Different pH dependency of mitomycin C activity in monolayer and three-dimensional cultures

Previous studies by other investigators have shown an enhancement of mitomycin C (MMC) activity at acidic extracellular pH (pHe) in monolayer cultures of human cells. The goal of the present study was to determine if the efficacy of intravesical MMC therapy in patients treated for superficial bladder cancer can be enhanced by using acidified dosing solutions. We evaluated (a) the effect of pHe on MMC activity in patient bladder tumors in vitro, and (b) the pH dependency of MMC activity in 2-dimensional monolayer and 3-dimensional multilayer cultures of human bladder RT4 tumor cells.

Patient bladder tumors were maintained as 3-dimensional histocultures. RT4 cells were harvested and maintained as monolayer cultures or as 3-dimensional cell pellets on a collagen gel matrix. The cell pellets were 300-450 cell layers and 4,000-5,000 microns in diameter. Tumors or cells were incubated for 2 hr with MMC-containing media at pHe of 5, 6, and 7.4. The drug effect was measured by the inhibition of DNA precursor (thymidine) incorporation. The stability of MMC as a function of pHe was determined. About 24% of MMC was degraded following 2 hr exposure at pHe 5 and < or = 2% at pHe 6 and 7.4.

The drug concentrations required to inhibit thymidine incorporation by 50% (IC50) were corrected for the degraded MMC at acidic pHe. The results showed no pH-dependent MMC activity in human patient bladder tumors nor in RT4 multilayer cultures; the IC50 values were about 10 micrograms/ml at all three pHe. In contrast, the monolayer RT4 cultures showed a pH-dependent MMC cytotoxicity; the IC50 were 0.1, 0.8 and 1.2 micrograms/ ml at pHe 5, 6 and 7.4, respectively (p < 0.05). Pre-incubation of multilayered RT4 cultures in acidic pH medium for 8 hr enhanced the MMC activity; the IC50 was reduced by about 5 fold at pHe about 3 fold at pHe 6. Similar pH-dependent MMC activity was found when multilayers were pre-treated for 1 hr with 0.5 microgram/ml nigericin, a proton ionophore known to cause the intracellular pH (pHi) to equilibrate with pHe.

These data suggest that the difference in the pH dependency of MMC activity in the monolayer and multilayer systems was due to the different experimental conditions. The time lag for pHi to equilibrate with pHe in the multilayer systems and the instability of MMC at low pHe imply that the efficacy of intravesical MMC therapy is unlikely to be enhanced by using acidic dosing solution.