The Lysosomal Sequestration of Tyrosine Kinase Inhibitors and Drug Resistance

Eliska Ruzickova, Nikola Skoupa, Petr Dolezel, Dennis A Smith, Petr Mlejnek, Eliska Ruzickova, Nikola Skoupa, Petr Dolezel, Dennis A Smith, Petr Mlejnek

Abstract

The Lysosomal sequestration of weak-base anticancer drugs is one putative mechanism for resistance to chemotherapy but it has never been directly proven. We addressed the question of whether the lysosomal sequestration of tyrosine kinase inhibitors (TKIs) itself contributes to the drug resistance in vitro. Our analysis indicates that lysosomal sequestration of an anticancer drug can significantly reduce the concentration at target sites, only when it simultaneously decreases its extracellular concentration due to equilibrium, since uncharged forms of weak-base drugs freely diffuse across cellular membranes. Even though the studied TKIs, including imatinib, nilotinib, and dasatinib, were extensively accumulated in the lysosomes of cancer cells, their sequestration was insufficient to substantially reduce the extracellular drug concentration. Lysosomal accumulation of TKIs also failed to affect the Bcr-Abl signaling. Cell pre-treatment with sunitinib significantly enhanced the lysosomal accumulation of the TKIs used; however, without apparent lysosomal biogenesis. Importantly, even increased lysosomal sequestration of TKIs neither decreased their extracellular concentrations nor affected the sensitivity of Bcr-Abl to TKIs. In conclusion, our results clearly show that the lysosomal sequestration of TKIs failed to change their concentrations at target sites, and thus, can hardly contribute to drug resistance in vitro.

Keywords: drug resistance; extracellular space; extralysosomal space; lysosomal sequestration; target sites; tyrosine kinase inhibitors.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Weak-base drug distribution within the cell in vitro. Our model includes several theoretical assumptions: (i) uncharged forms of weakly basic drugs can freely diffuse across cellular membranes [5,20]; (ii) only two interactions are considered, the Henderson–Hasselbach equilibrium ↑ ↓ and the passive diffusion of uncharged molecules ↔; (iii) the extra and intracellular (extralysosomal space) pHs are equal. Color saturation corresponds to the drug concentration. (a) Drug distribution without lysosomal sequestration. (This may occur, for example, after the addition of BafA1, a vacuolar ATPase inhibitor.) Uncharged molecules can freely diffuse across cell membranes. (b) Drug distribution with lysosomal sequestration. Uncharged molecules can freely diffuse only across the lysosomal membrane. Under such conditions, the lysosomal sequestration of a drug can dramatically reduce its extralysosmal concentration (drug concentration at target sites), and thus mediate drug resistance. However, such a model is not applicable since it does not fulfil theoretical assumptions. In fact, the diffusion of uncharged molecules across the plasma membrane would dissipate the gradient between extra and intracellular space. (c) Drug distribution with lysosomal sequestration. Uncharged molecules can freely diffuse across cell membranes. The lysosomal sequestration of a drug that reduces drug concentration at target sites (= in extralysosomal space) and simultaneously decreases the extracellular drug level can mediate resistance to this drug. This is the only model to fulfil theoretical assumptions.
Figure 1
Figure 1
Weak-base drug distribution within the cell in vitro. Our model includes several theoretical assumptions: (i) uncharged forms of weakly basic drugs can freely diffuse across cellular membranes [5,20]; (ii) only two interactions are considered, the Henderson–Hasselbach equilibrium ↑ ↓ and the passive diffusion of uncharged molecules ↔; (iii) the extra and intracellular (extralysosomal space) pHs are equal. Color saturation corresponds to the drug concentration. (a) Drug distribution without lysosomal sequestration. (This may occur, for example, after the addition of BafA1, a vacuolar ATPase inhibitor.) Uncharged molecules can freely diffuse across cell membranes. (b) Drug distribution with lysosomal sequestration. Uncharged molecules can freely diffuse only across the lysosomal membrane. Under such conditions, the lysosomal sequestration of a drug can dramatically reduce its extralysosmal concentration (drug concentration at target sites), and thus mediate drug resistance. However, such a model is not applicable since it does not fulfil theoretical assumptions. In fact, the diffusion of uncharged molecules across the plasma membrane would dissipate the gradient between extra and intracellular space. (c) Drug distribution with lysosomal sequestration. Uncharged molecules can freely diffuse across cell membranes. The lysosomal sequestration of a drug that reduces drug concentration at target sites (= in extralysosomal space) and simultaneously decreases the extracellular drug level can mediate resistance to this drug. This is the only model to fulfil theoretical assumptions.
