ER-targeted Bcl-2 and inhibition of ER-associated caspase-12 rescue cultured immortalized cells from ethanol toxicity
| Autor: | D. Blaine Moore, Barret J. Myers, Andreea G. Balan, Jansi L. Maganti |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Health (social science)
Cell Survival Recombinant Fusion Proteins Green Fluorescent Proteins CHO Cells Pharmacology Endoplasmic Reticulum Transfection Toxicology Biochemistry Behavioral Neuroscience Cricetulus Cricetinae Animals MTT assay Viability assay Enzyme Inhibitors Caspase 12 Caspase Cell Line Transformed Ethanol biology Chinese hamster ovary cell General Medicine Caspase Inhibitors Genes bcl-2 Mitochondria Microscopy Fluorescence Proto-Oncogene Proteins c-bcl-2 Neurology Apoptosis Toxicity biology.protein Immortalised cell line |
| Zdroj: | Alcohol. 44:553-563 |
| ISSN: | 0741-8329 |
| DOI: | 10.1016/j.alcohol.2010.07.003 |
| Popis: | Alcohol abuse, known for promoting apoptosis in the liver and nervous system, is a major public health concern. Despite significant morbidity and mortality resulting from ethanol consumption, the precise cellular mechanism of its toxicity remains unknown. Previous work has shown that wild-type Bcl-2 is protective against ethanol. The present study investigated whether protection from ethanol toxicity involves mitochondrial Bcl-2 or endoplasmic reticulum (ER) Bcl-2, and whether mitochondria-associated or ER-associated caspases are involved in ethanol toxicity. Chinese hamster ovary (CHO695) cells were transiently transfected with cDNA constructs encoding wild-type Bcl-2, mitochondria-targeted Bcl-2, or ER-targeted Bcl-2. MTT assay was used to measure cell viability in response to ethanol. Ethanol treatments of 1 and 2.5 M reduced cell viability at 5, 10, and 24 h. Wild-type Bcl-2, localized both to mitochondria and ER, provided significant rescue for CHO695 cells treated with 1M ethanol for 24 h, but did not rescue toxicity at 2.5 M. ER-targeted Bcl-2, however, provided significant and robust rescue following 24 h of 1 and 2.5 M ethanol. Mitochondria-targeted Bcl-2 offered no protection at any ethanol concentration and generally reduced cell viability. To follow up these experiments, we used a peptide inhibitor approach to investigate which caspases were responsible for ethanol-induced apoptosis. Caspase-9 and caspase-12 are known to be downstream of mitochondria and the ER, respectively. CHO695 cells were treated with a pan-caspase inhibitor, a caspase-9 or caspase-12 inhibitor along with 1.5 M ethanol, followed by MTT cell viability assay. Treatment with the pan-caspase inhibitor provided significant rescue from ethanol, whereas inhibition of caspase-9 did not. Inhibition of ER-associated caspase-12, however, conferred significant protection from ethanol toxicity, similar to the pan inhibitor. These findings are consistent with our transfection data and, taken together, suggest a significant role for the ER in ethanol toxicity. |
| Databáze: | OpenAIRE |
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