Regulation of Autophagy and Cell Death in Breast Carcinoma Cells
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University of Toledo Health Science Campus / OhioLINK
2010
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=mco1276005638 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-mco12760056382021-08-03T05:38:54Z Regulation of Autophagy and Cell Death in Breast Carcinoma Cells Koterba, Kristen L. Biomedical Research Cellular Biology Molecular Biology autophagy Beclin-1 Rottlerin LC3-II Caspase-independent cell death mitochondrial membrane potential breast carcinoma cells <p>Mammalian intracellular protein homeostasis is maintained by a process called macroautophagy, or autophagy, and is a major protein degradation mechanism induced by cellular stress such as nutrient starvation. The discovery of key autophagy genes as well as signaling pathways involved in the regulation of autophagy has elucidated the essential protein machinery involved in autophagosome formation, fusion, and degradation. However, the specific mechanism as to how these genes and kinase pathways regulate autophagy is largely unknown. In addition to normal cellular physiology, autophagy has been implicated in the pathophysiology of cancer. This is evidenced by an increase in the frequency of spontaneous malignancies by heterozygous disruption of the autophagy gene Beclin-1; suggesting that Beclin-1 functions as a tumor suppressor gene. We show that Beclin-1 co-immunoprecipitates with the Class III PI3K hVps34 and its myristoylated protein adapter p150, and that this interaction requires amino acids 80-107 of Beclin-1. We also demonstrate that upon autophagic stimulation, the Beclin-1-hVps34-p150 complex is recruited to the membrane fraction in MCF-7 cells.</p><p>Rottlerin is a cytotoxic compound reported to increase the presence of autophagic vacuoles in the cytoplasm of pancreatic cells, membrane association of GFP-tagged LC3 to autophagosomes, and a marked induction of LC3-II protein, all important hallmarks of autophagy. Additionally, inhibition of Beclin-1 expression by RNAi was shown to inhibit Rottlerin-induced autophagy. Rottlerin was initially reported to specifically inhibit Protein Kinase C delta (PKCδ), and has also been shown to uncouple mitochondrial oxidative phosphorylation. The second portion of the dissertation aimed to explore the hypothesis that the cytotoxic effects of Rottlerin are related to its regulation of autophagy. Our results demonstrate that Rottlerin causes an accumulation of the autophagosomal marker LC3-II in MCF-7 and ZR75 breast cancer cells. This increase in accumulation of LC3-II was caused mainly by an impairment of the autophagy pathway at the lysosomal degradation step. However, a modest increase in autophagosome biogenesis contributes to the accumulation of LC3-II. Our studies concluded that exposure of breast cancer cells to Rottlerin induces caspase-independent cell death by reducing mitochondrial membrane potential (MMP), stimulating ER stress, and inhibiting autophagosome fusion with lysosomes, thereby rendering the autophagy survival pathway ineffective.</p> 2010-06-10 English text University of Toledo Health Science Campus / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=mco1276005638 http://rave.ohiolink.edu/etdc/view?acc_num=mco1276005638 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
| collection |
NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Biomedical Research Cellular Biology Molecular Biology autophagy Beclin-1 Rottlerin LC3-II Caspase-independent cell death mitochondrial membrane potential breast carcinoma cells |
| spellingShingle |
Biomedical Research Cellular Biology Molecular Biology autophagy Beclin-1 Rottlerin LC3-II Caspase-independent cell death mitochondrial membrane potential breast carcinoma cells Koterba, Kristen L. Regulation of Autophagy and Cell Death in Breast Carcinoma Cells |
| author |
Koterba, Kristen L. |
| author_facet |
Koterba, Kristen L. |
| author_sort |
Koterba, Kristen L. |
| title |
Regulation of Autophagy and Cell Death in Breast Carcinoma Cells |
| title_short |
Regulation of Autophagy and Cell Death in Breast Carcinoma Cells |
| title_full |
Regulation of Autophagy and Cell Death in Breast Carcinoma Cells |
| title_fullStr |
Regulation of Autophagy and Cell Death in Breast Carcinoma Cells |
| title_full_unstemmed |
Regulation of Autophagy and Cell Death in Breast Carcinoma Cells |
| title_sort |
regulation of autophagy and cell death in breast carcinoma cells |
| publisher |
University of Toledo Health Science Campus / OhioLINK |
| publishDate |
2010 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=mco1276005638 |
| work_keys_str_mv |
AT koterbakristenl regulationofautophagyandcelldeathinbreastcarcinomacells |
| _version_ |
1719422986495197184 |