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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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Effect of ROS scavengers on 15d-PGJ2 induced cytoplasmic vacuolation, cell death, ER stress and LC3 induction and processingA. Effect of ROS scavengers, NAC, NMPG, TEMPOL and Trolox on 15d-PGJ2 induced cytoplasmic vacuolation as revealed by phase contrast images. B. Effect of ROS scavengers on cytotoxic effects of 15d-PGJ2 as measured by clonogenic assay. C. Immunoblots showing the effect of NAC, NMPG, TEMPOL and Trolox on the expression of ER stress markers Bip, Chop and processing and induction of LC3. D. Effect of above ROS scavengers on the accumulation of ubiquitinated proteins as revealed by western blotting with anti-ubiquitin antibody.
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Figure 6: Effect of ROS scavengers on 15d-PGJ2 induced cytoplasmic vacuolation, cell death, ER stress and LC3 induction and processingA. Effect of ROS scavengers, NAC, NMPG, TEMPOL and Trolox on 15d-PGJ2 induced cytoplasmic vacuolation as revealed by phase contrast images. B. Effect of ROS scavengers on cytotoxic effects of 15d-PGJ2 as measured by clonogenic assay. C. Immunoblots showing the effect of NAC, NMPG, TEMPOL and Trolox on the expression of ER stress markers Bip, Chop and processing and induction of LC3. D. Effect of above ROS scavengers on the accumulation of ubiquitinated proteins as revealed by western blotting with anti-ubiquitin antibody.

Mentions: The anti-neoplastic effects of 15d-PGJ2 were earlier shown to be exerted through ROS production (Chen et al 2002). Therefore, we tested the effect of several antioxidants on 15d-PGJ2 induced vacuolation and cell death in HCT116 cells. To our surprise, ROS scavengers TEMPOL, a Superoxide dismutase mimetic, and Trolox, a vitamin E analog, failed to block 15d-PGJ2 induced cytoplasmic vacuolation and cell death in HCT116 colon cancer cells (Fig. 6A and 6B). In sharp contrast, the thiol antioxidants N-α-Acetyl-L-cysteine (NAC) and N-mercapto-propionyl-glycine (NMPG) prevented 15d-PGJ2 induced cytoplasmic vacuolation in HCT116 cells (Fig. 6A) and restored their ability to survive and form clones in a clonogenic assay (Fig. 6B). Moreover, these thiol antioxidants, but not TEMPOL and Trolox, also significantly prevented 15d-PGJ2 induced expression of ER stress markers Bip and CHOP (Fig. 6C), and reduced the accumulation of ubiquitinated proteins (Fig. 6D). Furthermore, both NAC and NMPG but not TEMPOL and Trolox blocked 15d-PGJ2 induced expression and processing of LC3 (Fig. 6C). These results indicate that the effects of 15d-PGJ2 are probably mediated through its ability to covalently modify free sulfhydryl groups on proteins and not through ROS production.


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

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

Effect of ROS scavengers on 15d-PGJ2 induced cytoplasmic vacuolation, cell death, ER stress and LC3 induction and processingA. Effect of ROS scavengers, NAC, NMPG, TEMPOL and Trolox on 15d-PGJ2 induced cytoplasmic vacuolation as revealed by phase contrast images. B. Effect of ROS scavengers on cytotoxic effects of 15d-PGJ2 as measured by clonogenic assay. C. Immunoblots showing the effect of NAC, NMPG, TEMPOL and Trolox on the expression of ER stress markers Bip, Chop and processing and induction of LC3. D. Effect of above ROS scavengers on the accumulation of ubiquitinated proteins as revealed by western blotting with anti-ubiquitin antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2717022&req=5

Figure 6: Effect of ROS scavengers on 15d-PGJ2 induced cytoplasmic vacuolation, cell death, ER stress and LC3 induction and processingA. Effect of ROS scavengers, NAC, NMPG, TEMPOL and Trolox on 15d-PGJ2 induced cytoplasmic vacuolation as revealed by phase contrast images. B. Effect of ROS scavengers on cytotoxic effects of 15d-PGJ2 as measured by clonogenic assay. C. Immunoblots showing the effect of NAC, NMPG, TEMPOL and Trolox on the expression of ER stress markers Bip, Chop and processing and induction of LC3. D. Effect of above ROS scavengers on the accumulation of ubiquitinated proteins as revealed by western blotting with anti-ubiquitin antibody.
Mentions: The anti-neoplastic effects of 15d-PGJ2 were earlier shown to be exerted through ROS production (Chen et al 2002). Therefore, we tested the effect of several antioxidants on 15d-PGJ2 induced vacuolation and cell death in HCT116 cells. To our surprise, ROS scavengers TEMPOL, a Superoxide dismutase mimetic, and Trolox, a vitamin E analog, failed to block 15d-PGJ2 induced cytoplasmic vacuolation and cell death in HCT116 colon cancer cells (Fig. 6A and 6B). In sharp contrast, the thiol antioxidants N-α-Acetyl-L-cysteine (NAC) and N-mercapto-propionyl-glycine (NMPG) prevented 15d-PGJ2 induced cytoplasmic vacuolation in HCT116 cells (Fig. 6A) and restored their ability to survive and form clones in a clonogenic assay (Fig. 6B). Moreover, these thiol antioxidants, but not TEMPOL and Trolox, also significantly prevented 15d-PGJ2 induced expression of ER stress markers Bip and CHOP (Fig. 6C), and reduced the accumulation of ubiquitinated proteins (Fig. 6D). Furthermore, both NAC and NMPG but not TEMPOL and Trolox blocked 15d-PGJ2 induced expression and processing of LC3 (Fig. 6C). These results indicate that the effects of 15d-PGJ2 are probably mediated through its ability to covalently modify free sulfhydryl groups on proteins and not through ROS production.

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