Abstract
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.
Highlights
Hypoxia is a common feature of the tumor microenvironment [1] and contributes to tumor progression and resistance to therapy [2,3]
A third possibility is that efflux transporters could be responsible for the low Ci values, but in preliminary experiments we found no effect of overexpression from breast cancer resistance protein (BCRP) or P-glycoprotein (Pgp) efflux transporters (Figure S8) on sensitivity to 4a or 7a (or SN30000 (2)) in MDCK-11 cell lines under anoxia, and no effect from the pan-inhibition of monocarboxylate transporters (MCT) by phenylpyruvate [51]
In this study we illustrate the concept of designing prodrugs by superimposing selectivity for acidosis on study selectivity for hypoxia in orderoftodesigning exploit these two important features selectivity of the tumor
Summary
Hypoxia is a common feature of the tumor microenvironment [1] and contributes to tumor progression and resistance to therapy [2,3]. Low extracellular pH (pHe) results from the high glycolytic rates and slow clearance of metabolic acids in poorly perfused regions in tumors [4,5,6,7,8]. Acidosis is a potentially exploitable difference between normal tissues and tumors that can be utilized in drug design. The reversal of the pH gradient across the plasma membrane in regions of acidosis reflects active proton export by Molecules 2019, 24, 2524; doi:10.3390/molecules24142524 www.mdpi.com/journal/molecules
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