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. 2018 May 23;9(6):614.
doi: 10.1038/s41419-018-0571-4.

Docetaxel enhances lysosomal function through TFEB activation

Jianbin Zhang  1 , Jigang Wang  2   3 , Yin Kwan Wong  4 , Xin Sun  5 , Yun Chen  5 , Liming Wang  4 , Liu Yang  6 , Liqin Lu  5 , Han-Ming Shen  4 , Dongsheng Huang  7
Affiliations

Docetaxel enhances lysosomal function through TFEB activation

Jianbin Zhang et al. Cell Death Dis. .

Abstract

Docetaxel is an effective and commonly used chemotherapeutic drug for cancer. Autophagy has been reported to be involved in the anticancer mechanism of docetaxel. However, the effect of docetaxel on lysosomal function remains elusive. In the present study, we first found that docetaxel treatment enhances autophagic flux in different cancer cells. Moreover, docetaxel treatment activates lysosomal function and promotes its fusion with autophagosome. Second, doctaxel treatment activates TFEB (transcription factor EB), a key nuclear transcription factor in control of lysosome biogenesis and function. We found that docetaxel promotes TFEB nuclear translocation and increases its transcriptional activity while knockdown of TFEB impairs lysosomal activation by docetaxel. Thirdly, TFEB activation by docetaxel is mediated by ROS (reactive oxygen species) generation and scavenging of ROS suppresses TFEB activity and lysosomal function in docetaxel-treated cells. Finally, inhibition of lysosomal function leads to increased docetaxel-induced cell death, suggesting that lysosomal activation protects against docetaxel-mediated apoptosis. Taken together, our results provide novel insights into the regulatory mechanisms of docetaxel on lysosomes, which could facilitate the development of novel potential cancer therapeutic agents via lysosomal inhibition.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Docetaxel activates lysosomal function and induces autophagy in cancer cells.
a AGS cells were treated with docetaxel (10 nM) with or without CQ (chloroquine) for 12 h. Cells were then harvested for western blotting to examine LC3-II levels. α-tubulin served as a loading control. b Hela cells stably expressing GFP-LC3 were treated with docetaxel (10 nM, 12 h) and then cells were examined by confocal microscopy (scale bar 20 μm).The number of GFP-LC3 puncta was quantified (right panel). ** P < 0.01 c, d AGS cells were treated with docetaxel (10 nM) for 12 h followed by loading with LysoTracker Red DND-99 (50 nM) or LysoSensor Green DND-189 (1 μM) for 15 min. Fluorescence intensity was measured under confocal microscope (scale bar 10 μm) and quantified using flow cytometry (right panel). **P < 0.01 (e) AGS cells were treated with docetaxel (10 or 25 nM) for 12 h. AO staining was performed and analyzed using flow cytometry. The numeric data are presented as means ± SD from three independent experiments. Student’s t test, *P < 0.05, **P < 0.01. f AGS cells were treated with docetaxel (10 nM) for different time points (6, 12, or 24 h) and western blotting was performed to detect EGFR protein level. α-tubulin was used as loading control
Fig. 2
Fig. 2. Docetaxel promotes the fusion of autophagosome and lysosome in different cancer cells.
a AGS cells were treated with docetaxel (10 nM) for 12 h. After immunostaining with SQSTM1 (Alexa Fluor 488, green) and LAMP1 (Alexa Fluor 594, red), cells were examined by fluorescence microscopy (scale bar 20 μm). The colocalization of SQSTM1and LAMP1 was calculated and statistically analyzed (right panel). *P < 0.05. b Hela cells stably expressing GFP-LC3 were treated with docetaxel (10 nM, 12 h). After staining with LysoTracker Red, cells were examined by confocal microscopy (scale bar 20 μm).The colocalization of GFP-LC3 puncta (green) and lysosome (red) was calculated and statistically analyzed (right panel). *P < 0.05. c, d Docetaxel increased the RFP signal in the L929-tfLC3 cells. Cells were treated with docetaxel (10 nM) for 12 h and then cells were examined under confocal microscope (scale bar 20 μm). The ratio of RFP to GFP was calculated and statistically analyzed. *P < 0.05. e AGS cells were treated with docetaxel (10 nM, 12 h) in the presence or absence of thapsigargin (100 nM). After loading with Magic RedTM for cathepsin B, cell fluorescence was measured by flow cytometry. *P < 0.05; **P < 0.01
