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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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Exposure of MCF-7 cells to mitoxantrone induces an increase in gene expression of the established lysosomal enzyme markers GNS and CTSDMCF-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).
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Figure 5: Exposure of MCF-7 cells to mitoxantrone induces an increase in gene expression of the established lysosomal enzyme markers GNS and CTSDMCF-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).

Mentions: We next explored the factors regulating hydrophobic weak base drug-induced lysosomal biogenesis. In this respect, TFEB, a master transcription regulator, was previously shown to trigger lysosomal biogenesis upon its translocation from the cytoplasm to the nucleus [14]. To examine the impact of hydrophobic weak base drug exposure on the possible translocation of TFEB from the cytoplasm to the nucleus, three human malignant and non-malignant cell lines were transfected with FLAG-tagged TFEB using both stable and transient transfection approaches. MCF-7 cells were stably transfected with FLAG-tagged TFEB, resulting in the stable transfectant MCF-7-TFEB-FLAG; these were exposed to mitoxantrone or doxorubicin for 24 hr, followed by immunofluorescence microscopy using an anti-FLAG antibody. HeLa and HEK 293 cells were transiently transfected with FLAG-tagged TFEB; twenty four hours later, these cells were exposed to sunitinib, mitoxantrone or doxorubicin for 24 hr followed by immunofluorescent staining using an anti-FLAG antibody (Fig. 4). Prior to drug exposure, FLAG-tagged TFEB was predominantly localized in the cytoplasm in all three cell lines studied, hence being in agreement with previous studies [14, 16]. In contrast, upon exposure to mitoxantrone, doxorubicin, sunitinib, or the lysosomotropic agent chloroquine which was used in MCF-7 cells as a positive control, a marked increase in the translocation of FLAG-tagged TFEB from the cytoplasm to the nucleus was observed. Hence, these findings support our hypothesis that exposure to hydrophobic weak base drugs induces translocation of TFEB to the nucleus and consequent lysosomal biogenesis. To rule out the possibility that transfection reagents induce alterations in subcellular localization of TFEB, we employed a different transfection technique using a linear polyethylenimine-based transfection reagent (JetPEI™); the results showed the same translocation of TFEB from the cytoplasm to the nucleus upon drug exposure in all cell lines studied (data not shown). To confirm that this nuclear translocation of TFEB was associated with an actual transactivation of lysosomal gene expression, we performed quantitative real-time PCR analysis to determine the gene expression levels of glucosamine (N-Acetyl)-6-sulfatase (GNS) and cathepsin D (CTSD); GNS and CTSD are bona fide lysosomal genes in the CLEAR pathway that were previously shown to be up-regulated upon activation of lysosomal biogenesis as part of the TFEB-regulated CLEAR gene network [16]. As expected from the marked nuclear localization of TFEB upon exposure to lipophilic weak base drugs, the gene expression levels of both GNS (Fig. 5A) and CTSD (Fig. 5B) were significantly elevated after 24 hr exposure of MCF-7 cells to mitoxantrone. These results provide the first direct evidence for hydrophobic weak base chemotherapeutic drug-induced increase in lysosome number in cancer cells, hence indicating that single dose exposure to lipophilic weak base drugs, such as mitoxantrone, triggers enhanced lysosomal biogenesis in cancer cells.


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

Zhitomirsky B, Assaraf YG - Oncotarget (2015)

Exposure of MCF-7 cells to mitoxantrone induces an increase in gene expression of the established lysosomal enzyme markers GNS and CTSDMCF-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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359223&req=5

Figure 5: Exposure of MCF-7 cells to mitoxantrone induces an increase in gene expression of the established lysosomal enzyme markers GNS and CTSDMCF-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).
Mentions: We next explored the factors regulating hydrophobic weak base drug-induced lysosomal biogenesis. In this respect, TFEB, a master transcription regulator, was previously shown to trigger lysosomal biogenesis upon its translocation from the cytoplasm to the nucleus [14]. To examine the impact of hydrophobic weak base drug exposure on the possible translocation of TFEB from the cytoplasm to the nucleus, three human malignant and non-malignant cell lines were transfected with FLAG-tagged TFEB using both stable and transient transfection approaches. MCF-7 cells were stably transfected with FLAG-tagged TFEB, resulting in the stable transfectant MCF-7-TFEB-FLAG; these were exposed to mitoxantrone or doxorubicin for 24 hr, followed by immunofluorescence microscopy using an anti-FLAG antibody. HeLa and HEK 293 cells were transiently transfected with FLAG-tagged TFEB; twenty four hours later, these cells were exposed to sunitinib, mitoxantrone or doxorubicin for 24 hr followed by immunofluorescent staining using an anti-FLAG antibody (Fig. 4). Prior to drug exposure, FLAG-tagged TFEB was predominantly localized in the cytoplasm in all three cell lines studied, hence being in agreement with previous studies [14, 16]. In contrast, upon exposure to mitoxantrone, doxorubicin, sunitinib, or the lysosomotropic agent chloroquine which was used in MCF-7 cells as a positive control, a marked increase in the translocation of FLAG-tagged TFEB from the cytoplasm to the nucleus was observed. Hence, these findings support our hypothesis that exposure to hydrophobic weak base drugs induces translocation of TFEB to the nucleus and consequent lysosomal biogenesis. To rule out the possibility that transfection reagents induce alterations in subcellular localization of TFEB, we employed a different transfection technique using a linear polyethylenimine-based transfection reagent (JetPEI™); the results showed the same translocation of TFEB from the cytoplasm to the nucleus upon drug exposure in all cell lines studied (data not shown). To confirm that this nuclear translocation of TFEB was associated with an actual transactivation of lysosomal gene expression, we performed quantitative real-time PCR analysis to determine the gene expression levels of glucosamine (N-Acetyl)-6-sulfatase (GNS) and cathepsin D (CTSD); GNS and CTSD are bona fide lysosomal genes in the CLEAR pathway that were previously shown to be up-regulated upon activation of lysosomal biogenesis as part of the TFEB-regulated CLEAR gene network [16]. As expected from the marked nuclear localization of TFEB upon exposure to lipophilic weak base drugs, the gene expression levels of both GNS (Fig. 5A) and CTSD (Fig. 5B) were significantly elevated after 24 hr exposure of MCF-7 cells to mitoxantrone. These results provide the first direct evidence for hydrophobic weak base chemotherapeutic drug-induced increase in lysosome number in cancer cells, hence indicating that single dose exposure to lipophilic weak base drugs, such as mitoxantrone, triggers enhanced lysosomal biogenesis in cancer cells.

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