The role of cytoplasmic-to-lysosomal pH gradient in hydrophobic weak base drug sequestration in lysosomes

Hydrophobic weak base chemotherapeutics are known to markedly accumulate in lysosomes via a mechanism based on protonation and entrapment known as lysosomal drug sequestration. We have recently shown that lysosomal compartmentalization of these anticancer drugs can prevent them from exerting their cytotoxic activity by abolishing accessibility to their target sites, resulting in multidrug resistance. Consistently, we as well as others have recently demonstrated that lysosomal sequestration of the receptor tyrosine kinase inhibitor sunitinib is a determinant of intrinsic resistance in human cancer cells. We specifically found that the number of sunitinib sequestering lysosomes tightly correlated with intrinsic resistance to sunitinib in various naive tumor cell lines of distinct tissue lineage. We further demonstrated that lysosomal accumulation of several hydrophobic weak base drugs triggers activation of lysosomal biogenesis via translocation of the master regulator TFEB from the cytoplasm to the nucleus. This resulted in activation of the Coordinated Lysosomal Expression and Regulation (CLEAR) gene network. This drug-induced activation of lysosomal biogenesis brought about a marked increase in lysosome number per cell. Here we demonstrate the role of alterations in the cytoplasm-to-lysosome pH gradient in lysosomal sequestration of anticancer drugs. We specifically show that MCF-7 breast cancer cells which display a markedly diminished cytoplasm-to-lysosome pH gradient due to both decreased lysosomal acidification and acidification of cytoplasmic pH, are devoid of hydrophobic weak base drug sequestration in lysosomes. The latter finding was of particular significance as MCF-7 cells were found to harbor a relatively high number of lysosomes per cell. We further show that disruption of lysosomal acidification using bafilomycin A1, an inhibitor of vacuolar H + ATPase (V-ATPase), prevents lysosomal drug sequestration when performed prior to hydrophobic weak base drug exposure. Moreover, such lysosomal alkalinization performed after lysosomal drug sequestration, results in massive drug release from lysosomes into the cytoplasm, hence restoring drug accessibility to the cellular target site. These findings have significant implications for the overcoming of lysosome-dependent cancer chemoresistance.