Objectives: We tested the effect of PABA when combined with TTN on ovarian cancer cell proliferation, and generation of reactive oxygen species (ROS) as a marker for mitochondrial-mediated apoptosis. We also examined the effect of duration of exposure to PABA. Methods: SKOV-3 cells were maintained in DMEM media with 10% FBS. 5x103 cells were plated in 96 well plates overnight. Cells were treated with PABA at 100 ug/ml for either 7 or 18 days and were then treated with varying concentrations of TTN (0 to 5uM). At the end of treatment (24 hours), cell proliferation and viability was measured using a Cell Proliferation Assay Kit (Chemicon International, Inc, Temecula, CA). For measurement of ROS, PABA- treated and PABA-free cells were exposed to either 1 or 2.5 uM TTN. Treated cells were incubated in 5 uM dihydroethidine (Molecular Probes, Eugene, OR) at 37°C for 15 min, harvested by trypsinization and washed. ROS generation was determined by measuring the number of ethidium-positive cells via FACS analysis. Mixed model analysis of variance was used to assess the impact of the treatment components on the fraction of cells surviving compared to untreated cells. Results: For both 7 day and 18 day exposure to PABA, and at all concentrations of TTN, PABA caused significantly greater growth inhibition (GI) in the SKOV-3 cells compared to TTN treatment alone (p=0.043). However, the length of exposure to PABA had no significant effect (p=0.807) on GI. There was no statistically significant interaction (p>0.39) between TTN and either PABA (presence versus absence) or length of exposure to PABA (7 versus 18 days) suggesting an absence of synergism between PABA and TTN in SKOV-3 cells. At 1 uM TTN, the GI achieved with 7 days PABA exposure was improved by 18% over TTN alone and for 18 days’exposure by 21% over TTN alone. Five-week treatment of the cells with PABA even without TTN increased the percent of ethidium-positive cells 36-fold from 6.71 ± 0.95 (S.D.) without PABA to 240.24 ± 13.31 with PABA. Treatment with TTN showed no effect on ROS generation. Conclusions: PABA, which has been shown to upregulate cdc25a (Peter Brooks, unpublished data), increases the growth inhibition caused by TTN in ovarian cancer cells. This is associated with an increase in mitochondrial stress in these cells as indicated by an increased ROS generation. This implies that addition of PABA to TTN chemotherapy in ovarian cancer might improve tumor response through mitochondrial-mediated apoptosis. Supported by DOD OC010016 and the Chemotherapy Foundation.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]