Figure 2
Figure 2
Lysosomal sequestration of TKIs. (a) Absolute accumulation of IM in lysosomes of cancer cells. (b) Relative accumulation of IM in lysosomes of cancer cells. (c) Absolute accumulation of NIL in lysosomes of cancer cells. (d) Relative accumulation of NIL in lysosomes of cancer cells. ND—not determined (due to limited NIL solubility). (e) Absolute accumulation of DAS in lysosomes of cancer cells. (f) Relative accumulation of DAS in lysosomes of cancer cells. Columns represent the means and standard deviations of four independent experiments. * denotes a significant change in the intralysosomal IM content (p < 0.05) between the indicated cell lines. # denotes significant change in the intralysosomal content of the appropriate TKI (p < 0.05) between the groups indicated. ## denotes a very significant change in the intralysosomal content of the appropriate TKI (p < 0.01) between the groups indicated.
Figure 3
Figure 3
Lysosomal sequestration of TKIs and extracellular drug concentration. (a) The effect of lysosomal sequestration of IM on its extracellular concentration. (b) The effect of lysosomal sequestration of NIL on its extracellular concentration. ND—not determined (due to limited NIL solubility). (c) The effect of lysosomal sequestration of DAS on its extracellular concentration. Columns represent the means and standard deviations of four independent experiments. * denotes a significant change in the decrease in extracellular IM content (p < 0.05) between the indicated cell lines. ## denotes a very significant change in the decrease in extracellular IM content (p < 0.01) between the indicated groups.
Figure 3
Figure 3
Lysosomal sequestration of TKIs and extracellular drug concentration. (a) The effect of lysosomal sequestration of IM on its extracellular concentration. (b) The effect of lysosomal sequestration of NIL on its extracellular concentration. ND—not determined (due to limited NIL solubility). (c) The effect of lysosomal sequestration of DAS on its extracellular concentration. Columns represent the means and standard deviations of four independent experiments. * denotes a significant change in the decrease in extracellular IM content (p < 0.05) between the indicated cell lines. ## denotes a very significant change in the decrease in extracellular IM content (p < 0.01) between the indicated groups.
Figure 4
Figure 4
The effect of the lysosomal sequestration of TKIs on the inhibition of Bcr-Abl signaling. Cells were incubated with various TKI concentrations in the absence or presence BafA1. After 6 h, cell extracts were analyzed for CrkL phosphorylation (Tyr207) and Bcr-Abl phosphorylation (Bcr (Tyr177)) using western blot analysis. (a) Cells incubated wit IM ± BafA1 (typical analysis). (b) Quantitative analysis of western blots using densitometry. (c) Cells incubated wit NIL ± BafA1 (typical analysis). (d) Quantitative analysis of western blots using densitometry. (e) Cells incubated wit DAS ± BafA1 (typical analysis). (f) Quantitative analysis of western blots using densitometry. Columns represent the means and standard deviations of four independent experiments.
Figure 4
Figure 4
The effect of the lysosomal sequestration of TKIs on the inhibition of Bcr-Abl signaling. Cells were incubated with various TKI concentrations in the absence or presence BafA1. After 6 h, cell extracts were analyzed for CrkL phosphorylation (Tyr207) and Bcr-Abl phosphorylation (Bcr (Tyr177)) using western blot analysis. (a) Cells incubated wit IM ± BafA1 (typical analysis). (b) Quantitative analysis of western blots using densitometry. (c) Cells incubated wit NIL ± BafA1 (typical analysis). (d) Quantitative analysis of western blots using densitometry. (e) Cells incubated wit DAS ± BafA1 (typical analysis). (f) Quantitative analysis of western blots using densitometry. Columns represent the means and standard deviations of four independent experiments.
