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 (IC₅₀ ∼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.