Polyphyllin I induces mitophagic and apoptotic cell death in human breast cancer cells by increasing mitochondrial PINK1 levels - PubMed (original) (raw)

. 2017 Feb 7;8(6):10359-10374.

doi: 10.18632/oncotarget.14413.

Ruo-Qiu Fu 1, Han-Ming Shen 3, Jing Zhou 1, Xiao-Ye Hu 1, Yan-Xia Liu 1, Yu-Nong Li 1, Hong-Wei Zhang 1, Xin Liu 1, Yan-Hao Zhang 1, Cheng Huang 4, Rong Zhang 2, Ning Gao 1

Affiliations

Polyphyllin I induces mitophagic and apoptotic cell death in human breast cancer cells by increasing mitochondrial PINK1 levels

Guo-Bing Li et al. Oncotarget. 2017.

Abstract

The molecular mechanisms underlying the anti-breast cancer effects of polyphyllin I, a natural compound extracted from Paris polyphylla rhizomes, are not fully understood. In the present study, we found that polyphyllin I induces mitochondrial translocation of DRP1 by dephosphorylating DRP1 at Ser637, leading to mitochondrial fission, cytochrome c release from mitochondria into the cytosol and, ultimately apoptosis. Polyphyllin I also increased the stabilization of full-length PINK1 at the mitochondrial surface, leading to the recruitment of PARK2, P62, ubiquitin, and LC3B-II to mitochondria and culminating in mitophagy. PINK1 knockdown markedly suppressed polyphyllin I-induced mitophagy and enhanced polyphyllin I-induced, DRP1-dependent mitochondrial fission and apoptosis. Furthermore, suppression of DRP1 by mdivi-1 or shRNA inhibited PINK1 knockdown/polyphyllin I-induced mitochondrial fragmentation and apoptosis, suggesting that PINK1 depletion leads to excessive fission and, subsequently, mitochondrial fragmentation. An in vivo study confirmed that polyphyllin I greatly inhibited tumor growth and induced apoptosis in MDA-MB-231 xenografts, and these effects were enhanced by PINK1 knockdown. These data describe the mechanism by which PINK1 contributes to polyphyllin I-induced mitophagy and apoptosis and suggest that polyphyllin I may be an effective drug for breast cancer treatment.

Keywords: DRP1; PINK1; mitochondrial fission; mitophagy; polyphyllin I.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1

Figure 1. Polyphyllin I induces apoptosis via the mitochondrial pathway in MDA-MB-231 cells

A-B. MDA-MB-231 cells were treated with various concentrations of polyphyllin I (PPI) for 9 h or with 8 μM PPI for different periods of time as indicated. Cells were stained using an Annexin V-FITC Apoptosis Detection Kit I or rhodamine 123, apoptosis and mitochondrial (Mt) membrane potential were detected with flow cytometry. Data are presented as mean ± SD (*P< 0.05 or **P< 0.01 vs. the control). C. Whole-cell lysates (WCL), mitochondrial (Mito), and cytosolic (Cyto) fractions were prepared and subjected to western blot analysis using antibodies against cleaved-PARP (C-PARP), cleaved-CASP9 (C-CASP9), cleaved-CASP3 (C-CASP3), and cytochrome c (Cyto C). Tubulin (whole-cell lysates) and COX IV (mitochondrial fraction) were used as the loading controls. D. MDA-MB-231 cells were transfected with RFP-mito plasmids and then exposed to PPI (8 μM) for 12 h. After immunostaining with Cyto C (Alexa Fluor 488, green), cells were examined by confocal microscopy. Scale bars: 10 μm.

Figure 2

Figure 2. Polyphyllin I induces mitochondrial fission and mitophagy

A. MDA-MB-231 cells were exposed to 8 μM PPI for different periods of time as indicated or treated with 20 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone) for 9 h. Mitochondria were then stained using MitoTracker Red CMXRos and observed under a confocal microscope with a live cell imaging chamber. Scale bars: 10 μm. B. Average mitochondrial length was determined for 30 cells in each experiment; 3 independent experiments are included. Data are presented as mean ± SD (*P<0.01 vs. the control; ##P< 0.01 vs. the control). C. MDA-MB-231 cells were cotransfected with RFP-mito and GFP-LC3 and treated with 8 μM PPI or 20 μM CCCP for 9 h. After immunostaining with LAMP1 (Alexa Fluor 647, blue), cells were examined by confocal microscopy. Scale bars: 10 μm. D. The percentage of cells in which mitophagy occurred was determined using 30 cells from each experiment; 3 independent experiments are included. Cells with more than five RFP-Mito, LC3, and LAMP1 colocalization puncta were designated mitophagy-positive. Data are presented as mean ± SD (*P< 0.01 vs. the control). E. MDA-MB-231 cells were treated with 8 μM PPI for 9 h and imaged using a transmission electron microscope. Scale bars: 2 μm. Mitochondria and mitophagosomes were marked by white arrows. F-G. MDA-MB-231 cells were treated with 8 μM PPI for different periods of time as indicated or with 20 μM CCCP for 9 h. Mitochondrial fractions and whole-cell lysates were then prepared and subjected to western blot analysis.

