Different pH Dependency of Mitomycin C Activity in Monolayer and Three-Dimensional Cultures

Abstract

Purpose. Previous studies by other investigators have shown an enhancement of mitomycin C (MMC) activity at acidic extracellular pH (pH_e) 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 pH_e 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.

Methods. 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 µm in diameter. Tumors or cells were incubated for 2 hr with MMC-containing media at pH_eof 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 pH_e was determined. About 24% of MMC was degraded following 2 hr exposure at pH_e 5 and ≤ 2% at pH_e 6 and 7.4.

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

Conclusions. 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 pH_i to equilibrate with pH_e in the multilayer systems and the instability of MMC at low pH_e imply that the efficacy of intravesical MMC therapy is unlikely to be enhanced by using acidic dosing solution.