CoChat
Sign Up
Cited by 60

The DOI matches its Crossref record; 10 of the 10 references it cites were found in a registry or an index.

Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

Gonzalez, Yanira, Aryal, Baikuntha, Chehab, Leena, Rao, V. Ashutosh·2014·Oncotarget
DOI 10.18632/oncotarget.1715PMC PMC4039229
Open this paper in CoChat — free account

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.18632/oncotarget.1715

Cites

10 of 10 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Knockdown of Atg7 inhibits MitoQ-induced autophagy in breast cancer cells. MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 48 hr to downregulate Beclin-1 or Atg7. Cells were treated with (A) 10 nM Beclin-1 or (B) 10 nM Atg7 siRNA. Following 48 hr of (C) Beclin siRNA or (D) Atg7 siRNA treatment, cells were treated with MitoQ (1 or 5 μM) for 24 hr. Non-targeting pool (NTP) siRNA was used as a control. MitoQ-induced autophagy was measured using the levels of the autophagosome-associated LC3-II protein as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (E) Representative confocal images and (F) LC3-II intensity per cell was used to confirm autophagy. n = 50 cells. Scale bar = 10 μm. Error bars represent S.D. *statistical significance compared with NTP siRNA cells (p<0.001).
Figure 1 Knockdown of Atg7 inhibits MitoQ-induced autophagy in breast cancer cells. MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 48 hr to downregulate Beclin-1 or Atg7. Cells were treated with (A) 10 nM Beclin-1 or (B) 10 nM Atg7 siRNA. Following 48 hr of (C) Beclin siRNA or (D) Atg7 siRNA treatment, cells were treated with MitoQ (1 or 5 μM) for 24 hr. Non-targeting pool (NTP) siRNA was used as a control. MitoQ-induced autophagy was measured using the levels of the autophagosome-associated LC3-II protein as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (E) Representative confocal images and (F) LC3-II intensity per cell was used to confirm autophagy. n = 50 cells. Scale bar = 10 μm. Error bars represent S.D. *statistical significance compared with NTP siRNA cells (p<0.001).CC BY · PubMed Central
Figure 2 Knockout of Atg7 inhibits autophagy and increases apoptosis following MitoQ treatment in mouse embryonic fibroblasts (A) Levels of Atg7 were confirmed in wild type and Atg7−/−mouse embryonic fibroblast (MEF) cells. (B) Levels of LC3-II were used to compare autophagy induced by MitoQ (1 or 5 μM) following 24 hr of treatment in Atg7+/+ and Atg7−/− MEF cells. Rapamycin (10 μM) was used as a positive control. (C) Annexin V and propidium iodide staining was used to measure induction of apoptosis by 5 μM MitoQ following 24 and 48 hr of treatment. Vp16 (1 μM) was used as a positive control. *statistical significance (p<0.05) compared with normalized control of Atg7+/+or Atg7−/−.
Figure 2 Knockout of Atg7 inhibits autophagy and increases apoptosis following MitoQ treatment in mouse embryonic fibroblasts (A) Levels of Atg7 were confirmed in wild type and Atg7−/−mouse embryonic fibroblast (MEF) cells. (B) Levels of LC3-II were used to compare autophagy induced by MitoQ (1 or 5 μM) following 24 hr of treatment in Atg7+/+ and Atg7−/− MEF cells. Rapamycin (10 μM) was used as a positive control. (C) Annexin V and propidium iodide staining was used to measure induction of apoptosis by 5 μM MitoQ following 24 and 48 hr of treatment. Vp16 (1 μM) was used as a positive control. *statistical significance (p<0.05) compared with normalized control of Atg7+/+or Atg7−/−.CC BY · PubMed Central
