Inhibition of ER-Stress Associated Caspase-12 Rescues Cultured Cells from Ethanol Toxicity
Myers, Barret J.
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Alcohol abuse is known to promote cell death in many tissues, including the liver and nervous system, however its precise sub-cellular mechanism remains unclear. Ethanol induces apoptosis in cells via a caspase-dependent mechanism. Control of caspase activity, and thus apoptosis, is directly modulated by the Bcl-2 family of proteins, which includes both pro- and anti-apoptotic members. Anti-apoptotic Bcl-2 protects cells from apoptosis by inhibiting caspase activation. Bcl-2 is known to be localized both to mitochondria and the ER and overexpression of ER-localized Bcl-2 has been shown to significantly rescue cells from ethanol-induced apoptosis. Caspase-9 and caspase-12 are thought to be downstream of mitochondria and the endoplasmic reticulum respectively, however it is unknown which of these caspases is activated in ethanol-induced apoptosis. Chinese hamster ovary cells (CHO695) were treated with a pan-caspase inhibitor, a caspase-9 inhibitor or a caspase-12 inhibitor, and then treated with 1.5 M ethanol in order to determine the sub-cellular mechanism of rescue from ethanol toxicity. An MTT assay was used to measure cell viability in response to ethanol treatment. Treatment with the pan-inhibitor provided CHO695 cells with significant rescue from ethanol toxicity. Caspase-12 inhibition conferred significant protection from ethanol toxicity to cells, while caspase-9 inhibition did not provide significant rescue. Caspase-12 and pan-caspase inhibition were not statistically different. This work suggests that ethanol-induced apoptosis does not occur via a mitochondrial stress pathway and caspase-9 activation, but rather through an ER-stress pathway and subsequent caspase-12 activation. Future work should clarify the role of the ER in ethanol toxicity contributing to both liver and neuronal cell death associated with alcohol abuse.