Clusterin is a heterodimeric, disulfide-linked 70-80 kDa glycoprotein that is induced during regression of most, if not all, hormone-dependent epithelial tissues. These studies describe the biogenesis and intracellular trafficking of clusterin in MCF-7 cells before and after the initiation of apoptosis with antiestrogens and TNFa. Under physiological conditions, clusterin is modified in the endoplasmic reticulum (ER), and proteolytically cleaved in the Golgi to generate discrete a and b chains prior to secretion. Treatment with TNFa or the antiestrogen, ICI 182,780, induces apoptosis in MCF-7 cells and leads to substantial changes in the activity of Golgi-resident enzymes, significantly altering the biogenesis of clusterin. This leads to the appearance of a 50-53 kDa uncleaved, nonglycosylated, disulfide-linked isoform of clusterin that accumulates in the nucleus. While clusterin contains a cryptic SV-40-like nuclear localization signal, mutation of this sequence does not affect the nuclear accumulation of the disulfide-linked nuclear isoform. Confocal microscopy demonstrates that the nuclear accumulation of clusterin is coincident with DNA fragmentation. These data suggest that, at least in secretory epithelial cells, retrograde transport from the Golgi to the ER of a nonglycosylated, uncleaved isoform and the subsequent translocation of clusterin to the nucleus occur in dying cells.
Resveratrol, a natural phytoestrogen found in red wine and a variety of plants, is reported to have protective effects against lung cancer; however, there is little work directed toward the understanding of the mechanism of its action in this disease. In this study, we used a combination of experimental approaches to understand the biological activity and molecular mechanisms of resveratrol. Microarray gene expression profiling and high-throughput immunoblotting (PowerBlot) methodologies were employed to gain insights into the molecular mechanisms of resveratrol action in human lung cancer cells. In this report, we confirm the up-regulation of p53 and p21 and the induction of apoptosis by the activation of the caspases and the disruption of the mitochondrial membrane complex. We show the arrest of A549 cells in the G 1 phase of cell cycle in the presence of resveratrol and also report alterations in both gene and protein expressions of cyclin A, chk1, CDC27, and Eg5. Furthermore, the results indicated that resveratrol action is mediated via the transforming growth factor-B pathway, particularly through the Smad proteins. Results showed the down-regulation of the Smad activators 2 and 4 and the up-regulation of the repressor Smad 7 as a result of resveratrol treatment. Resveratrol is a potent inhibitor of A549 lung cancer cell growth, and our results suggest that resveratrol may be a promising chemopreventive or chemotherapeutic agent for lung cancer.
BackgroundClusterin is a secreted glycoprotein that is upregulated in a variety of cell lines in response to stress, and enhances cell survival. A second nuclear isoform of clusterin that is associated with cell death has also been identified. The aim of this study was to determine the role(s) of the secretory isoform in breast tumor progression and metastasis.MethodsTo investigate the role of secretory clusterin in the biology of breast cancer tumor growth and resistance to therapy we have engineered an MCF-7 cell line (MCF-7CLU) that over-expresses clusterin. We have measured the in vitro effects of clusterin over-expression on cell cycle, cell death, and sensitivity to TNFalpha and tamoxifen. Using an orthotopic model of breast cancer, we have also determined the effects of over-expression of clusterin on tumor growth and metastatic progression.ResultsIn vitro, over-expression of secretory clusterin alters the cell cycle kinetics and decreases the rate of cell death, resulting in the enhancement of cell growth. Over-expression of secretory clusterin also blocks the TNFalpha-mediated induction of p21 and abrogates the cleavage of Bax to t-Bax, rendering the MCF-7CLU cells significantly more resistant to the cytokine than the parental cells. Orthotopic primary tumors derived from MCF-7CLU cells grow significantly more rapidly than tumors derived from parental MCF-7 cells and, unlike the parental cells, metastasize frequently to the lungs.ConclusionsThese data suggest that secretory clusterin, which is frequently up-regulated in breast cancers by common therapies, including anti-estrogens, may play a significant role in tumor growth, metastatic progression and subsequent drug resistance in surviving cells.
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