone accumulation, examination of mitoxantrone accumulation in both cell lines by confocal laser microscopy revealed that the EPG85-257RN cells accumulate less mitoxantrone at steady state. From these results, we propose that mitoxantrone accumulation, along with alterations in topo-IIalpha expression, contribute to the resistance to mitoxantrone observed in these cells.
Kellner
U
U
Institute for General Pathology, Christian-Albrechts University, Kiel, Germany. ukellner@path.uni-kiel.de
Hutchinson
L
L
Seidel
A
A
Lage
H
H
Danks
M K
MK
Dietel
M
M
Kaufmann
S H
SH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D015153:Blotting, Western; D018736:DNA Adducts / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000032:analysis; D019008:Drug Resistance, Neoplasm; D019450:Genes, MDR / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008942:Mitoxantrone / Q000378:metabolism / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D018736:DNA Adducts; D012333:RNA, Messenger; D008942:Mitoxantrone; D004250:DNA Topoisomerases, Type II