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A novel role for MAP1 LC3 in nonautophagic cytoplasmic vacuolation death of cancer cells.

Kar R, Singha PK, Venkatachalam MA, Saikumar P - Oncogene (2009)

Bottom Line: Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation.Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in nonautophagic cytoplasmic vacuolation and cell death.Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2-induced cytoplasmic vacuolation and cell death, suggesting a novel role of LC3 in a death process other than autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.

ABSTRACT
Thiol reactive cyclopentenone prostaglandin, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ2), induced a novel, nonapoptotic and microtubule-associated protein 1 light chain 3 (MAP1 LC3) dependent but nonautophagic form of cell death in colon, breast and prostate cancer cell lines, characterized by extensive cytoplasmic vacuolation with dilatation of endoplasmic reticulum (ER). Disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and subsequent ER dilation, contributed to peroxisome proliferator-activated receptor gamma (PPARgamma)-independent cell death by 15d-PGJ2. Absence of intracellular organelles in these vacuoles, shown by electron microscopy and unique fragmentation of lamin B, suggested this form of cell death to be different from autophagy and apoptosis. Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation. Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in nonautophagic cytoplasmic vacuolation and cell death. Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2-induced cytoplasmic vacuolation and cell death, suggesting a novel role of LC3 in a death process other than autophagy.

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Increase of LC3 by 15d-PGJ2 is not through inhibition of lysosomal protease activityA. Cathepsin L activity in untreated HCT116 cell extracts was measured through hydrolysis of Z-Phe-Arg-AMC as described in materials and methods section. Extracts were preincubated with either 15d-PGJ2 or leupeptin before measuring the activity. B. Cells were treated with either leupeptin, or 15d-PGJ2 or 15d-PGJ2 with Wortmannin (WM), an autophagy inhibitor or 15d-PGJ2 with caspase inhibitor q-VD for 9h. Membrane bound fractions were collected and assayed for Cathepsin L activity. C. LC3 expression in HCT116 cells treated for 9h with either cathepsin inhibitor, leupeptin or 15d-PGJ2 or caspase inhibitor, qVD was analyzed by immunoblotting.
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Figure 8: Increase of LC3 by 15d-PGJ2 is not through inhibition of lysosomal protease activityA. Cathepsin L activity in untreated HCT116 cell extracts was measured through hydrolysis of Z-Phe-Arg-AMC as described in materials and methods section. Extracts were preincubated with either 15d-PGJ2 or leupeptin before measuring the activity. B. Cells were treated with either leupeptin, or 15d-PGJ2 or 15d-PGJ2 with Wortmannin (WM), an autophagy inhibitor or 15d-PGJ2 with caspase inhibitor q-VD for 9h. Membrane bound fractions were collected and assayed for Cathepsin L activity. C. LC3 expression in HCT116 cells treated for 9h with either cathepsin inhibitor, leupeptin or 15d-PGJ2 or caspase inhibitor, qVD was analyzed by immunoblotting.

Mentions: To find out whether expression of LC3 is related to decreased lysosomal function by 15d-PGJ2, we analyzed the effect of 15d-PGJ2, leupeptin, q-VD on Cathepsin B and L, lysosomal endopeptidases. Leupeptin completely blocked lysosomal cathepsin activity when it was added to either cell extracts (Fig. 8A) or intact cells (Fig. 8B), but caused only a slight increase in LC3 protein levels in the intact cells (Fig. 8C, lane 4). In contrast, 15d-PGJ2 did not affect cathepsin activity when it was added directly to cell extracts (Fig. 8A), caused only a ~50% decrease of cathepsin activity when it was added to intact cells (Fig. 8B), but caused large increases of LC3 protein in the intact cells (Fig. 8C, lane 2) suggesting that lysosomal inhibition did not effect LC3 induction. In fact, blockage of 15d-PGJ2 induced LC3 expression by transcription and translational inhibitors (supplemental Fig. 4) and MAPK inhibitors suggest alternative novel mechanisms to be responsible for LC3 upregulation.


