Cellular resistance to a nitric oxide releasing glutathione S-transferase P-activated prodrug, PABA/NO.
PABA/NO is a diazeniumdiolate selectively activated by glutathione S-transferase P (GSTP) to release nitric oxide (NO) and is a potent inducer of protein S-glutathionylation, a redox-sensitive post-translational modification of cysteine residues. Using a procedure that incrementally increased exposure of cells to PABA/NO, an acquired drug resistant human promyelocytic leukemia HL60 cell line (HL60(PABA)) that exhibited 1.9-fold resistance to the drug (IC(50) 15 μM vs ~8 μM for wild-type) was created. HL60(PABA) cells had a decreased growth rate attributable to altered cellular differentiation, as measured by increased expression of CD11b; decreased expression of CD14; decreased nuclear to cytoplasmic ratios and a condensation of nuclear chromatin. This was accompanied by alterations in both plasma and mitochondrial membrane potentials. Both GSTP expression and nitric oxide release were reduced two-fold, while increased expression levels of genes involved in the unfolded protein response (UPR) were evident in HL60(PABA) cells. Wild type cells treated with PABA/NO had increased levels of protein S-glutathionylation and JNK activation, while JNK was constitutively active in HL60(PABA) cells and these cells had reduced levels of S-glutathionylation. By removing PABA/NO from the growth medium, HL60(PABA) cells reverted to sensitivity within 21 days suggesting that resistance was not genetically stable. Mechanistically, PABA/NO resistance is mediated through reduced levels of GSTP resulting in reduced NO release and its subsequent alterations in cellular response to nitrosative stress.
Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, SC, USA.
Steven
S
Hutchens
Yefim
Y
Manevich
Lin
L
He
Kenneth D
KD
Tew
Danyelle M
DM
Townsend
D010129:4-Aminobenzoic Acid / Q000737:chemistry / Q000494:pharmacology; D001391:Azo Compounds / Q000737:chemistry / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism*; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology*; D056811:Unfolded Protein Response / Q000187:drug effects / Q000235:genetics; D062366:para-Aminobenzoates
D001391:Azo Compounds; C485370:O(2)-(2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate; D011355:Prodrugs; D062366:para-Aminobenzoates; D009569:Nitric Oxide; D005982:Glutathione Transferase; D048031:JNK Mitogen-Activated Protein Kinases; D005978:Glutathione; D010129:4-Aminobenzoic Acid