1. Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time
Yoganand Balagurunathan, Joan S Lewis-Wambi, Coya Tapia, Emmanuelle Nicolas, Helen R Kim, V Craig Jordan, Amanda L Willis, Michael J Slifker, Pilar Ramos, Eric A Ross, Eric A Ariazi, Heather E Cunliffe, Catherine G N Sharma, Smitha Yerrum Proc Natl Acad Sci U S A . 2011 Nov 22;108(47):18879-86. doi: 10.1073/pnas.1115188108.
In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.
2. Down regulation of glutathione and glutamate cysteine ligase in the inflammatory response of macrophages
Abigail Florentino, Natalie Lyn, Henry Jay Forman, Hongqiao Zhang, Sarah Jiuqi Zhang, Andrew Li, Kelvin J A Davies Free Radic Biol Med . 2020 Oct;158:53-59. doi: 10.1016/j.freeradbiomed.2020.06.017.
Glutathione (GSH) plays critical roles in the inflammatory response by acting as the master substrate for antioxidant enzymes and an important anti-inflammatory agent. In the early phase of the inflammatory response of macrophages, GSH content is decreased due to the down regulation of the catalytic subunit of glutamate cysteine ligase (GCLC). In the current study we investigated the underlying mechanism for this phenomenon. In human THP1-differentiated macrophages, GCLC mRNA had a half-life of 4 h under basal conditions, and it was significantly reduced to less than 2 h upon exposure to lipopolysaccharide (LPS), suggesting an increased decay of GCLC mRNA in the inflammatory response. The half-life of GCLC protein was >10 h under basal conditions, and upon LPS exposure the degradation rate of GCLC protein was significantly increased. The pan-caspase inhibitor Z-VAD-FMK but not the proteasome inhibitor MG132, prevented the down regulation of GCLC protein caused by LPS. Both caspase inhibitor Z-LEVD-FMK and siRNA of caspase-5 abrogated LPS-induced degradation of GCLC protein. In addition, supplement with γ-GC, the GCLC product, efficiently restored GSH content and suppressed the induction of NF-κB activity by LPS. In conclusion, these data suggest that GCLC down-regulation in the inflammatory response of macrophages is mediated through both increased mRNA decay and caspase-5-mediated GCLC protein degradation, and γ-GC is an efficient agent to restore GSH and regulate the inflammatory response.
3. Distinct mechanism of cell death is responsible for tunicamycin-induced ER stress in SK-N-SH and SH-SY5Y cells
Tetsuro Oda, Yoshihisa Ito, Yasuhiro Kosuge, Kumiko Ishige, Motoki Arakawa Neurosci Res . 2008 Jan;60(1):29-39. doi: 10.1016/j.neures.2007.09.005.
In order to elucidate underlying mechanism of cell death pathways in neuronal cells in humans, we studied responsible pathways involved in the endoplasmic reticulum (ER) stress-induced cell death in neuroblastoma cells, SK-N-SH and its neuroblast-type subclone SH-SY5Y cells. A time-dependent induction of ER chaperons, glucose regulated protein (GRP)78 and GRP94, was observed after treatment with tunicamycin (TM), and cell death was also induced concomitantly in both cells. Although the pro-caspase-12-like protein was defined in both cells, a decrease in the protein was observed in only SH-SY5Y cells after exposure to TM. In contrast, pro-caspase-4 was detected in only SK-N-SH cells, and the cleaved-form was induced by the treatment with TM. A caspase-4 inhibitor, Z-LEVD-FMK attenuated TM-induced cell death in SK-N-SH cells. Calpain- and caspase-3-mediated proteolysis of alpha II-spectrin was also increased after the treatment with TM in both cells. A calpain inhibitor, calpeptin, repressed TM-induced cell death in only SK-N-SH cells. GADD153/C/EBP homologous protein (CHOP) was significantly induced after exposure to TM in only SH-SY5Y cells and RNA interference to GADD153/CHOP repressed TM-induced cell death. These results demonstrate that induction of GADD153/CHOP plays a pivotal role in mechanism of ER stress-induced cell death in SH-SY5Y cells, on the other hand, cleavage of pro-caspase-4 by activation of calpain play a crucial role in SK-N-SH cells. It is also suggested that the relevance of caspase-4 to ER stress is cell-specific even between human-origin cell lines.
