thapsigargin and Prostatic-Neoplasms

Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned.

Topics: Antineoplastic Agents; Apoptosis; Calicheamicins; Cell Proliferation; Delayed-Action Preparations; Drug Carriers; Drug Design; Drug Liberation; Enzyme Therapy; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Maytansine; Molecular Targeted Therapy; Peptide Hydrolases; Prodrugs; Prostatic Neoplasms; Thapsigargin; Toxins, Biological

The skin irritating principle from Thapsia garganica was isolated, named thapsigargin and the structure elucidated. By inhibiting the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) thapsigargin provokes apoptosis in almost all cells. By conjugating thapsigargin to peptides, which are only substrates for either prostate specific antigen (PSA) or prostate specific membrane antigen (PSMA) prodrugs were created, which selectively affect prostate cancer cells or neovascular tissue in tumors. One of the prodrug is currently tested in clinical phase II. The prodrug under clinical trial has been named mipsagargin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apiaceae; Apoptosis; Cell Proliferation; Enzyme Inhibitors; Humans; Male; Mice; Molecular Structure; Neovascularization, Pathologic; Prodrugs; Prostatic Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Soft Tissue Neoplasms; Thapsigargin

Due to the propensity of relapse and resistance with prolonged androgen deprivation therapy (ADT), there is a growing interest in developing non-hormonal therapeutic approaches as alternative treatment modalities for hormone refractory prostate cancer (HRPC). Although the standard treatment for HRPC consists of a combination of ADT with taxanes and anthracyclines, the clinical use of chemotherapeutics is limited by systemic toxicity stemming from nondiscriminatory drug exposure to normal tissues. In order to improve the tumor selectivity of chemotherapeutics, various targeted prodrug approaches have been explored. Antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT) strategies leverage tumor-specific antigens and transcription factors for the specific delivery of cytotoxic anticancer agents using various prodrug-activating enzymes. In prostate cancer, overexpression of tumor-specific proteases such as prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) is being exploited for selective activation of anticancer prodrugs designed to be activated through proteolysis by these prostate cancer-specific enzymes. PSMA- and PSA-activated prodrugs typically comprise an engineered high-specificity protease peptide substrate coupled to a potent cytotoxic agent via a linker for rapid release of cytotoxic species in the vicinity of prostate cancer cells following proteolytic cleavage. Over the past two decades, various such prodrugs have been developed and they were effective at inhibiting prostate tumor growth in rodent models; several of these prodrug approaches have been advanced to clinical trials and may be developed into effective therapies for HRPC.

Topics: Animals; Antineoplastic Agents; Clinical Trials as Topic; Doxorubicin; Drug Therapy; Humans; Male; Nitric Oxide; Paclitaxel; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Recurrence; Thapsigargin; Vinblastine

Available chemotherapeutics take advantage of the fast proliferation of cancer cells. Consequently slow growth makes androgen refractory prostate cancer resistant towards available drugs. No treatment is available at the present, when the cancer has developed metastases outside the prostate (T4 stage). Cytotoxins killing cells irrespective of the phase of the cell cycle will be able to kill slowly proliferating prostate cancer cells. Lack of selectivity, however, prevents their use as systemic drugs. Prostate cancer cells secrete characteristic proteolytic enzymes, e.g. PSA and hK2, with unusual substrate specificity. Conjugation of cytotoxins with peptides, which are selective substrates for PSA or hK2, will afford prodrugs, from which the active drug only will be released in close vicinity of the cancer cells. Based on this strategy prodrugs targeted at prostate cancer cells have been constructed and evaluated as potential drugs for prostate cancer. The potency of the thapsigargins as apoptotic agents make these naturally occurring sesquiterpene lactones attractive lead compounds. Intensive studies on structure-activity relationships and chemistry of the thapsigargins have enabled construction of potent derivatives enabling conjugation with peptides. Studies on the mechanism of action of the thapsigargins have revealed that the cytoxicity is based on their ability to inhibit the intracellular sarco-/endoplasmtic calcium pump.

Topics: Antineoplastic Agents; Cell Proliferation; Drug Design; Drug Screening Assays, Antitumor; Humans; Male; Prostatic Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Structure-Activity Relationship; Thapsigargin

Prostate cancer is uniformly fatal once it has spread outside of the prostate gland. Prostate cancers have a remarkably low proliferative rate, which may in part explain their relative unresponsiveness to conventional antiproliferative chemotherapy. New therapies for prostate cancer that activate proliferation independent cell death are therefore needed. The endoplasmic reticulum (ER) has emerged as an organelle that plays a major role in cell signaling pathways, cellular response to stress and cellular activation of apoptosis. In this review, the SERCA pump is identified as an ER protein whose normal function is required by all cells and represents a potential therapeutic target for cancer therapy. Sustained SERCA inhibition by agents such as thapsigargin results in activation of ER-stress response and simultaneous activation of apoptotic pathways within the ER and the mitochondria. Due to the SERCA pump's critical role in normal cellular metabolism, agents like thapsigargin directed toward inhibiting SERCA function would likely produce significant toxicity to normal cells and, therefore, must be selectively targeted to cancer sites. The cytotoxicity of thapsigargin can be attenuated, however by coupling to a targeting peptide to produce an inactive prodrug that is only activated by prostate cancer specific proteases such as the serine protease prostate-specific antigen (PSA). PSA-activated thapsigargin prodrugs have been characterized that are selectively toxic to PSA-producing prostate cancer cells in vitro and in vivo. These prodrugs are currently undergoing preclinical evaluation as potential targeted therapy for prostate cancer.

Topics: Apoptosis; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Male; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Androgen is a major growth factor in the normal prostate and determines the overall number of prostate cells. Metastatic prostate cancer, while initially responsive to androgen ablation, eventually becomes hormone-refractory and resistant to many treatments. Unfortunately, there are very few agents in the preclinical stage with a seemingly promising future for hormone-refractory prostate cancer (HRPC) that are actually taken through the complete drug development process, including US Food and Drug Administration approval. Many novel strategies under investigation for treating HRPC target metastatic prostate cancer cells that are neither androgen-dependent nor in the proliferative state. Examples of therapies that target this so-called "Achilles' heel" of HRPC include immune therapy, gene therapy, angiogenesis inhibition, and activation of programmed cell death. Unique properties of HRPC allow for the development of novel treatments that target prostate-specific antigen (PSA), human glandular kallikrein-2, or prostate-specific membrane antigen. An inactive prodrug with a thapsigargin analogue, a sesquiterpene lactone from the plant Thapsia garganica, is currently under investigation specifically for the targeted therapy of HRPC. Preclinical data suggest the PSA-targeting abilities of this novel therapy are associated with a nearly complete cessation of tumour growth with minimal toxicity.

Topics: Androgens; Apoptosis; Drug Design; Humans; Male; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Technology, Pharmaceutical; Thapsigargin

Topics: Apoptosis; Gene Expression; Humans; