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. 2016 Nov 15;7(46):75659-75671.
doi: 10.18632/oncotarget.12318.

Curcumin targets the TFEB-lysosome pathway for induction of autophagy

Jianbin Zhang  1   2   3 , Jigang Wang  2 , Jian Xu  3 , Yuanqiang Lu  4 , Jiukun Jiang  4 , Liming Wang  2 , Han-Ming Shen  2   5 , Dajing Xia  3
Affiliations

Curcumin targets the TFEB-lysosome pathway for induction of autophagy

Jianbin Zhang et al. Oncotarget. .

Abstract

Curcumin is a hydrophobic polyphenol derived from the herb Curcumalonga and its wide spectrum of pharmacological activities has been widely studied. It has been reported that Curcumin can induce autophagy through inhibition of the Akt-mTOR pathway. However, the effect of Curcumin on lysosome remains largely elusive. In this study, we first found that Curcumin treatment enhances autophagic flux in both human colon cancer HCT116 cells and mouse embryonic fibroblasts (MEFs). Moreover, Curcumin treatment promotes lysosomal function, evidenced by the increased lysosomal acidification and enzyme activity. Second, Curcumin is capable of suppressing the mammalian target of rapamycin (mTOR). Interestingly, Curcumin fails to inhibit mTOR and to activate lysosomal function in Tsc2-/-MEFs with constitutive activation of mTOR, indicating that Curcumin-mediated lysosomal activation is achieved via suppression of mTOR. Third, Curcumin treatment activates transcription factor EB (TFEB), a key nuclear transcription factor in control of autophagy and lysosome biogenesis and function, based on the following observations: (i) Curcumin directly binds to TFEB, (ii) Curcumin promotes TFEB nuclear translocation; and (iii) Curcumin increases transcriptional activity of TFEB. Finally, inhibition of autophagy and lysosome leads to more cell death in Curcumin-treated HCT116 cells, suggesting that autophagy and lysosomal activation serves as a cell survival mechanism to protect against Curcumin-mediated cell death. Taken together, data from our study provide a novel insight into the regulatory mechanisms of Curcumin on autophagy and lysosome, which may facilitate the development of Curcumin as a potential cancer therapeutic agent.

Keywords: Curcumin; TFEB; autophagy; lysosome; mTOR.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors confirm that there are no conflicts of interest.

Figures

Figure 1
Figure 1. Curcumin induces autophagy
A. HCT116 cells were treated with Curcumin (5, 10 or 20 μM) for 12 hours. B. HCT116 cells were treated with Curcumin (10 μM, 12 hrs) in the presence or absence of bafilomycin A1 (Baf, 15 nM). Cells were harvested for western blotting to determine LC3-II and p62 level. β-actin was used as a loading control. C. and D. Mouse embryonic fibroblasts (MEFs) with stable expression of GFP-LC3 were treated with Curcumin (20 μM, 12 hrs) with or without bafilomycin A1 (Baf, 15 nM) and GFP-LC3 puncta in treated cells was measured and quantified by confocal microscopy (Scale bar 10 μm). All values are means ± SD at least 3 independent experiments. Student's t test, * P < 0.05.
Figure 2
Figure 2. Curcumin activates lysosomal function
A. HCT116 cells were treated with Curcumin (10 μM) at indicated concentration for 12 hours and then stained with LysoTracker Red DND-99 (50 nM) for 15 minutes. Fluorescence intensity of treated cells was measured by confocal microscopy (left) or flow cytometry (right). The numeric data are presented as means ± SD from 2 independent experiments. Student's t-test, * P < 0.05. B. and C. as indicated in A., after 12 hour treatment, AO staining and Magic Red (Cathepsin B) were performed and analysed using flow cytometry. D. HCT116 cells were treated with Curcumin (10 μM) for different time (6, 12 or 24 hrs) and EGFR was analysed using western blotting. β-actin was used as a loading control.
Figure 3
Figure 3. Activation of lysosomal function by Curcumin is mTOR-dependent
A. HCT116 cells were treated with Curcumin (10 μM) as indicated. B. Tsc2+/+ and Tsc2−/− MEFs were treated with Curcumin (20 μM) for 12 hours. Cells were harvested and lysed for western blotting to determine phospho-Akt (Ser473) and phospho-S6 (Ser235/236). β-actin was used as a loading control. C. as indicated in (B), LysoTracker Red staining was performed using confocal microscopy (Scale bar 10 μm) while MagicRed Cathepsin B D. and AO staining E. were also performed and were treated with Curcumin (20 μM) for 12 hours and analysed in Tsc2+/+ and Tsc2−/− MEFs using flow cytometry. All values are means ± SD at least 3 independent experiments and analysed using Student's t test (*P < 0.05).
Figure 4
Figure 4. Curcumin directly targets TFEB for activation
A. HCT116 cells were treated with 10 μM Curcumin for 12 hours and cell lysates were prepared followed by immunoblotting for TFEB and β-actin (up panel). HCT116 cells were labeled with Curcumin-probe (30 μM) for 4 hours and western blotting was performed to validate Curcumin-probe targeted TFEB (down panel). B. Enhanced TFEB nuclear translocation in response to Curcumin treatment (10 μM; 12 hours). Live imaging of GFP-TFEB (green) and DAPI (blue) in HCT116 cells showed an enrichment of the GFP-TFEB signal in the nuclear. Five fields containing 20 to 30 cells were analyzed for TFEB nuclear localization. Scale bar, 10 μm. C. HCT116 cells were treated with 10 μM Curcumin as indicated. Cytosolic and nuclear fraction from control and Curcumin-treated cells were probed for TFEB. The same membrane was then stripped and reprobed for α-tubulin or Lamin AC as loading control. D. HCT116 cells were transient transfected with the TFEB-3x Flag (kindly provided by Dr. A Ballabio) and then treated with 10 μM Curcumin for 12 hours. Cells were lysed and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting for 14-3-3. TFEB was also determined using anti-FLAG antibody. E. HCT116 cells were transiently transfected with the TFEB-luc reporter construct (kindly provided by Dr. A Ballabio). After 24 hours, the cells were treated with Curcumin (10 μM) for another 12 hours and the relative luciferase activity was measured. RLU refers to relative luciferase units. Error bars represent the standard deviation from two independent experiments. F. HCT116 cells were treated with Curcumin (10 μM) for 12 hours and cells were harvested for RNA extraction. Real-time PCR was performed to determine mRNA level changes in the known TFEB target genes, such as Lamp1, Atp6v1a, Uvrag and Atg9b. Gapdh was used as a loading control. All values are means ± SD at least 3 independent experiments. Student's t test, * P < 0.05.
Figure 5
Figure 5. Lysosomal inhibition sensitizes curcumin-induced cell death
HCT116 cells were treated with Curcumin (20 μM), with or without bafilomycin A1 (Baf, 25 nM) or CA-074 (25 μM) for 24 hours. A. Morphological changes of HCT116 cells with respective treatments were examined and photographed with an inverted microscope (Scale bar 200 μm). B. Cell pellets were subsequently collected and cell death was quantified using propidium iodide (PI) exclusion staining. Statistical significance (* P < 0.05) is indicated in the bar chart. C. Cells were then harvested for protein analysis. Cell lysates were resolved in SDS-PAGE and probed with specific antibody against PARP1.
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