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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.

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Effects of exogenous p62-overexpression. (a) H23 cells were transfected with indicated siRNAs for 3 days. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6) compared with control cells. (b) H23 cells were cultured in 6-well plates and transfected with indicated volumes of p62 expression vector. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. (c) H23 cells were transfected with indicated vectors, and cell viability was measured. (d) H23 cells were transfected with indicated vectors. After transfection, these cells were treated with HBSS for 24 h. The cells were stained with trypan blue, and the ratio of dead cells was measured. *P < 0.05 (n = 3). (e) PC9 cells were transfected with indicated vectors and siRNAs. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6).
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fig06: Effects of exogenous p62-overexpression. (a) H23 cells were transfected with indicated siRNAs for 3 days. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6) compared with control cells. (b) H23 cells were cultured in 6-well plates and transfected with indicated volumes of p62 expression vector. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. (c) H23 cells were transfected with indicated vectors, and cell viability was measured. (d) H23 cells were transfected with indicated vectors. After transfection, these cells were treated with HBSS for 24 h. The cells were stained with trypan blue, and the ratio of dead cells was measured. *P < 0.05 (n = 3). (e) PC9 cells were transfected with indicated vectors and siRNAs. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6).

Mentions: We then examined the roles of p62 using p62 low-expressing (Fig.1a) H23 cells. p62-silencing slightly suppressed cell viability of H23 cells (Fig.6a). We then constructed p62 expression vector and transfected in H23 cells. p62 expression was increased by the transfection of p62 expression vector in a concentration dependent manner, but cell viability was not changed (Fig.6b,c). Because p62 inhibition also induced autophagy via mTOR inactivation (Fig.2a), we then hypothesized that exogenous p62-overexpression could influence the autophagy-activated cells. Figure6(d) demonstrated that the starvation-induced cell death was attenuated by p62-overexpression, which suggested that starvation-induced autophagy caused autophagic cell death in p62 low-expressing H23 cells, and exogenous p62-overexpression contributed to autophagy maturation. In addition, exogenous p62-overexpression decreased the effects of p62-silencing in PC9 cells (Fig.6e).


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 exogenous p62-overexpression. (a) H23 cells were transfected with indicated siRNAs for 3 days. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6) compared with control cells. (b) H23 cells were cultured in 6-well plates and transfected with indicated volumes of p62 expression vector. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. (c) H23 cells were transfected with indicated vectors, and cell viability was measured. (d) H23 cells were transfected with indicated vectors. After transfection, these cells were treated with HBSS for 24 h. The cells were stained with trypan blue, and the ratio of dead cells was measured. *P < 0.05 (n = 3). (e) PC9 cells were transfected with indicated vectors and siRNAs. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig06: Effects of exogenous p62-overexpression. (a) H23 cells were transfected with indicated siRNAs for 3 days. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6) compared with control cells. (b) H23 cells were cultured in 6-well plates and transfected with indicated volumes of p62 expression vector. After transfection, protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. (c) H23 cells were transfected with indicated vectors, and cell viability was measured. (d) H23 cells were transfected with indicated vectors. After transfection, these cells were treated with HBSS for 24 h. The cells were stained with trypan blue, and the ratio of dead cells was measured. *P < 0.05 (n = 3). (e) PC9 cells were transfected with indicated vectors and siRNAs. After transfection, cell viability was measured, and protein homogenates were isolated and immunoblotting was used to determine the levels of p62 and β-actin expression. *P < 0.05 (n = 6).
Mentions: We then examined the roles of p62 using p62 low-expressing (Fig.1a) H23 cells. p62-silencing slightly suppressed cell viability of H23 cells (Fig.6a). We then constructed p62 expression vector and transfected in H23 cells. p62 expression was increased by the transfection of p62 expression vector in a concentration dependent manner, but cell viability was not changed (Fig.6b,c). Because p62 inhibition also induced autophagy via mTOR inactivation (Fig.2a), we then hypothesized that exogenous p62-overexpression could influence the autophagy-activated cells. Figure6(d) demonstrated that the starvation-induced cell death was attenuated by p62-overexpression, which suggested that starvation-induced autophagy caused autophagic cell death in p62 low-expressing H23 cells, and exogenous p62-overexpression contributed to autophagy maturation. In addition, exogenous p62-overexpression decreased the effects of p62-silencing in PC9 cells (Fig.6e).

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