Yen W C, Schmittgen T, Au J L
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus 43210, USA.
Pharm Res. 1996 Dec;13(12):1887-91. doi: 10.1023/a:1016053729362.
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.
其他研究者之前的研究表明，在人细胞单层培养中，酸性细胞外pH值（pHe）可增强丝裂霉素C（MMC）的活性。本研究的目的是确定使用酸化给药溶液是否能提高浅表性膀胱癌患者膀胱内MMC治疗的疗效。我们评估了（a）pHe对患者膀胱肿瘤中MMC活性的体外影响，以及（b）MMC活性在人膀胱RT4肿瘤细胞的二维单层和三维多层培养中的pH依赖性。
将患者膀胱肿瘤维持为三维组织培养。收获RT4细胞并将其维持为单层培养或在胶原凝胶基质上作为三维细胞团块。细胞团块为300 - 450个细胞层，直径为4000 - 5000微米。将肿瘤或细胞与含MMC的培养基在pHe为5、6和7.4的条件下孵育2小时。通过抑制DNA前体（胸苷）掺入来测量药物效果。确定MMC作为pHe函数的稳定性。在pHe 5下暴露2小时后，约24%的MMC降解，在pHe 6和7.4下<或 = 2%。
针对酸性pHe下降解的MMC对抑制胸苷掺入50%（IC50）所需的药物浓度进行了校正。结果显示，在人类患者膀胱肿瘤和RT4多层培养中，MMC活性不存在pH依赖性；在所有三个pHe下，IC50值约为10微克/毫升。相比之下，RT4单层培养显示出MMC细胞毒性的pH依赖性；在pHe 5、6和7.4时，IC50分别为0.1、0.8和1.2微克/毫升（p < 0.05）。将RT4多层培养在酸性pH培养基中预孵育8小时可增强MMC活性；在pHe约为5时，IC50降低约5倍，在pHe 6时降低约3倍。当多层用0.5微克/毫升尼日利亚菌素（一种已知可使细胞内pH（pHi）与pHe平衡的质子离子载体）预处理1小时时，发现了类似的pH依赖性MMC活性。
这些数据表明，MMC活性在单层和多层系统中pH依赖性的差异是由于不同的实验条件。多层系统中pHi与pHe平衡的时间滞后以及MMC在低pHe下的不稳定性意味着使用酸性给药溶液不太可能提高膀胱内MMC治疗的疗效。
