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G9a Inhibition Induces Autophagic Cell Death via AMPK/mTOR Pathway in Bladder Transitional Cell Carcinoma.

Li F, Zeng J, Gao Y, Guan Z, Ma Z, Shi Q, Du C, Jia J, Xu S, Wang X, Chang L, He D, Guo P - PLoS ONE (2015)

Bottom Line: The present study aimed at examining the potential role of autophagy in the anti-proliferation effect of G9a inhibition on TCC T24 and UMUC-3 cell lines in vitro.In addition, pre-inhibiting AMPK by Compound C attenuated autophagy together with the anti-proliferation effect induced by G9a inhibition while pre-activating AMPK by AICAR enhanced them.In conclusion, our results indicate that G9a inhibition induces autophagy through activating AMPK/mTOR pathway and the autophagy induced positively contributes to the inhibition of cell proliferation in TCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.

ABSTRACT
G9a has been reported to highly express in bladder transitional cell carcinoma (TCC) and G9a inhibition significantly attenuates cell proliferation, but the underlying mechanism is not fully understood. The present study aimed at examining the potential role of autophagy in the anti-proliferation effect of G9a inhibition on TCC T24 and UMUC-3 cell lines in vitro. We found that both pharmaceutical and genetical G9a inhibition significantly attenuated cell proliferation by MTT assay, Brdu incorporation assay and colony formation assay. G9a inhibition induced autophagy like morphology as determined by transmission electron microscope and LC-3 fluorescence assay. In addition, autophagy flux was induced by G9a inhibition in TCC cells, as determined by p62 turnover assay and LC-3 turnover assay. The autophagy induced positively contributed to the inhibition of cell proliferation because the growth attenuation capacity of G9a inhibition was reversed by autophagy inhibitors 3-MA. Mechanically, AMPK/mTOR pathway was identified to be involved in the regulation of G9a inhibition induced autophagy. Intensively activating mTOR by Rheb overexpression attenuated autophagy and autophagic cell death induced by G9a inhibition. In addition, pre-inhibiting AMPK by Compound C attenuated autophagy together with the anti-proliferation effect induced by G9a inhibition while pre-activating AMPK by AICAR enhanced them. In conclusion, our results indicate that G9a inhibition induces autophagy through activating AMPK/mTOR pathway and the autophagy induced positively contributes to the inhibition of cell proliferation in TCC cells. These findings shed some light on the functional role of G9a in cell metabolism and suggest that G9a might be a therapeutic target in bladder TCC in the future.

No MeSH data available.


Related in: MedlinePlus

G9a inhibition induces autophagic vacuoles in T24 and UMUC-3.(A and B) Morphological changes of T24 and UMUC-3 cells after treatment of 1.5 μM BIX-01294 for 24 h or UMUC-3 cells transfected by shG9a #1 for 72 h by inverted phase contrast microscopy (×100 and ×200). Arrows point to cytoplasmic vacuole accumulation. (C) Representative electron micrographs of T24 cells treated with 1 μM BIX-01294 for 24 h and 72 h transfected UMUC-3 cells (×8000 and ×40,000). Arrows point to distinct autophagic structures. (D) Examples of cells transiently transfected with ptfLC-3 plasmid and treated with 1 μM BIX-01294 for 24 h or transfected with shG9a #1 for 72h under fluorescence microscope (×200). Yellow arrows point to autophagosomes while red arrows point to autolysosomes. (E) Quantification of the number of autophagosomes (yellow LC-3 puncta) and autolysosomes (red LC-3 puncta) per cell. Rectangle indicates the magnifying picture right to it in the same treatment group. *P < 0.01.
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pone.0138390.g004: G9a inhibition induces autophagic vacuoles in T24 and UMUC-3.(A and B) Morphological changes of T24 and UMUC-3 cells after treatment of 1.5 μM BIX-01294 for 24 h or UMUC-3 cells transfected by shG9a #1 for 72 h by inverted phase contrast microscopy (×100 and ×200). Arrows point to cytoplasmic vacuole accumulation. (C) Representative electron micrographs of T24 cells treated with 1 μM BIX-01294 for 24 h and 72 h transfected UMUC-3 cells (×8000 and ×40,000). Arrows point to distinct autophagic structures. (D) Examples of cells transiently transfected with ptfLC-3 plasmid and treated with 1 μM BIX-01294 for 24 h or transfected with shG9a #1 for 72h under fluorescence microscope (×200). Yellow arrows point to autophagosomes while red arrows point to autolysosomes. (E) Quantification of the number of autophagosomes (yellow LC-3 puncta) and autolysosomes (red LC-3 puncta) per cell. Rectangle indicates the magnifying picture right to it in the same treatment group. *P < 0.01.

Mentions: We noticed that TCC cells in which G9a was inhibited showed morphological features of cytoplasmic vacuole accumulation which resembled autophagy under an inverted microscope (Fig 4A and 4B). These disorganized vacuoles were confirmed to be autophagy related by transmission electron microscopy (TEM) as the ultrastructure of the cells was observed. G9a inhibition significantly increased autophagic double-membrane compartments containing lamellar structures (Fig 4C).


