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. 2015 Jan 20;6(2):1143-56.
doi: 10.18632/oncotarget.2732.

Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance

Benny Zhitomirsky  1 , Yehuda G Assaraf  1
Affiliations

Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance

Benny Zhitomirsky et al. Oncotarget. .

Erratum in

Abstract

Multidrug resistance (MDR) is a primary hindrance to curative cancer chemotherapy. In this respect, lysosomes were suggested to play a role in intrinsic MDR by sequestering protonated hydrophobic weak base chemotherapeutics away from their intracellular target sites. Here we show that intrinsic resistance to sunitinib, a hydrophobic weak base tyrosine kinase inhibitor known to accumulate in lysosomes, tightly correlates with the number of lysosomes accumulating high levels of sunitinib in multiple human carcinoma cells. Furthermore, exposure of cancer cells to hydrophobic weak base drugs leads to a marked increase in the number of lysosomes per cell. Non-cytotoxic, nanomolar concentrations, of the hydrophobic weak base chemotherapeutics doxorubicin and mitoxantrone triggered rapid lysosomal biogenesis that was associated with nuclear translocation of TFEB, the dominant transcription factor regulating lysosomal biogenesis. This resulted in increased lysosomal gene expression and lysosomal enzyme activity. Thus, treatment of cancer cells with hydrophobic weak base chemotherapeutics and their consequent sequestration in lysosomes triggers lysosomal biogenesis, thereby further enhancing lysosomal drug entrapment and MDR. The current study provides the first evidence that drug-induced TFEB-associated lysosomal biogenesis is an emerging determinant of MDR and suggests that circumvention of lysosomal drug sequestration is a novel strategy to overcome this chemoresistance.

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Figures

Figure 1
Figure 1. Lysosome-mediated protection of nuclear DNA from hydrophobic weak base cytotoxic agents
MCF-7 breast cancer cells and A549/K1.5 non-small cell lung cancer cells were incubated for 1 hr with 100 nM LysoTracker red (red fluorescence) and 10 μM of the imidazoacridinone C-1330 (green fluorescence). Fluorescence microscopy analysis was performed using a Zeiss inverted Cell-Observer Axiovert 200 microscope (Carl Zeiss, Oberkochen, Germany).
Figure 2
Figure 2. The correlation between lysosome number and intrinsic resistance to the hydrophobic weak base tyrosine kinase inhibitor sunitinib, but not to the hydrophilic thymidylate synthase inhibitors 5-fluorouracil and pemetrexed
Seven human carcinoma cell lines of diverse tissue lineage were analyzed for sunitinib (A), 5-fluorouracil (B), and pemetrexed (C) cytotoxicity using a colorimetric XTT cell proliferation assay (IC50 values are shown on the Y axis), as well as for lysosome number per cell. The latter was performed by monitoring the number of green fluorescent lysosomes (i.e. sunitinib-accumulating lysosomes and acidic vesicles; shown on the X axis) determined using the fluorescence microscope InCell analyzer, preceded by lysosome staining with 10 μM sunitinib (green fluorescence) for 30 min, and computationally analyzed using the InCell investigator software.
Figure 3
Figure 3. Hydrophobic weak base drugs induce lysosomal biogenesis in MCF-7 breast cancer cells
MCF-7 cells were exposed for 72 hr to a single dose of 100 nM mitoxantrone, doxorubicin, sunitinib and C-1330 as well as 100 μM chloroquine. Following drug exposure, cells were stained with 100 nM LysoTracker red for 1 hr and LysoTracker red fluorescence was determined using an InCell analyzer fluorescence microscope (A). We also examined the dose-dependent increase in lysosomal content after exposure of MCF-7 cells to a single dose of mitoxantrone for 72 hr. MCF-7 cells were exposed to increasing concentrations of mitoxantrone (1–100 nM) for 72 hr. Cells were then stained with LysoTracker red and the red fluorescence was determined by fluorescence microscopy. Lysosome number, size and fluorescence intensity were determined using an InCell investigator software (B). Statistical significance is denoted by *(p < 0.05) and **(p < 0.01).
Figure 4
Figure 4. Translocation of TFEB-FLAG from the cytoplasm to the nucleus after exposure of malignant and non-malignant human cells to doxorubicin, mitoxantrone and sunitinib
Stably transfected MCF-7 TFEB-3XFLAG cells were exposed to 0.5 μM doxorubicin, 0.5 μM mitoxantrone or 100 μM chloroquine for 24 hr. In an independent set of experiments, 24 hr after transient transfection of HEK293 and HeLa cells with FLAG-tagged TFEB using electroporation, The cells were exposed to 0.5 μM doxorubicin, mitoxantrone, or sunitinib for 24 hr. Cells were then fixed, stained with the DNA dye DAPI (blue fluorescence), incubated with an anti-FLAG antibody (green fluorescence) and analyzed by a fluorescence microscope. The first row represents the drug-free control cells.
Figure 5
Figure 5. Exposure of MCF-7 cells to mitoxantrone induces an increase in gene expression of the established lysosomal enzyme markers GNS and CTSD
MCF-7 cells were exposed to various concentrations of mitoxantrone for 24 hr. Then, GNS (A) and CTSD (B) gene expression levels were determined using quantitative real time PCR. Statistical significance is denoted by *(p < 0.05) and **(p < 0.01).
Figure 6
Figure 6. Mitoxantrone induces an increase in lysosomal enzyme activity in a drug dose-dependent manner
β-Hexosaminidase activity was determined in MCF-7 cells after their exposure to increasing concentrations of mitoxantrone for 24 hr (A) or 72 hr (B). Statistical significance is denoted by *(p < 0.05).
Figure 7
Figure 7. A schematic summary model for hydrophobic weak base drug-induced lysosome-mediated drug resistance
Hydrophobic weak base drugs enter the lysosomes by simple diffusion; undergo protonation in the acidic lysosomal lumen, thereby becoming irreversibly sequestered in lysosomes and acidic intracellular vesicles such as late endosomes. In turn, lysosomal drug sequestration triggers TFEB-mediated lysosomal biogenesis, resulting in a significant increase in the number of lysosomes per cell. Increased lysosome number per cell enhances the efficiency of lysosomal drug sequestration, with lysosomes acting as a sink pulling hydrophobic weak base drugs away from their cellular target sites, thereby resulting in MDR.
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