Abstract
Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in multidrug-resistant tumors where a stressful micro-environment exists.
Highlights
Multidrug resistance (MDR)5 is the principal cause of cancer cell resistance to chemotherapeutic drug treatment [1, 2]
Glucose Variation-induced Stress Increased the Expression of HIF-1␣ and Pgp in MDR Cells—Recently we reported that glucose variation-induced stress, mediated by low or high levels of glucose, increases total tumor cell Pgp expression and MDR [21]
We hypothesized that increased Pgp expression could result in an increase of this transporter in the lysosomal compartment that may contribute to lysosomal sequestration of cytotoxic chemotherapeutics and MDR
Summary
Multidrug resistance (MDR) is the principal cause of cancer cell resistance to chemotherapeutic drug treatment [1, 2]. It has been demonstrated that Pgp functions to efflux drugs out of the cell when present on the plasma membrane and serves a functional intracellular role in the lysosomal membrane to induce resistance [7] In this case, lysosomal membrane Pgp transports substrates into the organelle, which acts as a “safe house” to prevent cytotoxic chemotherapeutics, such as doxorubicin (DOX; Fig. 1Ai), reaching their intracellular targets, e.g. the nucleus [7]. There have been reports of several drugs that are more effective against MDR cells than their drug-sensitive counterparts (8 –11) One such agent, namely the thiosemicarbazone, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1Aii), a Pgp substrate, has been shown to utilize Pgp on the lysosomal membrane to increase its transport and accumulation in this organelle, resulting in enhanced cytotoxicity [12]. Endocytosis is important to consider as a mediator of protein redistribution from the cell surface to intracellular organelles that occurs as a protective response under stress stimuli
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