Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1142, New Zealand.
Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Symonds St, Auckland 1142, New Zealand.
Molecules. 2019 Jul 10;24(14):2524. doi: 10.3390/molecules24142524.
Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH gradient across the plasma membrane in cells that regulate intracellular pH (pHi) has potential to drive the selective uptake of weak acids at low extracellular pH (pHe). Here, we investigate the dual targeting of low pHe and hypoxia, another key feature of tumor microenvironments. We prepared eight bioreductive prodrugs based on the benzotriazine di-oxide (BTO) nucleus by appending alkanoic or aminoalkanoic acid sidechains. The BTO acids showed modest selectivity for both low pHe (pH 6.5 versus 7.4, ratios 2 to 5-fold) and anoxia (ratios 2 to 8-fold) in SiHa and FaDu cell cultures. Related neutral BTOs were not selective for acidosis, but had greater cytotoxic potency and hypoxic selectivity than the BTO acids. Investigation of the uptake and metabolism of representative BTO acids confirmed enhanced uptake at low pHe, but lower intracellular concentrations than expected for passive diffusion. Further, the modulation of intracellular reductase activity and competition by the cell-excluded electron acceptor WST-1 suggests that the majority of metabolic reductions of BTO acids occur at the cell surface, compromising the engagement of the resulting free radicals with intracellular targets. Thus, the present study provides support for designing bioreductive prodrugs that exploit pH-dependent partitioning, suggesting, however, that that the approach should be applied to prodrugs with obligate intracellular activation.
细胞外酸化是肿瘤微环境的一个重要特征，但尚未成功应用于癌症治疗。调节细胞内 pH 值 (pHi) 的细胞膜两侧 pH 梯度的逆转有可能驱动弱酸性物质在低细胞外 pH 值 (pHe) 下的选择性摄取。在这里，我们研究了低 pHe 和缺氧的双重靶向，这是肿瘤微环境的另一个关键特征。我们通过附加烷酸或氨基酸侧链，以苯并三嗪二氧化物 (BTO) 核为基础制备了八种生物还原前药。BTO 酸在 SiHa 和 FaDu 细胞培养物中对低 pHe（pH 6.5 与 7.4，比值为 2 至 5 倍）和缺氧（比值为 2 至 8 倍）均具有适度的选择性。相关的中性 BTO 对酸中毒没有选择性，但比 BTO 酸具有更大的细胞毒性和缺氧选择性。对代表性 BTO 酸的摄取和代谢的研究证实了在低 pHe 下的摄取增强，但细胞内浓度低于预期的被动扩散。此外，细胞排斥电子受体 WST-1 对细胞内还原酶活性和竞争的调节表明，BTO 酸的大多数代谢还原发生在细胞表面，从而损害了生成的自由基与细胞内靶标的结合。因此，本研究为设计利用 pH 依赖性分配的生物还原前药提供了支持，但表明该方法应适用于需要强制性细胞内激活的前药。
