CLU blocks HDACI-mediated killing of neuroblastoma

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation

Matched pairs of human prostate stromal cells display differential tropic effects on LNCaP prostate cancer cells

Prostate stromal cells may play binary roles in the process of prostate cancer development. As the first to be encountered by infiltrating prostate cancer cells, prostate stromal cells form the first defense line against prostate cancer progression and metastasis. However, interaction between prostate cancer and stromal cells may facilitate the formation of a tumor microenvironment favoring cancer cell growth and survival. To establish an experimental system for studying the interaction between cancer and stromal cells, we isolated three matched pairs of normal and cancer-associated human prostate stromal clones. In this report, we describe the morphologic and behavioral characteristics of these cells and their effect on LNCaP prostate cancer cells in co-culture. Unlike LNCaP prostate cancer cells, the isolated prostate stromal clones are large fibroblast-like cells with a slow proliferation rate. Growth and survival of these clones are not affected by androgens. The stromal cells display high resistance to serum starvation, while cancer-associated stromal clones have differentiated survival ability. In co-culture experiments, the stromal cells protected some LNCaP prostate cancer cells from death by serum starvation, and cancer-associated stromal clones showed more protection. This work thus established a panel of valuable human prostate stromal cell lines, which could be used in co-culture to study the interaction between prostate cancer and prostate stromal cells

Prostate response to prolactin in sexually active male rats

BACKGROUND: The prostate is a key gland in the sexual physiology of male mammals. Its sensitivity to steroid hormones is widely known, but its response to prolactin is still poorly known. Previous studies have shown a correlation between sexual behaviour, prolactin release and prostate physiology. Thus, here we used the sexual behaviour of male rats as a model for studying this correlation. Hence, we developed experimental paradigms to determine the influence of prolactin on sexual behaviour and prostate organization of male rats. METHODS: In addition to sexual behaviour recordings, we developed the ELISA procedure to quantify the serum level of prolactin, and the hematoxilin-eosin technique for analysis of the histological organization of the prostate. Also, different experimental manipulations were carried out; they included pituitary grafts, and haloperidol and ovine prolactin treatments. Data were analyzed with a One way ANOVA followed by post hoc Dunnet test if required. RESULTS: Data showed that male prolactin has a basal level with two peaks at the light-dark-light transitions. Consecutive ejaculations increased serum prolactin after the first ejaculation, which reached the highest level after the second, and started to decrease after the third ejaculation. These normal levels of prolactin did not induce any change at the prostate tissue. However, treatments for constant elevations of serum prolactin decreased sexual potency and increased the weight of the gland, the alveoli area and the epithelial cell height. Treatments for transient elevation of serum prolactin did not affect the sexual behaviour of males, but triggered these significant effects mainly at the ventral prostate. CONCLUSION: The prostate is a sexual gland that responds to prolactin. Mating-induced prolactin release is required during sexual encounters to activate the epithelial cells in the gland. Here we saw a precise mechanism controlling the release of prolactin during ejaculations that avoid the detrimental effects produced by constant levels. However, we showed that minor elevations of prolactin which do not affect the sexual behaviour of males, produced significant changes at the prostate epithelium that could account for triggering the development of hyperplasia or cancer. Thus, it is suggested that minute elevations of serum prolactin in healthy subjects are at the etiology of prostate abnormal growth

Firefly Luciferase and Rluc8 Exhibit Differential Sensitivity to Oxidative Stress in Apoptotic Cells

Over the past decade, firefly Luciferase (fLuc) has been used in a wide range of biological assays, providing insight into gene regulation, protein-protein interactions, cell proliferation, and cell migration. However, it has also been well established that fLuc activity can be highly sensitive to its surrounding environment. In this study, we found that when various cancer cell lines (HeLa, MCF-7, and 293T) stably expressing fLuc were treated with staurosporine (STS), there was a rapid loss in bioluminescence. In contrast, a stable variant of Renilla luciferase (RLuc), RLuc8, exhibited significantly prolonged functionality under the same conditions. To identify the specific underlying mechanism(s) responsible for the disparate sensitivity of RLuc8 and fLuc to cellular stress, we conducted a series of inhibition studies that targeted known intracellular protein degradation/modification pathways associated with cell death. Interestingly, these studies suggested that reactive oxygen species, particularly hydrogen peroxide (H2O2), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H2O2 to HeLa cells also led to a reduction in fLuc bioluminescence, while H2O2 scavengers stabilized fLuc activity. Comparatively, RLuc8 was far less sensitive to ROS. These observations suggest that fLuc activity can be substantially altered in studies where ROS levels become elevated and can potentially lead to ambiguous or misleading findings

Effects of clusterin over-expression on metastatic progression and therapy in breast cancer

<p>Abstract</p> <p>Background</p> <p>Clusterin 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.</p> <p>Methods</p> <p>To 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 <it>in vitro </it>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.</p> <p>Results</p> <p>In 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.</p> <p>Conclusions</p> <p>These 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.</p