Figure 3

Figure 3. Polyphyllin I triggers PINK1/PARK2-dependent mitophagy

A. MDA-MB-231 cells were treated with 8 μM PPI for different periods of time as indicated, and PARK2, P62, and ubiquitin (UB) levels in mitochondrial fractions were determined by western blot. B. Cells were cotransfected with GFP-UB and RFP-mito and treated with 8 μM PPI for 9 h, after which PARK2 (Alexa Fluor 405, pink) and P62 (Alexa Fluor 647, blue) immunostaining was detected using confocal microscopy. C. Cells cotransfected with GFP-UB and RFP-LC3 were treated with 8 μM PPI for 9 h, after which PARK2 (Alexa Fluor 405, pink) and TOMM20 (Alexa Fluor 647, blue) immunostaining was detected using confocal microscopy. Scale bars: 10 μm. D-E. MDA-MB-231 cells were treated with 8 μM PPI for different periods of time as indicated; whole-cell lysates were then separated on 8% SDS-PAGE gels and analyzed by western blot using the anti-PINK1 antibody. Relative full-length (∼63 kDa) and cleaved (∼52 kDa) PINK1 levels were quantified by densitometry and normalized to Tubulin. The results were expressed as a percentage of control, which was set at 100%. Data are presented as mean ± SD (*P< 0.01 vs. the control). F. Cells were treated with 8 μM PPI for different periods of time as indicated, and whole-cell lysates were then subjected to western blot analysis. G. Cells were treated with 8 μM PPI for 9 h, after which mitochondrial fractions were prepared and subjected to immunoprecipitation using anti-PINK1 antibody; associated PARK2 was detected using immunoblotting. H. RFP-mito-expressing MDA-MB-231 cells were treated with 8 μM PPI for 9 h, and PINK1 (Alexa Fluor 488, green) and PARK2 (Alexa Fluor 405, pink) immunostaining were evaluated using confocal microscopy. Scale bars: 10 μm.

Figure 4

Figure 4. PINK1 knockdown combined with polyphyllin I treatment blocks mitophagy and increases mitochondrial fission and apoptosis

A. MDA-MB-231 cells stably expressing non-target shRNA (shCon) or PINK1 shRNA (shPINK1) were lysed and analyzed by western blot. B-C. shCon and shPINK1 cells were treated with or without 8 μM PPI for 6 h; whole-cell lysates and mitochondrial fractions were then prepared and subjected to western blot analysis. D-E. shCon and shPINK1 cells were cotransfected with RFP-mito and GFP-LC3, and then treated with 8 μM PPI for 6 h. LAMP1 (Alexa Fluor 647, blue) immunostaining was then detected using confocal microscopy. Scale bars: 10 μm. The percentage of cells in which mitophagy occurred was determined using 30 cells from each experiment; 3 independent experiments are included. The cells with more than five RFP-Mito, LC3, and LAMP1 colocalization puncta were designated mitophagy-positive. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI). F-G. shCon and shPINK1 cells were transfected with RFP-mito, and then treated with 8 μM PPI for 6 h. Mitochondria were observed using confocal microscopy. Scale bars: 10 μm. The average mitochondrial length was quantified as previously described. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI). H. shCon and shPINK1 cells were treated with 8 μM PPI for 6 h, and DRP1 levels in mitochondrial fractions were determined by immunoblotting. I. Cells were treated with or without 8 μM PPI for 6 h, and apoptosis was then measured by flow cytometry. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treatment with PPI). J-K. Whole-cell lysates, mitochondrial (Mito), and cytosolic (Cyto) fractions were prepared and subjected to western blot analysis.

