A cellular and molecular investigation of the action of PMX464, a putative thioredoxin inhibitor, in normal and colorectal cancer cell lines.
OBJECTIVE
PMX464 is a novel benzothiazole substituted cyclohexadienone reportedly targeting the thioredoxin (Trx1)/thioredoxin reductase (TrxR1) system. We have previously shown that PMX464 has enhanced hypoxic anti-proliferative effects in colorectal tumour cells, with some non-tumour cells (quiescent endothelium and fibroblasts) being relatively resistant. The current study aimed to validate the Trx1 system as a molecular target of PMX464 in tumour cells and to investigate the differential sensitivities of normal cells at the molecular level.


METHODS
Proliferation, clonogenic survival, protein expression and function, cell cycle and apoptosis assays were conducted using colorectal tumour (HT29), endothelial (HUVEC) and fibroblast (MRCV) cells treated with PMX464 under normoxic and hypoxic (1% O(2)) conditions.


RESULTS
Protein and enzyme assays showed that PMX464, in HT29, inhibited Trx1 function without altering expression and that inhibition correlated with decreased proliferation and survival, and was more marked under hypoxia. In contrast, although hypoxic HUVEC were sensitive, in terms of proliferation and survival, inhibition of Trx1 function was not observed. Quiescent HUVEC and MRCVs (that have undetectable Trx1 protein) were relatively resistant. The effect on HT29 cells was essentially due to cell cycle inhibition, as apoptosis was modest. Anti-proliferative effects were lost after a lag period, suggesting a reversible phenomenon.


CONCLUSIONS
The Trx1 system is an important target in tumour cells and can be inhibited by PMX464. Quiescent HUVEC and fibroblasts are relatively resistant conferring a therapeutic benefit when targeting Trx1.
Mukherjee
A
A
Division of Clinical Oncology, School of Molecular Medical Sciences, University of Nottingham, Nottingham University Hospitals, Hucknall Road, Nottingham, UK.
Huber
K
K
Evans
H
H
Lakhani
N
N
Martin
S
S
D017209:Apoptosis / Q000187:drug effects; D052160:Benzothiazoles / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy*; D003512:Cyclohexanones / Q000494:pharmacology*; D016503:Drug Delivery Systems; D004730:Endothelium, Vascular; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D013879:Thioredoxins / Q000037:antagonists & inhibitors*; D014471:Umbilical Veins
C515226:AW 464; D052160:Benzothiazoles; D003512:Cyclohexanones; D013879:Thioredoxins