Department of Cellular Biology and Anatomy: Faculty Research and
Presentations
http://hdl.handle.net/10675.2/862
2024-03-28T08:48:22ZInsulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim
http://hdl.handle.net/10675.2/827
Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim
Periyasamy-Thandavan, Sudharsan; Takhar, Suchreet; Singer, Adam; Dohn, Michael Robert; Jackson, William Hutch; Welborn, April Eve; LeRoith, Derek; Marrero, Mario; Thangaraju, Muthusamy; Huang, Shuang; Schoenlein, Patricia Veronica
Introduction: In this pre-clinical in vitro study conducted in estrogen receptor positive (ER+) breast cancer cells, we have characterized the effects of insulin-like growth factor I (IGF-1) on the cytostatic and cytotoxic action of antiestrogen treatment when used as a single agent or in combination with the antiprogestin mifepristone (MIF). Our goal was to identify new molecular targets to improve the efficacy of hormonal therapy in breast cancer patients that have a poor response to hormonal therapy, in part, due to high circulating levels of unbound insulinIGF-1.; Methods: IGF-1-mediated effects on cytostasis and apoptotic cell death were determined with cell counts conducted in the presence and absence of trypan blue; enzyme-linked immunosorbent assays to determine the intracellular levels of cleaved cytokeratin 18, a marker of epithelial cancer cell apoptosis; and immunoblot analysis to determine the levels of cleaved poly-ADP ribose polymerase (PARP) and lamin A that result from caspase-dependent apoptosis. Cytotoxicity was further characterized by determination of the levels of reactive oxygen species (ROS) and the percent of mitochondrial membrane depolarization in cell populations treated with the different hormones in the presence and absence of IGF-1. Small molecule inhibitors of the dual-specificity protein kinase MEK1, MEK1 siRNA, Bim siRNA, and vectors overexpressing MEK1 wild type and mutant, dominant negative cDNA were used to identify key IGF-1 downstream prosurvival effectors.; Results: IGF-1, at physiologically relevant levels, blocked the cytotoxic action(s) of the antiestrogens 4-hydroxytamoxifen (4-OHT) and tamoxifen (TAM) when used as single agents or in combination with the antiprogestin MIF. The antiapoptotic action of IGF-1 was mediated primarily through the action of MEK1. MEK1 expression reduced the levels of ROS and mitochondrial membrane depolarization induced by the hormonal treatments via a mechanism that involved the phosphorylation and proteasomal turnover of the proapoptotic BH3-only Bcl-2 family member Bim. Importantly, small-molecule inhibitors of MEK1 circumvented the prosurvival action of IGF-1 by restoring Bim to levels that more effectively mediated apoptosis in ER+ breast cancer cells.; Conclusion: his study provides strong support for the use of MEK1 inhibitors in combination with hormonal therapy to effectively affect cytostasis and activate a Bim-dependent apoptotic pathway in ER+ breast cancer cells. We discuss that MEK1 blockade may be a particularly effective treatment for women with high circulating levels of IGF-1, which have been correlated to a poor prognosis.
2012-03-19T00:00:00ZPalmitoleate Induces Hepatic Steatosis but Suppresses Liver Inflammatory Response in Mice
http://hdl.handle.net/10675.2/802
Palmitoleate Induces Hepatic Steatosis but Suppresses Liver Inflammatory Response in Mice
Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Ong, Kuok Teong; Woo, Shih-Lung; Walzem, Rosemary L.; Mashek, Douglas G.; Dong, Hui; Lu, Fuer; Wei, Lai; Huo, Yuqing; Wu, Chaodong
The interaction between fat deposition and inflammation during obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD). The present study examined the effects of palmitoleate, a monounsaturated fatty acid (16⠶1n7), on liver metabolic and inflammatory responses, and investigated the mechanisms by which palmitoleate increases hepatocyte fatty acid synthase (FAS) expression. Male wild-type C57BL/6J mice were supplemented with palmitoleate and subjected to the assays to analyze hepatic steatosis and liver inflammatory response. Additionally, mouse primary hepatocytes were treated with palmitoleate and used to analyze fat deposition, the inflammatory response, and sterol regulatory element-binding protein 1c (SREBP1c) activation. Compared with controls, palmitoleate supplementation increased the circulating levels of palmitoleate and improved systemic insulin sensitivity. Locally, hepatic fat deposition and SREBP1c and FAS expression were significantly increased in palmitoleate-supplemented mice. These pro-lipogenic events were accompanied by improvement of liver insulin signaling. In addition, palmitoleate supplementation reduced the numbers of macrophages/Kupffer cells in livers of the treated mice. Consistently, supplementation of palmitoleate decreased the phosphorylation of nuclear factor kappa B (NF-κB, p65) and the expression of proinflammatory cytokines. These results were recapitulated in primary mouse hepatocytes. In terms of regulating FAS expression, treatment of palmitoleate increased the transcription activity of SREBP1c and enhanced the binding of SREBP1c to FAS promoter. Palmitoleate also decreased the phosphorylation of NF-κB p65 and the expression of proinflammatory cytokines in cultured macrophages. Together, these results suggest that palmitoleate acts through dissociating liver inflammatory response from hepatic steatosis to play a unique role in NAFLD.
2012-06-29T00:00:00ZAutomated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish
http://hdl.handle.net/10675.2/775
Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish
Walker, Steven L.; Ariga, Junko; Mathias, Jonathan R.; Coothankandaswamy, Veena; Xie, Xiayang; Distel, Martin; Koster, Reinhard W.; Parsons, Michael J.; Bhalla, Kapil N.; Saxena, Meera T.; Mumm, Jeff S.
Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed,
2012-01-04T00:00:00ZCell Membrane Disruption Stimulates NO/PKG Signaling and Potentiates Cell Membrane Repair in Neighboring Cells
http://hdl.handle.net/10675.2/725
Cell Membrane Disruption Stimulates NO/PKG Signaling and Potentiates Cell Membrane Repair in Neighboring Cells
Togo, Tatsuru
McNeil, Paul L.
Resealing of a disrupted plasma membrane at the micron-diameter range requires Ca2+-regulated exocytosis. Repeated membrane disruptions reseal more quickly than the initial wound, and this potentiation of membrane resealing persists for at least 24 hours after the initial wound. Long-term potentiation of membrane resealing requires CREB-dependent gene expression, which is activated by the PKC- and p38 MAPK-dependent pathway in a wounded cell. The present study demonstrates that membrane resealing is potentiated in both wounded and neighboring cells in MDCK cells. Wounding of cells expressing CREB133, a mutant variant of CREB, does not show the potentiated response of cell membrane resealing in either wounded or neighboring cells. Furthermore, wounding of cells induces CREB phosphorylation, not only in wounded cells, but also in neighboring cells. Inhibition of the nitric oxide/PKG signaling pathway suppresses CREB phosphorylation in neighboring cells, but not in wounded cells. The potentiation of membrane resealing in neighboring cells is suppressed if the nitric oxide/PKG pathway is inhibited during the initial wound. Together, these results suggest that the nitric oxide/PKG pathway stimulates CREB phosphorylation in neighboring cells so that subsequent cell membrane disruptions of the neighboring cells reseal more quickly.
2012-08-07T00:00:00Z