Androgen receptor activation by G(s) signaling in prostate cancer cells

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated alpha subunit of heterotrimeric guanine nucleotide-binding G(s) protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G alpha(s) activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G alpha(s) and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.     

Independent signals determine the subcellular localization of NEP in prostate cancer cells

NEP (Neutral endopeptidase 24.11) is a cell surface enzyme that hydrolyzes bioactive neuropeptides implicated in the transition from androgen-dependent prostate cancer (PC) to androgen-independent PC. We report the cloning and sequence analyses of NEP cDNAs from human androgen-responsive LNCaP PC cells and prostatic stromal cells. To investigate the functional role of a nuclear localization sequence (NLS) detected within the N-terminus and of an endoplasmic reticulum retention signal within the C-terminus, NEP-GFP expression vectors were constructed containing the whole NEP gene, fragments encoding the N-terminus/C-terminus of the protein (5(')NEP-GFP/3(')NEP-GFP), and 5(')NEP-GFP constructs lacking the NLS. 3(')NEP-GFP transfected cells showed plasma membrane/cytoplasmic fluorescence whereas the 5(')NEP-GFP fusion protein was also detected in the nucleus. The omission of the NLS resulted in no reduction in nuclear and an increase in cytoplasmic staining. The results suggest that the analyzed structural motifs determine the subcellular distribution of NEP in epithelial LNCaP PC cells and stromal prostatic cells and therefore could be responsible for the altered cellular localization of NEP observed in PC.     

Dithiocarbamate prodrugs activated by prostate specific antigen to target prostate cancer

Disulfiram in conjunction with copper has been shown to be a potent anticancer agent. However, disulfiram’s therapeutic potential in prostate cancer is hindered by off-target effects due to its reactive and nucleophilic thiol-containing component, diethyldithiocarbamate (DTC). To minimize undesirable reactivity, we have strategically blocked the thiol moiety in DTC with a cleavable p-aminobenzyl (pAB) group linked to peptide substrates recognized by prostate specific antigen (PSA). Here we report the synthesis and evaluation in cancer cell models of two PSA-activatable prodrugs: HPD (Ac-HSSKLQL-pAB-DTC and RPD (RSSYYSL-pAB-DTC). In vitro exposure to PSA was found to trigger activation of HPD and RPD to release diethyldithiocarbamate, and both prodrugs were found to induce toxicity in prostate cancer cells, with HPD showing the most promising selectivity. With copper supplementation, the IC₅₀ of HPD was 1.4 µM in PSA-expressing LNCaP cells, and 11 µM in PC3 cells that do not express PSA. These studies demonstrate the utility of using peptide recognition handles to direct the activity of dithiocarbamate prodrugs for selective cytotoxicity of cancer cells.     

Estrogen induced synthesis of specific proteins in human breast cancer cells

Human breast cancer cells in tissue culture (MCF-7) were pretreated with the antiestrogen nafoxidine to arrest cellular proliferation and then were given estradiol to release this block and stimulate DNA synthesis and cell division. During this period of growth stimulation intracellular proteins, labeled by a double isotope method, were analyzed on SDS-polyacrylamide gel electrophoresis. Estradiol directly increases the rates of synthesis of specific proteins which migrate on SDS-gels at molecular weights of 24,000 and 36,000. Nafoxidine-pretreatment alone does not induce these same proteins, and no changes in the rates of specific protein synthesis occur in cells grown on control medium for the same length of time as on estradiol. Induced synthesis of these proteins is observed only during the period of estrogen stimulation of cell proliferation following pretreatment with nafoxidine. We do not detect induction when cells are incubated with estradiol without antiestrogen-pretreatment. Since rescue of antiestrogen growth inhibition is also the only condition under which MCF-7 cell division can be reproducibly stimulated by estrogen, these proteins may be related to estrogen effects on cellular proliferation.     

Antioxidant system activation in prostate cancer

The activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and the levels of copper, zinc, and malondialdehyde were determined in 21 age-, sex-, and body-mass-index-matched prostate cancer patients; 50 patients diagnosed with benign prostatic obstruction (BPO) were compared to 50 healthy male subjects acting as controls. The patients were divided into two groups depending on the stage of the disease (group 1 [organ-confined] and group II [advanced disease]) and into three subgroups according to differentiation criteria: subgroup A (n=5, Gleason sum 2–4, well differentiated); subgroup B (n=9, Gleason sum 5–7, moderately differentiated), and subgroup C (n=7, Gleason sum 8–10, poorly differentiated). The MDA levels were higher and the antioxidant activity and Zn levels lower in the prostate cancer groups than in the healthy control and BPO groups. These results confirm the value of therapies aimed at increasing the antioxidant capacity and encourage the use of plasma and erythrocyte Zn levels in the differential diagnosis of BPO and prostate cancer. The MDA levels can be used in the diagnosis and follow-up of prostate cancer.     

Calcium signals and phospholipid methylation in eukaryotic cells.

Rat basophil leukaemic (2H3) cells, mast cells and mouse thymocytes respond to stimulation by specific ligands with an increase in the free cytosolic Ca2+ concentration. The time courses of these Ca signals and the biological responses have been compared with changes in phospholipid metabolism. Increased phosphoinositide metabolism coincides with the Ca signals and the responses in each cell system, whereas any increase in phospholipid methylation during the response is less than one molecule per receptor and at least 5-50-fold less than the increases reported previously. Furthermore, no significant changes were detected in the concentration of S-adenosylmethionine, the methyl-group donor in the synthesis of methylated phospholipids. The hypothesis that phospholipid methylation is obligatory for receptor-mediated Ca signals is not supported by these data and requires critical re-evaluation.     

ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS enhanced the expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function.     

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.     

Calcium cytotoxicity sensitizes prostate cancer cells to standard-of-care treatments for locally advanced tumors

Therapy resistance is a major roadblock in oncology. Exacerbation of molecular dysfunctions typical of cancer cells have proven effective in twisting oncogenic mechanisms to lethal conditions, thus offering new therapeutic avenues for cancer treatment. Here, we demonstrate that selective agonists of Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), a cation channel characteristic of the prostate epithelium frequently overexpressed in advanced stage III/IV prostate cancers (PCa), sensitize therapy refractory models of PCa to radio, chemo or hormonal treatment. Overall, our study demonstrates that pharmacological-induced Ca²⁺ cytotoxicity is an actionable strategy to sensitize cancer cells to standard therapies.Subject terms: Targeted therapies, Prostate cancer     

A specific molecular beacon probe for the detection of human prostate cancer cells

The small-molecule, water-soluble molecular beacon probe 1 is hydrolyzed by the lysate and living cells of human prostate cancer cell lines (LNCaP), resulting in strong green fluorescence. In contrast, probe 1 does not undergo significant hydrolysis in either the lysate or living cells of human nontumorigenic prostate cells (RWPE-1). These results, corroborated by UV-Vis spectroscopy and fluorescent microscopy, reveal that probe 1 is a sensitive and specific fluorogenic and chromogenic sensor for the detection of human prostate cancer cells among nontumorigenic prostate cells and that carboxylesterase activity is a specific biomarker for human prostate cancer cells.     

Cell death of prostate cancer cells by specific amino acid restriction depends on alterations of glucose metabolism

Selective amino acid restriction targets mitochondria resulting in DU145 and PC3 prostate cancer cell death. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met) differentially modulates glucose metabolism, glycogen synthase kinase 3β (GSK3β), p53, and pyruvate dehydrogenase (PDH) in these two cell lines. In DU145 cells, Gln and Met restriction increase glucose consumption, but Tyr/Phe restriction does not. Addition of glucose to culture media diminishes cell death induced by Tyr/Phe‐restriction. Addition of pyruvate reduces cell death due to Tyr/Phe and Gln restriction. Tyr/Phe, Gln and Met restriction increase phosphorylation of GSK3β‐Ser⁹, phosphorylation of p53‐Ser¹⁵ and reduce the mitochondrial localization of PDH. Addition of glucose or pyruvate to cultures significantly reverses the alterations in GSK3β, p53 and PDH induced by amino acid restriction. In p53‐null PC3 cells, Tyr/Phe, Gln and Met restriction decreases glucose consumption, reduces phosphorylation of Akt‐Ser⁴⁷³, and increases phosphorylation of GSK3β‐Ser⁹. Addition of pyruvate or glucose reduces death of Met‐restricted cells. Addition of glucose increases phosphorylation of Akt‐Ser⁴⁷³ in amino acid‐restricted cells reduces phosphorylation of GSK3β‐Ser⁹ in Tyr/Phe and Gln restricted cells and increases phosphorylation of GSK3β‐Ser⁹ in Met restricted cells. Addition of pyruvate reduces phosphorylation of GSK3β‐Ser⁹ in all amino acid‐restricted cells. In summary, cell death induced by specific amino acid restriction is dependent on or closely related to the modulation of glucose metabolism. GSK3β (DU145 and PC3) and p53 (DU145) are crucial switches connecting metabolism and these signaling molecules to cell survival during amino acid restriction. J. Cell. Physiol. 224: 491–500, 2010. © 2010 Wiley‐Liss, Inc.     

Propapoptotic effects of NF-kappaB in LNCaP prostate cancer cells lead to serine protease activation

LNCaP prostate cancer cells are resistant to induction of apoptosis by gamma-irradiation and partially sensitive to TNF-alpha or FAS antibody, irradiation sensitizes cells to apoptosis induced by FAS antibody or TNF-alpha. LNCaP cell clones stably expressing IkappaBalpha super repressor were resistant to apoptosis induced by death ligands in the presence or absence of irradiation. IkappaBalpha super repressor expression also increased clonogenic survival after exposure to TNF-alpha+irradiation, but had no effect on survival after irradiation alone. IkappaBalpha super repressor expression blocked the increase of whole cell and cell surface FAS expression induced by TNF-alpha, but did not effect induction of FAS expression and cell surface FAS expression that resulted from irradiation. In cells expressing IkappaBalpha super repressor there was diminished activation of caspases-8 and -7 and diminished production of proscaspases-8 and -7, usually required for death induction in LNCaP cells. Peptide inhibitors of caspase activation complemented the IkappaBalpha super repressor inhibition of apoptosis, but peptide inhibitors of serine proteases had no effect on LNCaP cells expressing IkappaBalpha super repressor. Moreover, cleavage of a serine protease substrate was induced by treatment of LNCaP cells with TNF-alpha and irradiation. The data suggest that in LNCaP cells NF-kappaB mediates a proapoptotic pathway that leads to activation of proapoptotic serine proteases.