Limits...
An inhibition of p62/SQSTM1 caused autophagic cell death of several human carcinoma cells.

Nihira K, Miki Y, Ono K, Suzuki T, Sasano H - Cancer Sci. (2014)

Bottom Line: Electron microscopical analysis revealed the formation of autophagosomes with multilayer membranes caused by p62-silencing. p62 silencing-mediated reduced cell viability was restored by both genomic and pharmacological inhibition of autophagy but not that of apoptosis.These findings were also detected in several types of carcinoma cell lines including adenocarcinomas and squamous cell carcinomas.Results of our present study revealed that an inhibition of p62 resulted in the formation of mis-regulated autophagosomes with multilayer membranes and an autophagic cell death, and p62 can therefore be an attractive target for the development of anti-neoplastic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Tohoku University, Sendai, Japan.

Show MeSH

Related in: MedlinePlus

Effects of p62-silencing on morphological formation of autophagosomes. (a,b) PC9 cells were transfected with Control siRNA (upper left), sip62-1 (upper right and (b)) or sip62-2 (Lower right). Representative electron microscopical images of low and high magnifications were shown. N, nucleus. Triangles indicated autophagic vesicles. Scale bars, 5 μm (low magnification) or 0.5 μm (high magnification). (c) PC9 cells were transfected with indicated siRNAs for 3 days. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62, Atg5 and β-actin. Representative electron microscopical images were also shown. N, nucleus. Triangles indicated multilayer vesicles. Number of multilayer vesicles per each cell (at least 30 cells) was counted. Scale bars, 2 μm. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4317843&req=5

fig03: Effects of p62-silencing on morphological formation of autophagosomes. (a,b) PC9 cells were transfected with Control siRNA (upper left), sip62-1 (upper right and (b)) or sip62-2 (Lower right). Representative electron microscopical images of low and high magnifications were shown. N, nucleus. Triangles indicated autophagic vesicles. Scale bars, 5 μm (low magnification) or 0.5 μm (high magnification). (c) PC9 cells were transfected with indicated siRNAs for 3 days. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62, Atg5 and β-actin. Representative electron microscopical images were also shown. N, nucleus. Triangles indicated multilayer vesicles. Number of multilayer vesicles per each cell (at least 30 cells) was counted. Scale bars, 2 μm. *P < 0.05.

Mentions: We further elucidated the mechanisms of p62 silencing-induced autophagy by an ultrastructural examination. Figure3(a) showed representative electron microscopical images of siRNA-transfected PC9 cells. Autophagic vesicles were not detected in PC9 cells transfected with Control siRNA but when transfected with two sip62s, morphologically identified autophagic vesicles were clearly detected in cytoplasm (Fig.3a). The great majority of autophagic vesicles detected in p62-silenced PC9 cells formed multilayer structures but its average diameter was 0.5–1 μm, the same as representative autophagic vesicles. We also found the small number of the cells with large multilayer vesicles (5–15 μm) containing other multilayer vesicles (Fig.3b). Autophagy inhibition as a result of the transfection of siRNA targeting autophagy-related gene Atg5 reduced the number of multilayer autophagosomes (Fig.3c). These ultrastructural studies indicated that an inhibition of p62 induced the formation of autophagosomes with multilayer membranes.


An inhibition of p62/SQSTM1 caused autophagic cell death of several human carcinoma cells.

Nihira K, Miki Y, Ono K, Suzuki T, Sasano H - Cancer Sci. (2014)

Effects of p62-silencing on morphological formation of autophagosomes. (a,b) PC9 cells were transfected with Control siRNA (upper left), sip62-1 (upper right and (b)) or sip62-2 (Lower right). Representative electron microscopical images of low and high magnifications were shown. N, nucleus. Triangles indicated autophagic vesicles. Scale bars, 5 μm (low magnification) or 0.5 μm (high magnification). (c) PC9 cells were transfected with indicated siRNAs for 3 days. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62, Atg5 and β-actin. Representative electron microscopical images were also shown. N, nucleus. Triangles indicated multilayer vesicles. Number of multilayer vesicles per each cell (at least 30 cells) was counted. Scale bars, 2 μm. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317843&req=5

fig03: Effects of p62-silencing on morphological formation of autophagosomes. (a,b) PC9 cells were transfected with Control siRNA (upper left), sip62-1 (upper right and (b)) or sip62-2 (Lower right). Representative electron microscopical images of low and high magnifications were shown. N, nucleus. Triangles indicated autophagic vesicles. Scale bars, 5 μm (low magnification) or 0.5 μm (high magnification). (c) PC9 cells were transfected with indicated siRNAs for 3 days. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62, Atg5 and β-actin. Representative electron microscopical images were also shown. N, nucleus. Triangles indicated multilayer vesicles. Number of multilayer vesicles per each cell (at least 30 cells) was counted. Scale bars, 2 μm. *P < 0.05.
Mentions: We further elucidated the mechanisms of p62 silencing-induced autophagy by an ultrastructural examination. Figure3(a) showed representative electron microscopical images of siRNA-transfected PC9 cells. Autophagic vesicles were not detected in PC9 cells transfected with Control siRNA but when transfected with two sip62s, morphologically identified autophagic vesicles were clearly detected in cytoplasm (Fig.3a). The great majority of autophagic vesicles detected in p62-silenced PC9 cells formed multilayer structures but its average diameter was 0.5–1 μm, the same as representative autophagic vesicles. We also found the small number of the cells with large multilayer vesicles (5–15 μm) containing other multilayer vesicles (Fig.3b). Autophagy inhibition as a result of the transfection of siRNA targeting autophagy-related gene Atg5 reduced the number of multilayer autophagosomes (Fig.3c). These ultrastructural studies indicated that an inhibition of p62 induced the formation of autophagosomes with multilayer membranes.

Bottom Line: Electron microscopical analysis revealed the formation of autophagosomes with multilayer membranes caused by p62-silencing. p62 silencing-mediated reduced cell viability was restored by both genomic and pharmacological inhibition of autophagy but not that of apoptosis.These findings were also detected in several types of carcinoma cell lines including adenocarcinomas and squamous cell carcinomas.Results of our present study revealed that an inhibition of p62 resulted in the formation of mis-regulated autophagosomes with multilayer membranes and an autophagic cell death, and p62 can therefore be an attractive target for the development of anti-neoplastic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Tohoku University, Sendai, Japan.

Show MeSH
Related in: MedlinePlus