Figure 5

Figure 5. Suppression of DRP1 by mdivi-1 or shRNA blocks PINK1 depletion- and polyphyllin I-induced alterations in mitochondrial fission, mitophagy and apoptosis

A. shCon and shPINK1 cells were pretreated with Mdivi-1 (50 μM) for 2 h, followed by treatment with 8 μM PPI for 6 h. Mitochondrial fractions (Mito) were prepared and subjected to western blot analysis. B-C. RFP-mito-expressing cells were treated as indicated in (A), and fluorescence images were evaluated by confocal microscopy. Scale bars: 10 μm. Average mitochondrial length was quantified as previously described. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI, ##P< 0.01 compared to shPINK1 cells treated with PPI). D-E. Cells were treated as indicated in (A); apoptosis was then measured by flow cytometry, and whole-cell lysates were prepared and subjected to western blot analysis. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI, ##P< 0.01 compared to shPINK1 cells treated with PPI). F. MDA-MB-231 cells were infected with shCon or shPINK1 and/or shDRP1 lentivirus; after selection with puromycin, cells were lysed and analyzed by western blot using the anti-DRP1 antibody. G. Cells were treated with 8 μM PPI for 6 h, and mitochondrial fractions were then prepared and subjected to western blot analysis. H-I. Cells were transfected with RFP-mito and then treated with 8 μM PPI for 6 h. Mitochondria were examined using confocal microscopy. Scale bars: 10 μm. Average mitochondrial length was quantified as previously described. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI, ##P< 0.01 compared to shPINK1 cells treated with PPI). J-K. Apoptosis was measured by flow cytometry, and whole-cell lysates were prepared and subjected to western blot analysis. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI, ##P< 0.01 compared to shPINK1 cells treated with PPI).

Figure 6

Figure 6. PINK1 depletion suppresses mitophagy and promotes mitochondrial fragmentation by dephosphorylating DRP1 at Ser637

A. shCon and shPINK1 cells were exposed to PPI (8 μM) for 6 h, after which whole-cell lysates were prepared and subjected to western blot analysis. Relative protein levels from 3 independent experiments were quantified by densitometry and normalized to Tubulin; the results are expressed as percentage of control level, which was set at 100%. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells, ##P< 0.01 compared to shCon cells treated with PPI). B. Cells were pretreated with the calcineurin inhibitor FK506 (1 μM) for 2 h, followed by treatment with 8 μM PPI for 6 h; calcineurin activity was then measured as described in the Methods. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI, ##P< 0.01 compared to shPINK1 cells treated with PPI). C. Cells were pretreated with the PKA inhibitor H89 (50 μM) for 2 h, followed by treatment with PPI (8 μM) for 6 h; whole-cell lysates were then prepared and subjected to western blot using the anti-phospho-PKA substrate (RRXS/T) antibody. D. Cells were pretreated with FK506 (1 μM) or H89 (50 μM) for 2 h and then exposed to PPI (8 μM) for 6 h, after which whole-cell lysates (WCL) and mitochondrial fractions (Mito) were prepared and subjected to western blot analysis. E-G. Cells were cotransfected with RFP-Mito and GFP-LC3 and treated as described in (D); fluorescence images were then evaluated using confocal microscopy. Scale bars: 10 μm. Percentages of mitophagy-positive cells and average mitochondrial length were measured as previously described. Data are presented as mean ± SD (*P< 0.01 compared to shCon cells treated with PPI, ##P< 0.05 or ##P< 0.01 compared to shPINK1 cells treated with PPI).

Figure 7

Figure 7. Polyphyllin I suppressed, and PINK1 knockdown further suppressed, tumor growth in a MDA-MB-231 xenograft model

A. Tumor volumes were measured every week and differed at the end of the treatment period (*P < 0.01 compared to shCon cells treated with PPI, ##P< 0.01 compared to shPINK1 cells treated with PPI). B. Representative image of tumors from each group. C. Body weight changes in mice during the 6 weeks of PPI treatment. There were no differences in body weights between the PPI-treated shCon and vehicle-treated shCon groups or between the PPI-treated shPINK1 group and shCon groups. D. Representative tumor tissues were sectioned and subjected to H&E staining, TUNEL assay, and immunohistochemistry staining for C-CASP3. Scale bars: 50 μm. E. Representative tumor tissues from each group were prepared and subjected to western blot using anti-DRP1, -PARK2, -LC3B, -PINK1, and -C-CASP3 antibodies.

Figure 8

Figure 8. A proposed model for polyphyllin I-induced mitophagic and apoptotic cell death in human breast cancer cells

Polyphyllin I induced mitochondrial translocation of DRP1 by dephosphorylating DRP1 at Ser637, which lead to mitochondrial fission and cytochrome c release from the mitochondria into the cytosol, in turn activating caspases and promoting apoptosis. Meanwhile, polyphyllin I also increased stabilization of full-length PINK1 at the mitochondrial surface, leading to recruitment of PARK2, P62, ubiquitin, and LC3B-II to the mitochondria and culminating in mitophagy. Moreover, PINK1 knockdown markedly suppressed mitophagy and enhanced polyphyllin I-induced, DRP1-dependent mitochondrial fission and apoptosis.

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