G9a Inhibition Induces Autophagic Cell Death via AMPK/mTOR Pathway in Bladder Transitional Cell Carcinoma.

Li F, Zeng J, Gao Y, Guan Z, Ma Z, Shi Q, Du C, Jia J, Xu S, Wang X, Chang L, He D, Guo P - PLoS ONE (2015)

G9a inhibition induces autophagic vacuoles in T24 and UMUC-3.(A and B) Morphological changes of T24 and UMUC-3 cells after treatment of 1.5 μM BIX-01294 for 24 h or UMUC-3 cells transfected by shG9a #1 for 72 h by inverted phase contrast microscopy (×100 and ×200). Arrows point to cytoplasmic vacuole accumulation. (C) Representative electron micrographs of T24 cells treated with 1 μM BIX-01294 for 24 h and 72 h transfected UMUC-3 cells (×8000 and ×40,000). Arrows point to distinct autophagic structures. (D) Examples of cells transiently transfected with ptfLC-3 plasmid and treated with 1 μM BIX-01294 for 24 h or transfected with shG9a #1 for 72h under fluorescence microscope (×200). Yellow arrows point to autophagosomes while red arrows point to autolysosomes. (E) Quantification of the number of autophagosomes (yellow LC-3 puncta) and autolysosomes (red LC-3 puncta) per cell. Rectangle indicates the magnifying picture right to it in the same treatment group. *P < 0.01.
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getmorefigures.php?uid=PMC4580411&req=5

pone.0138390.g004: G9a inhibition induces autophagic vacuoles in T24 and UMUC-3.(A and B) Morphological changes of T24 and UMUC-3 cells after treatment of 1.5 μM BIX-01294 for 24 h or UMUC-3 cells transfected by shG9a #1 for 72 h by inverted phase contrast microscopy (×100 and ×200). Arrows point to cytoplasmic vacuole accumulation. (C) Representative electron micrographs of T24 cells treated with 1 μM BIX-01294 for 24 h and 72 h transfected UMUC-3 cells (×8000 and ×40,000). Arrows point to distinct autophagic structures. (D) Examples of cells transiently transfected with ptfLC-3 plasmid and treated with 1 μM BIX-01294 for 24 h or transfected with shG9a #1 for 72h under fluorescence microscope (×200). Yellow arrows point to autophagosomes while red arrows point to autolysosomes. (E) Quantification of the number of autophagosomes (yellow LC-3 puncta) and autolysosomes (red LC-3 puncta) per cell. Rectangle indicates the magnifying picture right to it in the same treatment group. *P < 0.01.
Mentions: We noticed that TCC cells in which G9a was inhibited showed morphological features of cytoplasmic vacuole accumulation which resembled autophagy under an inverted microscope (Fig 4A and 4B). These disorganized vacuoles were confirmed to be autophagy related by transmission electron microscopy (TEM) as the ultrastructure of the cells was observed. G9a inhibition significantly increased autophagic double-membrane compartments containing lamellar structures (Fig 4C).

Bottom Line: The present study aimed at examining the potential role of autophagy in the anti-proliferation effect of G9a inhibition on TCC T24 and UMUC-3 cell lines in vitro.In addition, pre-inhibiting AMPK by Compound C attenuated autophagy together with the anti-proliferation effect induced by G9a inhibition while pre-activating AMPK by AICAR enhanced them.In conclusion, our results indicate that G9a inhibition induces autophagy through activating AMPK/mTOR pathway and the autophagy induced positively contributes to the inhibition of cell proliferation in TCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.

ABSTRACT
G9a has been reported to highly express in bladder transitional cell carcinoma (TCC) and G9a inhibition significantly attenuates cell proliferation, but the underlying mechanism is not fully understood. The present study aimed at examining the potential role of autophagy in the anti-proliferation effect of G9a inhibition on TCC T24 and UMUC-3 cell lines in vitro. We found that both pharmaceutical and genetical G9a inhibition significantly attenuated cell proliferation by MTT assay, Brdu incorporation assay and colony formation assay. G9a inhibition induced autophagy like morphology as determined by transmission electron microscope and LC-3 fluorescence assay. In addition, autophagy flux was induced by G9a inhibition in TCC cells, as determined by p62 turnover assay and LC-3 turnover assay. The autophagy induced positively contributed to the inhibition of cell proliferation because the growth attenuation capacity of G9a inhibition was reversed by autophagy inhibitors 3-MA. Mechanically, AMPK/mTOR pathway was identified to be involved in the regulation of G9a inhibition induced autophagy. Intensively activating mTOR by Rheb overexpression attenuated autophagy and autophagic cell death induced by G9a inhibition. In addition, pre-inhibiting AMPK by Compound C attenuated autophagy together with the anti-proliferation effect induced by G9a inhibition while pre-activating AMPK by AICAR enhanced them. In conclusion, our results indicate that G9a inhibition induces autophagy through activating AMPK/mTOR pathway and the autophagy induced positively contributes to the inhibition of cell proliferation in TCC cells. These findings shed some light on the functional role of G9a in cell metabolism and suggest that G9a might be a therapeutic target in bladder TCC in the future.

No MeSH data available.


Related in: MedlinePlus