Tools and techniques to measure mitophagy using fluorescence microscopy

Dolman, Nick J.; Chambers, Kevin M.; Mandavilli, Bhaskar S.; Batchelor, Robert H.; Janes, Michael

doi:10.4161/auto.24001

Cited by 119 publications

(91 citation statements)

References 84 publications

(146 reference statements)

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“…For staining of mitochondrion and lysosomes, Mitotracker ® Green FM (Thermo Scientific), mitochondrion selective probes, and Lysotracker ® Red DND-99 (Thermo Scientific), lysosome selective probes, were added to living cells at the final concentrations of 0.1 and 0.025 µM, respectively. Mitophagy was assessed by the colocalization of Mitotracker and Lysotracker, as described elsewhere (42).…”

Section: Real-time Quantitative Reverse-transcription Polymerase Chaimentioning

confidence: 99%

Impaired mitophagy activates mtROS/HIF-1α interplay and increases cancer aggressiveness in gastric cancer cells under hypoxia

Shida

Kitajima

Nakamura

et al. 2016

International Journal of Oncology

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Abstract. Mitochondrial autophagy (mitophagy) is a selective form of autophagy and a critical step in excluding mitochondria damaged by stress, including hypoxia. This study aimed to determine whether the integrity of mitophagy affected production of the mitochondrial reactive oxygen species (mtROS), hypoxia inducible factor (HIF)-1α expression and aggressive characteristics in GC cells under hypoxia. Three GC cell lines, 44As3, 58As9 and MKN45, were investigated in this study. HIF-1α expression was induced in the three GC cell lines under hypoxia, with higher expression observed in 44As3 and 58As9 cells compared with MKN45 cells. Cell survival and invasion abilities under hypoxia were significantly stronger in 44As3 and 58As9 cells than MKN45 cells. Moreover, mtROS accumulated in a time-dependent manner in 44As3 and 58As9 cells, but not in MKN45 cells. ROS scavenger N-acetyl-Lcysteine (NAC) treatment resulted in strong attenuation of HIF-1α expression, whereas HIF-1α knockdown increased ROS production in the three GC cell lines under hypoxia. These results suggested that the mtROS/HIF-1α interplay affected the hypoxia-induced cancer aggressiveness. Assessment of mitophagy by LC3-I/II conversion, SQSTM1/p62 degradation and specific fluorescence markers demonstrated that hypoxiainduced mitophagy was observed only in MKN45 cells, while the process was impaired in the other two cell lines. Treatment with the autophagy inhibitor chloroquine conversely increased HIF-1α expression, mtROS generation, cell survival and invasion in hypoxic MKN45 cells. The present study revealed a novel mechanism in which the integrity of mitophagy might determine cancer aggressiveness via mtROS/HIF-1α interplay in GC cells under hypoxic conditions.

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Section: Real-time Quantitative Reverse-transcription Polymerase Chaimentioning

confidence: 99%

Impaired mitophagy activates mtROS/HIF-1α interplay and increases cancer aggressiveness in gastric cancer cells under hypoxia

Shida

Kitajima

Nakamura

et al. 2016

International Journal of Oncology

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“…In addition to examination of autophagy through Western blotting of LC3 cleavage (see above), the accumulation of was p62/SQSTM1 in autophagosomes was quantified using the Premo Autophagy Sensor GFP-p62 assay (Molecular Probes, OR, USA) [25]. Breast cancer cell lines were seeded in 24-well plates at a density of 2 x 10 5 cells per well, allowed to attach overnight, exposed to pGFP-62 induction mix in serum-free medium for 24 hours, and then exposed to test chemicals as required.…”

Section: 6! Autophagy Analysismentioning

confidence: 99%

Activation of the Farnesoid X-receptor in breast cancer cell lines results in cytotoxicity but not increased migration potential

et al. 2016

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“…4A, c, white arrow, and 4A, f). Colocalization staining of Mito-tracker and Lyso-tracker can be used to track mitophagy (27). We observed an increase of colocalization (yellow) staining in ch282-5-treated CT26 cells in an early phase (Fig.…”

Section: Ch282-5 Antitumor Effect Is Augmented By Disturbing Mitophagmentioning

confidence: 72%

A Novel Bioavailable BH3 Mimetic Efficiently Inhibits Colon Cancer via Cascade Effects of Mitochondria

Wang

Zhang

Yan

et al. 2016

Clinical Cancer Research

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Purpose: Gossypol and its analogs, through their ability to bind to and inactivate BH3 domain-containing antiapoptotic proteins, have been shown to inhibit the growth of various human cancer cells in culture and xenograft models. Here, we evaluated the antitumor efficacy of a novel gossypol derivative and BH3 mimetic ch282-5 (2-aminoethanesulfonic acid sodium-gossypolone) in colon cancer models. Several innovative combination strategies were also explored and elaborated.Experimental Design: Ch282-5 was synthesized by modifying the active aldehyde groups and R groups of gossypol according to a computer-aided drug design program. The stability of ch282-5 was examined by high-performance liquid chromatography, and cytotoxic effects of ch282-5 on colon cancer cells were assessed by MTS assay. Activation of mitochondrial apoptotic pathway by ch282-5 was evidenced with a series of molecular biology techniques. In vivo antitumor activity of ch282-5 and its combination with chloroquine, rapamycin, oxaliplatin, and ABT-263 was also evaluated in colon cancer xenograft models and experimental liver metastasis models.Results: Ch282-5 showed antiproliferative and pro-cell death activity against colon cancer cells both in vitro and in vivo, and the response to the drug correlated with inhibition of antiapoptotic Bcl-2 proteins, induction of mitochondria-dependent apoptotic pathway, and disruption of mitophagy and mTOR pathway. Ch282-5 also suppressed liver metastasis produced by intrasplenic injection of colon cancer cells. Furthermore, ch282-5 could potentiate the effectiveness of oxaliplatin and rescue ABT-263 efficacy by downregulation of Mcl-1 and elevation of platelet number.Conclusions: These findings provide a rational basis for clinical investigation of this highly promising BH3 mimetic in colon cancer.

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Tools and techniques to measure mitophagy using fluorescence microscopy

Cited by 119 publications

References 84 publications

Impaired mitophagy activates mtROS/HIF-1α interplay and increases cancer aggressiveness in gastric cancer cells under hypoxia

Impaired mitophagy activates mtROS/HIF-1α interplay and increases cancer aggressiveness in gastric cancer cells under hypoxia

Activation of the Farnesoid X-receptor in breast cancer cell lines results in cytotoxicity but not increased migration potential

A Novel Bioavailable BH3 Mimetic Efficiently Inhibits Colon Cancer via Cascade Effects of Mitochondria

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