Figure 4
Figure 4
The effect of the lysosomal sequestration of TKIs on the inhibition of Bcr-Abl signaling. Cells were incubated with various TKI concentrations in the absence or presence BafA1. After 6 h, cell extracts were analyzed for CrkL phosphorylation (Tyr207) and Bcr-Abl phosphorylation (Bcr (Tyr177)) using western blot analysis. (a) Cells incubated wit IM ± BafA1 (typical analysis). (b) Quantitative analysis of western blots using densitometry. (c) Cells incubated wit NIL ± BafA1 (typical analysis). (d) Quantitative analysis of western blots using densitometry. (e) Cells incubated wit DAS ± BafA1 (typical analysis). (f) Quantitative analysis of western blots using densitometry. Columns represent the means and standard deviations of four independent experiments.
Figure 5
Figure 5
The effect of SUN on the lysosomal sequestration capacity of cancer cells. Cells were cultured for 3 days in the presence of 1 µM SUN under standard conditions. Cells cultured in medium without SUN were taken as controls. The lysosomal sequestration capacity of cells was analyzed using flow cytometry upon staining with Lysotracker Red. Columns represent the means and standard deviations of four independent experiments. ** denotes a significant change in the mean fluorescence (p < 0.01) between respective SUN-stimulated and SUN-unstimulated cells.
Figure 6
Figure 6
Accumulation of TKIs in the lysosomes of SUN-stimulated cancer cells. Cells were cultured for 3 days in the presence of 1 µM SUN under standard conditions. Cells cultured in medium without SUN were taken as controls. (a) Accumulation of IM (absolute value). (b) Accumulation of IM (relative value). (c) Accumulation of NIL (absolute value). ND—not determined (due to limited NIL solubility). (d) Accumulation of NIL (relative value). (e) Accumulation of DAS (absolute value). (f) Accumulation of DAS (relative value). Columns represent the means and standard deviations of four independent experiments. * denotes a significant change in the intralysosomal IM content (p < 0.05) between the indicated cell lines. # denotes a significant change in the intralysosomal content of appropriate TKI (p < 0.05) between the groups indicated. ## denotes very significant change in the intralysosomal content of appropriate TKI (p < 0.01) between the groups indicated.
Figure 6
Figure 6
Accumulation of TKIs in the lysosomes of SUN-stimulated cancer cells. Cells were cultured for 3 days in the presence of 1 µM SUN under standard conditions. Cells cultured in medium without SUN were taken as controls. (a) Accumulation of IM (absolute value). (b) Accumulation of IM (relative value). (c) Accumulation of NIL (absolute value). ND—not determined (due to limited NIL solubility). (d) Accumulation of NIL (relative value). (e) Accumulation of DAS (absolute value). (f) Accumulation of DAS (relative value). Columns represent the means and standard deviations of four independent experiments. * denotes a significant change in the intralysosomal IM content (p < 0.05) between the indicated cell lines. # denotes a significant change in the intralysosomal content of appropriate TKI (p < 0.05) between the groups indicated. ## denotes very significant change in the intralysosomal content of appropriate TKI (p < 0.01) between the groups indicated.
Figure 7
Figure 7
The effect of SUN on the expression of lysosomal proteins in cancer cells. Cells were cultured for 3 days in the presence of 1 µM SUN under standard conditions. Cells cultured in medium without SUN were taken as controls. (a) Western blot analysis of LAMP1 (typical analysis). (b) Quantitative analysis of LAMP1 expression using densitometry. (c) Western blot analysis of LAMP2 (typical analysis). (d) Quantitative analysis of LAMP2 expression using densitometry. (e) Western blot analysis of vacuolar ATPase subunit B2 (typical analysis). (f) Quantitative analysis of vacuolar ATPase subunit B2 using densitometry. (g) Enzymatic activity of lysosomal APC. (h) Enzymatic activity of lysosomal GLB. Columns represent the means and standard deviations of four independent experiments.
Figure 7
Figure 7
The effect of SUN on the expression of lysosomal proteins in cancer cells. Cells were cultured for 3 days in the presence of 1 µM SUN under standard conditions. Cells cultured in medium without SUN were taken as controls. (a) Western blot analysis of LAMP1 (typical analysis). (b) Quantitative analysis of LAMP1 expression using densitometry. (c) Western blot analysis of LAMP2 (typical analysis). (d) Quantitative analysis of LAMP2 expression using densitometry. (e) Western blot analysis of vacuolar ATPase subunit B2 (typical analysis). (f) Quantitative analysis of vacuolar ATPase subunit B2 using densitometry. (g) Enzymatic activity of lysosomal APC. (h) Enzymatic activity of lysosomal GLB. Columns represent the means and standard deviations of four independent experiments.