A novel role for MAP1 LC3 in nonautophagic cytoplasmic vacuolation death of cancer cells.

Kar R, Singha PK, Venkatachalam MA, Saikumar P - Oncogene (2009)

Increase of LC3 by 15d-PGJ2 is not through inhibition of lysosomal protease activityA. Cathepsin L activity in untreated HCT116 cell extracts was measured through hydrolysis of Z-Phe-Arg-AMC as described in materials and methods section. Extracts were preincubated with either 15d-PGJ2 or leupeptin before measuring the activity. B. Cells were treated with either leupeptin, or 15d-PGJ2 or 15d-PGJ2 with Wortmannin (WM), an autophagy inhibitor or 15d-PGJ2 with caspase inhibitor q-VD for 9h. Membrane bound fractions were collected and assayed for Cathepsin L activity. C. LC3 expression in HCT116 cells treated for 9h with either cathepsin inhibitor, leupeptin or 15d-PGJ2 or caspase inhibitor, qVD was analyzed by immunoblotting.
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Related In: Results  -  Collection

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Figure 8: Increase of LC3 by 15d-PGJ2 is not through inhibition of lysosomal protease activityA. Cathepsin L activity in untreated HCT116 cell extracts was measured through hydrolysis of Z-Phe-Arg-AMC as described in materials and methods section. Extracts were preincubated with either 15d-PGJ2 or leupeptin before measuring the activity. B. Cells were treated with either leupeptin, or 15d-PGJ2 or 15d-PGJ2 with Wortmannin (WM), an autophagy inhibitor or 15d-PGJ2 with caspase inhibitor q-VD for 9h. Membrane bound fractions were collected and assayed for Cathepsin L activity. C. LC3 expression in HCT116 cells treated for 9h with either cathepsin inhibitor, leupeptin or 15d-PGJ2 or caspase inhibitor, qVD was analyzed by immunoblotting.
Mentions: To find out whether expression of LC3 is related to decreased lysosomal function by 15d-PGJ2, we analyzed the effect of 15d-PGJ2, leupeptin, q-VD on Cathepsin B and L, lysosomal endopeptidases. Leupeptin completely blocked lysosomal cathepsin activity when it was added to either cell extracts (Fig. 8A) or intact cells (Fig. 8B), but caused only a slight increase in LC3 protein levels in the intact cells (Fig. 8C, lane 4). In contrast, 15d-PGJ2 did not affect cathepsin activity when it was added directly to cell extracts (Fig. 8A), caused only a ~50% decrease of cathepsin activity when it was added to intact cells (Fig. 8B), but caused large increases of LC3 protein in the intact cells (Fig. 8C, lane 2) suggesting that lysosomal inhibition did not effect LC3 induction. In fact, blockage of 15d-PGJ2 induced LC3 expression by transcription and translational inhibitors (supplemental Fig. 4) and MAPK inhibitors suggest alternative novel mechanisms to be responsible for LC3 upregulation.

Bottom Line: Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation.Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in nonautophagic cytoplasmic vacuolation and cell death.Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2-induced cytoplasmic vacuolation and cell death, suggesting a novel role of LC3 in a death process other than autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.

ABSTRACT
Thiol reactive cyclopentenone prostaglandin, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ2), induced a novel, nonapoptotic and microtubule-associated protein 1 light chain 3 (MAP1 LC3) dependent but nonautophagic form of cell death in colon, breast and prostate cancer cell lines, characterized by extensive cytoplasmic vacuolation with dilatation of endoplasmic reticulum (ER). Disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and subsequent ER dilation, contributed to peroxisome proliferator-activated receptor gamma (PPARgamma)-independent cell death by 15d-PGJ2. Absence of intracellular organelles in these vacuoles, shown by electron microscopy and unique fragmentation of lamin B, suggested this form of cell death to be different from autophagy and apoptosis. Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation. Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in nonautophagic cytoplasmic vacuolation and cell death. Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2-induced cytoplasmic vacuolation and cell death, suggesting a novel role of LC3 in a death process other than autophagy.

Show MeSH
Related in: MedlinePlus