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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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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 pemetrexedSeven 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.
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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 pemetrexedSeven 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.

Mentions: Based on these results, we further hypothesized that cells harboring a higher number of lysosomes may exhibit an increased intrinsic resistance to hydrophobic weak base drugs that are prone to undergo marked sequestration in lysosomes. To test this hypothesis, several drug-naïve human tumor cell lines of epithelial origin (i.e. carcinoma) were used including: ovarian carcinoma (2008 and IGROV-1), non-small cell lung cancer (A549), cervical carcinoma (HeLa), nasopharyngeal carcinoma (KB-3-1), gastric carcinoma (EPG85-257P) as well as normal human embryonic kidney cells (HEK293). The different cell lines were tested for their sensitivity to the protein tyrosine kinase inhibitor sunitinib, which we have recently shown to highly accumulate in lysosomes [23]. We also quantified the number of lysosomes per cell in all these tumor cell lines using fluorescence microscopy. We observed a striking linear correlation (R2 = 0.92) between mean lysosome number per cell and the inherent resistance of the various tumor cell lines to sunitinib (Fig. 2A). These results suggest that increased accumulation of sunitinib in lysosomes confers upon the tumor cells an intrinsic resistance to this hydrophobic weak base drug. To determine whether this innate resistance is indeed dependent on lysosomal sequestration of the drug, the correlation of the lysosome number in the above tumor cell lines was also compared to the cytotoxic activity of two widely used anticancer drugs that do not undergo lysosomal sequestration, the fluoropyrimidine 5-fluorouracil (5-FU), and the antifolate pemetrexed (PMX), both of which inhibit thymidylate synthase, a key enzyme in pyrimidine nucleotide biosynthesis (Fig. 2B and 2C, respectively). No correlation whatsoever was observed between lysosome number per cell and tumor cell sensitivity to these established anti-tumor agents. These findings suggest that increased lysosome number per cell is likely to serve as a marker for inherent resistance to hydrophobic weak base drugs that are prone to undergo marked accumulation in lysosomes.


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

Zhitomirsky B, Assaraf YG - Oncotarget (2015)

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 pemetrexedSeven 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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 pemetrexedSeven 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.
Mentions: Based on these results, we further hypothesized that cells harboring a higher number of lysosomes may exhibit an increased intrinsic resistance to hydrophobic weak base drugs that are prone to undergo marked sequestration in lysosomes. To test this hypothesis, several drug-naïve human tumor cell lines of epithelial origin (i.e. carcinoma) were used including: ovarian carcinoma (2008 and IGROV-1), non-small cell lung cancer (A549), cervical carcinoma (HeLa), nasopharyngeal carcinoma (KB-3-1), gastric carcinoma (EPG85-257P) as well as normal human embryonic kidney cells (HEK293). The different cell lines were tested for their sensitivity to the protein tyrosine kinase inhibitor sunitinib, which we have recently shown to highly accumulate in lysosomes [23]. We also quantified the number of lysosomes per cell in all these tumor cell lines using fluorescence microscopy. We observed a striking linear correlation (R2 = 0.92) between mean lysosome number per cell and the inherent resistance of the various tumor cell lines to sunitinib (Fig. 2A). These results suggest that increased accumulation of sunitinib in lysosomes confers upon the tumor cells an intrinsic resistance to this hydrophobic weak base drug. To determine whether this innate resistance is indeed dependent on lysosomal sequestration of the drug, the correlation of the lysosome number in the above tumor cell lines was also compared to the cytotoxic activity of two widely used anticancer drugs that do not undergo lysosomal sequestration, the fluoropyrimidine 5-fluorouracil (5-FU), and the antifolate pemetrexed (PMX), both of which inhibit thymidylate synthase, a key enzyme in pyrimidine nucleotide biosynthesis (Fig. 2B and 2C, respectively). No correlation whatsoever was observed between lysosome number per cell and tumor cell sensitivity to these established anti-tumor agents. These findings suggest that increased lysosome number per cell is likely to serve as a marker for inherent resistance to hydrophobic weak base drugs that are prone to undergo marked accumulation in lysosomes.

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