Summary
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 (HL60PABA) that exhibited 1.9-fold resistance to the drug (IC50 ∼15 μM vs ∼8 μM for wild-type) was created. HL60PABA 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 HL60PABA cells. Wild type cells treated with PABA/NO had increased levels of protein S-glutathionylation and JNK activation, while JNK was constitutively active in HL60PABA cells and these cells had reduced levels of S-glutathionylation. By removing PABA/NO from the growth medium, HL60PABA 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.
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Abbreviations
- PABA/NO:
-
(O2- {2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate)
- GSH:
-
Glutathione reduced
- GSSG:
-
Glutathione oxidized
- ER-stress:
-
Endoplasmic reticulum stress
- GSTP:
-
Glutathione S-transferase P
- PDI:
-
Protein disulfide isomerase
- Tg:
-
Thapsigargin
- JNK:
-
c-Jun N-terminal kinase
- ROS-RNS:
-
Reactive oxygen and nitrogen species
- NO:
-
Nitric oxide
- TCEP:
-
Tris(2-carboxyethyl) phosphine
- UPR:
-
Unfolded protein response
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Acknowledgements
Supported by NCI National Cancer Institute grants CA08660 and CA117259. We thank Cameron McIlwain for her initial role in creating the drug resistant cell lines and the Drug Metabolism and Pharmacokinetics and Flow Cytometry Core Facilities of the Hollings Cancer Center.
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Hutchens, S., Manevich, Y., He, L. et al. Cellular resistance to a nitric oxide releasing glutathione S-transferase P-activated prodrug, PABA/NO. Invest New Drugs 29, 719–729 (2011). https://doi.org/10.1007/s10637-010-9407-5
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DOI: https://doi.org/10.1007/s10637-010-9407-5