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

Zhitomirsky B, Assaraf YG - Oncotarget (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.

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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Hydrophobic weak base drugs induce lysosomal biogenesis in MCF-7 breast cancer cellsMCF-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).
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Figure 3: Hydrophobic weak base drugs induce lysosomal biogenesis in MCF-7 breast cancer cellsMCF-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).

Mentions: Based on previous evidence that lysosomal stress can trigger lysosomal biogenesis [16], we further postulated that lysosomal entrapment of hydrophobic weak base drugs may also result in enhanced lysosomal biogenesis, leading to a further increase in the number of lysosomes per cell. Towards this end, MCF-7 cells which contain a low number of lysosomes per cell, were exposed for 24 hr to a single dose (100 nM) of several cytotoxic drugs that were previously shown to undergo lysosomal sequestration including doxorubicin, mitoxantrone, C-1330 and sunitinib [23–25]. The anti-malarial drug chloroquine (100 μM), a well-established lysosomotropic agent which we recently showed to induce a marked increase in lysosome size and number [25], was used here as positive control. A marked increase in the number of lysosomes per cell was observed for all of these hydrophobic weak base drugs by fluorescence microscopy (Fig. 3A). In order to demonstrate the dose-dependent relationship of the drug-induced increase in lysosome number, MCF-7 cells were exposed to increasing concentrations of mitoxantrone for 72 hr, followed by viable cell staining with LysoTracker red, and were assessed by fluorescence microscopy and computational analysis. The results of this computational analysis that are depicted in Fig. 3B were obtained based on an examination of 40 microscope fields, representing 500–1000 cells from each sample. This analysis revealed a 2.7-fold increase in the number of lysosomes per cell after an exposure to sub-cytotoxic mitoxantrone concentrations as low as 10 nM, representing less than 6% of the IC50 (which was 170 nM for a 72 hr mitoxantrone treatment) as determined by a colorimetric XTT cell proliferation assay. The maximal increase in lysosomal number per cell in MCF-7 cells was achieved with 30 nM mitoxantrone, which led to a 3.9-fold increase in the number of lysosomes per cell, along with a 1.9-fold increase in the mean size of lysosomes (determined as the cross-sectional lysosomal area) as well as a mean of 1.4-fold increase in lysosome fluorescence intensity (an indicator of lysosome ability to accumulate LysoTracker red). Collectively, these results indicate an estimated increase of up to 16.4-fold in the total volume of cell lysosomes upon exposure to 30 nM mitoxantrone.


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

Zhitomirsky B, Assaraf YG - Oncotarget (2015)

Hydrophobic weak base drugs induce lysosomal biogenesis in MCF-7 breast cancer cellsMCF-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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Hydrophobic weak base drugs induce lysosomal biogenesis in MCF-7 breast cancer cellsMCF-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).
Mentions: Based on previous evidence that lysosomal stress can trigger lysosomal biogenesis [16], we further postulated that lysosomal entrapment of hydrophobic weak base drugs may also result in enhanced lysosomal biogenesis, leading to a further increase in the number of lysosomes per cell. Towards this end, MCF-7 cells which contain a low number of lysosomes per cell, were exposed for 24 hr to a single dose (100 nM) of several cytotoxic drugs that were previously shown to undergo lysosomal sequestration including doxorubicin, mitoxantrone, C-1330 and sunitinib [23–25]. The anti-malarial drug chloroquine (100 μM), a well-established lysosomotropic agent which we recently showed to induce a marked increase in lysosome size and number [25], was used here as positive control. A marked increase in the number of lysosomes per cell was observed for all of these hydrophobic weak base drugs by fluorescence microscopy (Fig. 3A). In order to demonstrate the dose-dependent relationship of the drug-induced increase in lysosome number, MCF-7 cells were exposed to increasing concentrations of mitoxantrone for 72 hr, followed by viable cell staining with LysoTracker red, and were assessed by fluorescence microscopy and computational analysis. The results of this computational analysis that are depicted in Fig. 3B were obtained based on an examination of 40 microscope fields, representing 500–1000 cells from each sample. This analysis revealed a 2.7-fold increase in the number of lysosomes per cell after an exposure to sub-cytotoxic mitoxantrone concentrations as low as 10 nM, representing less than 6% of the IC50 (which was 170 nM for a 72 hr mitoxantrone treatment) as determined by a colorimetric XTT cell proliferation assay. The maximal increase in lysosomal number per cell in MCF-7 cells was achieved with 30 nM mitoxantrone, which led to a 3.9-fold increase in the number of lysosomes per cell, along with a 1.9-fold increase in the mean size of lysosomes (determined as the cross-sectional lysosomal area) as well as a mean of 1.4-fold increase in lysosome fluorescence intensity (an indicator of lysosome ability to accumulate LysoTracker red). Collectively, these results indicate an estimated increase of up to 16.4-fold in the total volume of cell lysosomes upon exposure to 30 nM mitoxantrone.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.

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.

Show MeSH
Related in: MedlinePlus