ER-targeted Bcl-2 and inhibition of ER-associated caspase-12 rescue cultured immortalized cells from ethanol toxicity

作者：Balan Andreea G; Myers Barret J; Maganti Jansi L; Moore D Blaine^*

来源：Alcohol, 2010, 44(6): 553-563.

DOI：10.1016/j.alcohol.2010.07.003

• Ethanol
• Bcl-2
• Endoplasmic reticulum
• Caspase-12
• Apoptosis

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 wildtype 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 1 M 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.

• 出版日期2010-9