Figure 2 (C) Annexin V and propidium iodide staining was used to measure induction of apoptosis by 5 μM MitoQ following 24 and 48 hr of treatment. Vp16 (1 μM) was used as a positive control. *statistical significance (p<0.05) compared with normalized control of Atg7+/+or Atg7−/−.
Figure 2 (C) Annexin V and propidium iodide staining was used to measure induction of apoptosis by 5 μM MitoQ following 24 and 48 hr of treatment. Vp16 (1 μM) was used as a positive control. *statistical significance (p<0.05) compared with normalized control of Atg7+/+or Atg7−/−.CC BY · PubMed Central
Figure 3 Autophagy-deficient cells have higher levels of ROS Cells were treated with 5 μM MitoQ for 0.5, 1, 6, or 24 hr. 10 μM hydroethidine (HE) was added for the last 0.5 hr of the MitoQ treatment. (A) Levels of ROS induced by MitoQ in MEF Atg7+/+or Atg7−/− were measured by detection of HE and its oxidation products by HPLC (left traces: absorption signals at 290 nm, righ traces: fluorescence signals, with excitation/emission settings: 356 nm/440 nm for 0-2.5 min and 490 nm/ 567 nm for 2.5-6 min). (B) Graphs show quantitative analysis of HE, 2-OH-E+, E+, and E+-E+levels in cell lysates after normalization to protein concentration. Error bars represent S.D. *statistical significance (p<0.05).
Figure 3 Autophagy-deficient cells have higher levels of ROS Cells were treated with 5 μM MitoQ for 0.5, 1, 6, or 24 hr. 10 μM hydroethidine (HE) was added for the last 0.5 hr of the MitoQ treatment. (A) Levels of ROS induced by MitoQ in MEF Atg7+/+or Atg7−/− were measured by detection of HE and its oxidation products by HPLC (left traces: absorption signals at 290 nm, righ traces: fluorescence signals, with excitation/emission settings: 356 nm/440 nm for 0-2.5 min and 490 nm/ 567 nm for 2.5-6 min). (B) Graphs show quantitative analysis of HE, 2-OH-E+, E+, and E+-E+levels in cell lysates after normalization to protein concentration. Error bars represent S.D. *statistical significance (p<0.05).CC BY · PubMed Central
Figure 4 Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7+/+or Atg7−/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p<0.05) compared with NTP siRNA cells.
Figure 4 Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7+/+or Atg7−/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p<0.05) compared with NTP siRNA cells.CC BY · PubMed Central
Figure 5 Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.
Figure 5 Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.CC BY · PubMed Central
Figure 6 Schematic model for the proposed autophagy mechanism induced by MitoQ We propose a feedback autophagy and cellular response to excessive oxidative stress following cytotoxic exposure to MitoQ in cancer cells. Please refer to the discussion section for a detailed description.
Figure 6 Schematic model for the proposed autophagy mechanism induced by MitoQ We propose a feedback autophagy and cellular response to excessive oxidative stress following cytotoxic exposure to MitoQ in cancer cells. Please refer to the discussion section for a detailed description.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 10 references