Fig. 3
Fig. 3. Docetaxel increases TFEB transcriptional activity.
a Docetaxel treatment enhanced TFEB nuclear translocation (10 nM; 12 h). Live-cell imaging of GFP-TFEB (green) and DAPI (blue) in AGS cells showed an enrichment of the GFP-TFEB signal in the nucleus (scale bar 20 μm). The ratio of nuclear to cytosolic TFEB was calculated and statistically analyzed. *P < 0.05. b The TFEB-luciferase reporter constructs were first transiently transfected into AGS cells and then cells were treated with docetaxel (10 nM) for 12 h. The relative luciferase activity was measured using Dual-Luciferase® Reporter assay. RLU stands for relative luciferase units. Error bars represent the standard deviation from two independent experiments. c AGS cells were treated with docetaxel (10 nM; 12 h) followed by cell harvesting and RNA extraction. Changes in mRNA levels of some known TFEB target genes were measured using real-time PCR. GAPDH served as a loading control. All values are means ± SD at least three independent experiments. Student’s t test, *P < 0.05; **P < 0.01. d As in (c), cells were harvested for western blotting to detect the ATP6V1A and UVRAG levels. α-tubulin was used as loading control
Fig. 4
Fig. 4. Knockdown of TFEB impairs lysosomal activation by docetaxel.
a, b AGS cells were transfected with scrambled or TFEB siRNA for 48 h and then treated with docetaxel (10 nM) for 12 h. After immunostaining with SQSTM1 (Alexa Fluor 488, green) and LAMP1 (Alexa Fluor 594, red), cells were examined by fluorescence microscopy (scale bar 20 μm). The colocalization of SQSTM1 and LAMP1 was calculated and statistically analyzed. *P < 0.05. c As in (a), at the indicated time, cells were loaded with Magic Red cathepsin B for 15 min. Fluorescence intensity of 10,000 cells per sample was measured by flow cytometry. *P < 0.05
Fig. 5
Fig. 5. Docetaxel enhances TFEB activity through ROS generation.
a AGS cells were treated with docetaxel (10 nM) with or without NAC (5 mM) for 12 h followed by incubation with CM-H2DCFDA. The cells’ fluorescence intensity was then measured using flow cytometry. *P < 0.05. b As described in (a), nuclear and cytosolic fractions from both control and docetaxel-treated cells were probed for TFEB and then quantified. The same membrane was then stripped and reprobed for α-tubulin or Lamin AC as loading control. c AGS cells were transiently transfected with the TFEB-luc reporter construct. After 24 h, the cells were treated with docetaxel (10 nM) for another 12 h with or without NAC (5 mM) and the relative luciferase activity was measured. RLU refers to relative luciferase units. *P < 0.05. d As in (a), after treatment, AGS cells were harvested for RNA extraction. Real-time PCR was used to determine mRNA level changes in known TFEB target genes. GAPDH served as a loading control. All values are means ± SD at least three independent experiments. Student’s t test, *P < 0.05
Fig. 6
Fig. 6. Lysosomal inhibition sensitizes docetaxel-induced cell death.
a AGS cells were transfected with scrambled or ATP6V1A siRNA for 48 h and then treated with docetaxel (10 nM; 12 h). After loading with LysoTracer Red, cells were examined under confocal microscope. Scale bar 20 μm. b, c As in (a), cells were treated with docetaxel (25 nM) for 24 h. Changes in AGS cell morphology following each treatment were examined and captured with an inverted microscope (scale bar 50 μm). Cell pellets were subsequently collected and quantification of cell death was performed using Annexin V staining. Statistical significance (*P < 0.05) is indicated in the bar chart. d As in (b), after treatment, cells were harvested and western blotting was performed to detect ATP6V1A and EGFR levels. α-tubulin was used as loading control. e The schematic model of the enhanced function of lysosome by docetaxel
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