Figure 7
Figure 7
The effect of SUN on the expression of lysosomal proteins in cancer cells. Cells were cultured for 3 days in the presence of 1 µM SUN under standard conditions. Cells cultured in medium without SUN were taken as controls. (a) Western blot analysis of LAMP1 (typical analysis). (b) Quantitative analysis of LAMP1 expression using densitometry. (c) Western blot analysis of LAMP2 (typical analysis). (d) Quantitative analysis of LAMP2 expression using densitometry. (e) Western blot analysis of vacuolar ATPase subunit B2 (typical analysis). (f) Quantitative analysis of vacuolar ATPase subunit B2 using densitometry. (g) Enzymatic activity of lysosomal APC. (h) Enzymatic activity of lysosomal GLB. Columns represent the means and standard deviations of four independent experiments.
Figure 8
Figure 8
Lysosomal sequestration of TKIs in SUN-stimulated cells and extracellular drug concentrations. (a) The effect of the lysosomal sequestration of IM in SUN-stimulated cells on its extracellular concentration. (b) The effect of the lysosomal sequestration of NIL in SUN-stimulated cells on its extracellular concentration. ND—not determined (due to limited NIL solubility). (c) The effect of the lysosomal sequestration of DAS in SUN-stimulated cells on its extracellular concentration. Columns represent the means and standard deviations of four independent experiments. * denotes a significant change in the decrease in extracellular IM content (p < 0.05) between the cell lines indicated. # denotes significant change in the decrease in extracellular IM content (p < 0.05) between the groups indicated. ## denotes very significant change in the decrease in extracellular IM content (p < 0.01) between the groups indicated.
Figure 9
Figure 9
The effect of the lysosomal sequestration of IM on Bcr-Abl signaling in SUN-stimulated cancer cells. SUN-stimulated cells were incubated with various TKIs concentration in the absence or presence BafA1. After 6 h, cell extracts were analyzed for the CrkL phosphorylation (Tyr207) and Bcr-Abl phosphorylation (Bcr (Tyr177)) using western blot analysis. (a) Cells incubated wit IM ± BafA1 (typical analysis). (b) Quantitative analysis of western blots using densitometry. (c) Cells incubated wit NIL ± BafA1 (typical analysis). (d) Quantitative analysis of western blots using densitometry. (e) Cells incubated wit DAS ± BafA1 (typical analysis). (f) Quantitative analysis of western blots using densitometry. Columns represent the means and standard deviations of four independent experiments.
Figure 9
Figure 9
The effect of the lysosomal sequestration of IM on Bcr-Abl signaling in SUN-stimulated cancer cells. SUN-stimulated cells were incubated with various TKIs concentration in the absence or presence BafA1. After 6 h, cell extracts were analyzed for the CrkL phosphorylation (Tyr207) and Bcr-Abl phosphorylation (Bcr (Tyr177)) using western blot analysis. (a) Cells incubated wit IM ± BafA1 (typical analysis). (b) Quantitative analysis of western blots using densitometry. (c) Cells incubated wit NIL ± BafA1 (typical analysis). (d) Quantitative analysis of western blots using densitometry. (e) Cells incubated wit DAS ± BafA1 (typical analysis). (f) Quantitative analysis of western blots using densitometry. Columns represent the means and standard deviations of four independent experiments.
Figure 9
Figure 9
The effect of the lysosomal sequestration of IM on Bcr-Abl signaling in SUN-stimulated cancer cells. SUN-stimulated cells were incubated with various TKIs concentration in the absence or presence BafA1. After 6 h, cell extracts were analyzed for the CrkL phosphorylation (Tyr207) and Bcr-Abl phosphorylation (Bcr (Tyr177)) using western blot analysis. (a) Cells incubated wit IM ± BafA1 (typical analysis). (b) Quantitative analysis of western blots using densitometry. (c) Cells incubated wit NIL ± BafA1 (typical analysis). (d) Quantitative analysis of western blots using densitometry. (e) Cells incubated wit DAS ± BafA1 (typical analysis). (f) Quantitative analysis of western blots using densitometry. Columns represent the means and standard deviations of four independent experiments.

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