Research works printed but not linked: 34

  1. Gibson, 2012, Reactive oxygen species regulation of autophagy in cancer: implications for cancer treatment, Free Radic Biol Med, 53, 1399, 10.1016/j.freeradbiomed.2012.07.011 · DOI
  2. Kimmelman, 2011, The dynamic nature of autophagy in cancer, Genes Dev, 25, 1999, 10.1101/gad.17558811 · DOI
  3. White, 2011, Principles and current strategies for targeting autophagy for cancer treatment, Clin Cancer Res, 17, 654, 10.1158/1078-0432.CCR-10-2634 · DOI
  4. Schulman, 2011, Atg8 transfer from Atg7 to Atg3: a distinctive E1-E2 architecture and mechanism in the autophagy pathway, Mol Cell, 44, 451, 10.1016/j.molcel.2011.08.034 · DOI
  5. Stenmark, 2012, p62 at the interface of autophagy, oxidative stress signaling, and cancer, Antioxid Redox Signal, 17, 786, 10.1089/ars.2011.4394 · DOI
  6. Johansen, 2007, p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy, The Journal of biological chemistry, 282, 24131, 10.1074/jbc.M702824200 · DOI · Paper page
  7. Johansen, 2005, p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death, J Cell Biol, 171, 603, 10.1083/jcb.200507002 · DOI · Paper page
  8. Yamamoto, 2011, Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution, Genes Cells, 16, 123, 10.1111/j.1365-2443.2010.01473.x · DOI · Paper page
  9. Kominami, 2010, The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1, Nat Cell Biol, 12, 213, 10.1038/ncb2021 · DOI · Paper page
  10. McMahon, 2009, NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer, Trends Biochem Sci, 34, 176, 10.1016/j.tibs.2008.12.008 · DOI
  11. Diaz-Meco, 2009, p62 at the crossroads of autophagy, apoptosis, and cancer, Cell, 137, 1001, 10.1016/j.cell.2009.05.023 · DOI · Paper page
  12. Komatsu, 2011, Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells, J Cell Biol, 193, 275, 10.1083/jcb.201102031 · DOI
  13. Ichimura, 2012, p62/SQSTM1/A170: physiology and pathology, Pharmacol Res, 66, 457, 10.1016/j.phrs.2012.07.004 · DOI
  14. Lee, 2013, Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy, Mol Cell, 51, 618, 10.1016/j.molcel.2013.08.003 · DOI
  15. Garcia-Manero, 2013, Oncogenic functions of the transcription factor Nrf2, Free radical biology & medicine
  16. Zhong, 2010, Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy, Autophagy, 6, 614, 10.4161/auto.6.5.12189 · DOI · Paper page
  17. Zhu, 2012, Hydrogen sulfide as an endogenous modulator in mitochondria and mitochondria dysfunction, Oxid Med Cell Longev, 2012, 878052
  18. Rhee, 2013, Sestrins activate Nrf2 by promoting p62-dependent autophagic degradation of Keap1 and prevent oxidative liver damage, Cell metabolism, 17, 73, 10.1016/j.cmet.2012.12.002 · DOI · Paper page
  19. Murphy, 1997, Selective targeting of bioactive compounds to mitochondria, Trends Biotechnol, 15, 326, 10.1016/S0167-7799(97)01068-8 · DOI · Paper page
  20. Kalyanaraman, 2012, Mitochondria-targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death, Cancer Res, 72, 2634, 10.1158/0008-5472.CAN-11-3928 · DOI
  21. Srinivasula, 2011, Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation, Proceedings of the National Academy of Sciences of the United States of America, 108, 1427, 10.1073/pnas.1014156108 · DOI
  22. Codogno, 2010, Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview, Am J Physiol Cell Physiol, 298, C776, 10.1152/ajpcell.00507.2009 · DOI
  23. Yamamoto, 2012, Keap1 degradation by autophagy for the maintenance of redox homeostasis, Proceedings of the National Academy of Sciences of the United States of America, 109, 13561, 10.1073/pnas.1121572109 · DOI · Paper page
  24. Ghidoni, 2008, Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells, Cell Death Differ, 15, 1318, 10.1038/cdd.2008.51 · DOI · Paper page
  25. Puyal, 2011, Beclin 1-independent autophagy contributes to apoptosis in cortical neurons, Autophagy, 7, 1115, 10.4161/auto.7.10.16608 · DOI
  26. Nanjundan, 2010, Arsenic trioxide induces a beclin-1-independent autophagic pathway via modulation of SnoN/SkiL expression in ovarian carcinoma cells, Cell Death Differ, 17, 1867, 10.1038/cdd.2010.53 · DOI
  27. Proikas-Cezanne, 2012, Canonical and non-canonical autophagy: variations on a common theme of self-eating?, Nat Rev Mol Cell Biol, 13, 7, 10.1038/nrm3249 · DOI
  28. He, 2009, Autophagy is essential for mitochondrial clearance in mature T lymphocytes, Journal of immunology, 182, 4046, 10.4049/jimmunol.0801143 · DOI
  29. Finkel, 2009, Mitochondrial dysfunction and oxidative stress mediate the physiological impairment induced by the disruption of autophagy, Aging, 1, 425, 10.18632/aging.100038 · DOI
  30. Zhang, 2010, A noncanonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction between Keap1 and p62, Mol Cell Biol, 30, 3275, 10.1128/MCB.00248-10 · DOI
  31. Shin, 2012, Assurance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade, EMBO Rep, 13, 150, 10.1038/embor.2011.246 · DOI
  32. Hannink, 2005, Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway, The Journal of biological chemistry, 280, 30091, 10.1074/jbc.M501279200 · DOI
  33. Kalyanaraman, 2008, Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine, Nat Protoc, 3, 8, 10.1038/nprot.2007.473 · DOI
  34. Kalyanaraman, 2012, Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses, The Journal of biological chemistry, 287, 2984, 10.1074/jbc.M111.309062 · DOI

Cited by

Sorted by citations

Metadata

Authors
Gonzalez, Y., Aryal, B., Chehab, L., & Rao, V. A.
Venue
Oncotarget
Publisher
Impact Journals, LLC
Published
2014-03-12
Volume
5
Issue
6
Pages
1526-1537
ISSN
1949-2553
DOI
10.18632/oncotarget.1715
PMC
PMC4039229
Cited by
60
References
10
Language
en

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review