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. 2017 Jun 8:8:351.
doi: 10.3389/fphar.2017.00351. eCollection 2017.

Tetrandrine, an Activator of Autophagy, Induces Autophagic Cell Death via PKC-α Inhibition and mTOR-Dependent Mechanisms

Vincent Kam Wai Wong  1 , Wu Zeng  1 , Juan Chen  2 , Xiao Jun Yao  1 , Elaine Lai Han Leung  1 , Qian Qian Wang  1 , Pauline Chiu  3 , Ben C B Ko  4 , Betty Yuen Kwan Law  1
Affiliations

Tetrandrine, an Activator of Autophagy, Induces Autophagic Cell Death via PKC-α Inhibition and mTOR-Dependent Mechanisms

Vincent Kam Wai Wong et al. Front Pharmacol. .

Abstract

Emerging evidence suggests the therapeutic role of autophagic modulators in cancer therapy. This study aims to identify novel traditional Chinese medicinal herbs as potential anti-tumor agents through autophagic induction, which finally lead to autophagy mediated-cell death in apoptosis-resistant cancer cells. Using bioactivity-guided purification, we identified tetrandrine (Tet) from herbal plant, Radix stephaniae tetrandrae, as an inducer of autophagy. Across a number of cancer cell lines, we found that breast cancer cells treated with tetrandrine show an increase autophagic flux and formation of autophagosomes. In addition, tetrandrine induces cell death in a panel of apoptosis-resistant cell lines that are deficient for caspase 3, caspase 7, caspase 3 and 7, or Bax-Bak respectively. We also showed that tetrandrine-induced cell death is independent of necrotic cell death. Mechanistically, tetrandrine induces autophagy that depends on mTOR inactivation. Furthermore, tetrandrine induces autophagy in a calcium/calmodulin-dependent protein kinase kinase-β (CaMKK-β), 5' AMP-activated protein kinase (AMPK) independent manner. Finally, by kinase profiling against 300 WT kinases and computational molecular docking analysis, we showed that tetrandrine is a novel PKC-α inhibitor, which lead to autophagic induction through PKC-α inactivation. This study provides detailed insights into the novel cytotoxic mechanism of an anti-tumor compound originated from the herbal plant, which may be useful in promoting autophagy mediated- cell death in cancer cell that is resistant to apoptosis.

Keywords: PKC-α; apoptosis-resistant; autophagy; mTOR; tetrandrine.

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Figures

Figure 1
Figure 1
Structure of tetrandine and fangchinoline.
Figure 2
Figure 2
Tetrandrine (Tet) induces autophagy in multiple cell lines. (A) A panel of cancer cells lines expressing GFP-LC3 were treated with the indicated compounds at 5 μM for 16 h. (B) Both ATG7+/+ wild-type and ATG7−/− deficient (KO) MEFs were transiently transfected with the GFP-LC3 plasmid for 24 h and then treated with DMSO (Ctrl) or 5 μM Tet for 16 h. The cells were then fixed for fluorescence imaging and cells counting. Magnification: x63. (C) Representative electron micrographs showing the ultrastructures of MCF-7 cells treated with Tet (5 μM) for 0–48 h. Magnification: x5600. Arrows, double-membraned autophagosomes. Magnification: x24000. (D) MCF-7 cells were treated with Tet (5 μM) and lysosomal protease inhibitors (E64d and pepstatin A) (10 μg/mL each), either alone or in combination, for 0–24 h. Cell lysates were analyzed by western blot for LC3 conversion. (E) Induction of p62 by Tet (5 μM) in MCF-7 cells. Cell lysates were analyzed by western blot for p62 and β-actin respectively. The results are representative of three independent experiments.
Figure 3
Figure 3
Tetrandrine induces autophagy through the mTOR-dependent pathway. (A) Quantitation on the percentage of CACNA1C gene knockdown MCF-7 cells with GFP-LC3 puncta formation after treatment with Tet (5 μM) for 16 h. (B) Bar chart showed the percentage of GFP-positive cells with GFP-LC3 puncta after 5 μM of Iso-Tet or Tet treatment in the presence or absence of STO-609 (25 μM), or (C) Bapta/AM (25 μM) for 16 h. (D) MCF-7 cells were treated with 0–20 μM of Tet for 16 h and the cell lysates were analyzed for p-p70S6K and p70S6K, (E) p-4EBP and 4EBP, (F) p-AMPK and AMPK, and β-actin respectively. Cells treated with alcar (1 mM) and rapamycin (rap) (300 nM) for 8 and 16 h respectively were used as positive control. Solid lines represent chopped image from the same gel under same exposure. (G) Percentage of GFP-positive cells with GFP-LC3 puncta formation after 5 μM of Iso-Tet or Tet treatment in the presence or absence of compound C (10 μM) for 16 h. Treated cells were fixed for fluorescence imaging and cells counting. Columns, means of three independent experiments; bars, SEM. ***p < 0.001.
Figure 4
Figure 4
Tetrandrine induces autophagy through PKC-α inhibition. (A) Kinase inhibition profile of Tet was determined by measuring the residual activity values of 300 wild-type protein kinases. Bar chart represents the identification of the kinases whose residual activity dropped below 50% when treated with 1 μM of tetrandrine. Receptor tyrosine kinase (EpHA5), protein-tyrosine kinase (FES), fibroblast growth factor receptor 2 (FGFR2) and PKC-α with residual activity dropped below 50% were shown. (B) The binding modes of (a) compound 28, (b) tetrandrine, or (c) fangchinoline with PKC-α, and (d) the aligned conformations of tetrandrine and fangchinoline in complex with PKC-α. (C) HeLa and MCF-7 cells were transfected with control negative siRNA sequence or PKC-α-siRNA together with GFP-LC3 plasmid for 24 h; cells were then fixed for fluorescence microscopic analysis, and (D) western blot analysis for targeting PKC-α, p-PKC-α and LC3-II in MCF-7 cells, respectively. Bar chart represents the percentage of cells with GFP-LC3 puncta formation. (E) MCF-7 cells transiently transfected with the GFP-LC3 plasmid for 24 h were treated with DMSO (Ctrl) or 5 μM of Tet in the presence or absence of PKC-α activator, PMA, with the indicated concentrations for 16 h. Arrows indicated cells with GFP-LC3 puncta formation. Bar chart represents the percentage cells with GFP-LC3 puncta formation. Columns, means of three independent experiments; bars, SEM. ***p < 0.001. (F) MCF-7 cells were treated with DMSO (Ctrl) or 5 μM Tet in the presence or absence of PMA (10 ng/ml), for 16 h before subjecting to western blot analysis.
Figure 5
Figure 5
Tetrandrine induces autophagic cell death in apoptosis resistant cells. (A) Cytotoxicity of Tet on ATG7 wild-type and ATG7−/− MEFs as measured by MTT assay, and (B) flow analysis after annexin V staining. (C) Cytotoxicity (IC50) of Tet on caspase 3/7 or caspase 3 and 7 knockout cells (left panel), caspase 8 knockout cells (middle panel), and Bax-Bak wild-type and DKO deficient MEFs cells (right panel). (D) Tet-mediated cell cytotoxicity (IC50) in Bax-Bak DKO MEFs with the presence of 10 μM necrostatin (Nec-1) for 24 h as measured by MTT assay, and (E) flow cytometry analysis after annexin V staining. Results shown are the means ± S.E.M. of three independent experiments. **p < 0.01; *p < 0.05.
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