title: Tumor cell resistance to topoisomerase II poisons: role for intracellular free calcium in the sensitization by inhibitors or calcium-calmodulin-dependent enzymes.
abstract: Tumor cell resistance to inhibitors of topoisomerase II (topo II) is associated frequently with the overexpression of P-glycoprotein (PGP), and strategies to overcome resistance are focused on restoring defects in drug accumulation. Inhibitors of calcium-calmodulin-dependent enzymes sensitize resistant tumor cells to the topo II poison etoposide (VP-16) by enhancing DNA damage and an apoptotic response. In the present study, we have investigated the consequences of buffering intracellular calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra(acetoxy-methyl) ester (BAPTA-AM) on the sensitizing effects of the calmodulin-dependent protein kinase II inhibitor 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-piperazine (KN-62) in etoposide-resistant human leukemia HL-60 (HL-60/ADR0.05) cells. In cells pretreated with 20 microM BAPTA-AM for 2 hr, extracellular ATP failed to trigger intracellular calcium transients, and no effects on the accumulation of VP-16 were apparent. Also, the effect of KN-62 in significantly (P=0.002 to 0.042) enhancing the accumulation of VP-16 in HL-60/ADR0.05 cells was unaffected due to pretreatment with BAPTA-AM. In contrast, pretreatment with BAPTA-AM reduced the DNA damage induced by VP-16, and significantly (P=0.038) reversed the enhancement by KN-62 of VP-16-stabilized topo II-mediated DNA cleavable complex formation. The pretreatment of HL-60/ADR0.05 cells with BAPTA-AM was also associated with the hypophosphorylation of topo IIalpha. Consistent with the ability of BAPTA-AM to circumvent the potentiation by KN-62 of VP-16-induced DNA damage, survival of cells treated with 40 microM VP-16 in the absence of KN-62 and 10 microM VP-16 in the presence of KN-62 was significantly (P=0.026 to 0.031) higher due to BAPTA-AM pretreatment. Results demonstrate that intracellular calcium transients could play a key role in the sensitization of etoposide-resistant tumor cells by inhibitors of calcium-calmodulin-dependent enzymes.
authors.affiliation: Experimental Therapeutics Program, Cancer Center, Cleveland Clinic Foundation, OH 44195, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Grabowski
authors.forename: G R
authors.initials: GR
authors.lastname: Dubyak
authors.forename: L
authors.initials: L
authors.lastname: Rybicki
authors.forename: H
authors.initials: H
authors.lastname: Hidaka
authors.forename: R
authors.initials: R
authors.lastname: Ganapathi
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D004249:DNA Damage; D019008:Drug Resistance, Neoplasm; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000493:pharmacokinetics / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D059005:Topoisomerase II Inhibitors
chemical_list: D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; C063302:KN 62; D005047:Etoposide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium