TLK286 is a novel anticancer prodrug that has shown significant activity in Phase 2 clinical trials in ovarian, non-small cell lung, breast and colorectal cancers. TLK286 is a glutathione analog designed to be activated by GST P1-1, an enzyme frequently elevated in ovarian, non-small cell lung and other cancers. We previously reported that continuous exposure of OVCAR-3 cells to TLK286 for 12 months did not produce resistance to TLK286 or standard cancer chemotherapeutics such as paclitaxel, carboplatin, or doxorubicin and led to a significant decrease in growth rate. Here we further explore the cellular and molecular mechanism underlying TLK286-induced growth inhibition after long-term exposure of OVCAR-3, human ovarian cancer, and short term-exposure of A549, human lung cancer cells. OVCAR-3 cells chronically exposed to TLK286 exhibited sustained morphologic changes similar to those observed with premature senescence. Staining for senescence-associated β-galactosidase (SA-β-gal) showed a marked increase in the proportion of positive cells. Following four day treatment with TLK286, A549 cells persistently arrested in the G2 phase of the cell cycle as demonstrated by FACS analysis for cellular DNA content and phosphorylated histone H3. At the molecular level, treatment with TLK286 led to the induction of the cdk inhibitor p21 and irreversible down regulation of cdc2 kinase, which is essential for the G2/M transition. TLK286-treated A549 cells had a flat, enlarged morphology and stained positive for SA-β-gal, indicating senescence. These data suggest that TLK286 can induce a premature senescence phenotype that may play a role in the inability of cells to develop resistance as well as potentially contributing to the in vivo anti-cancer activity.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]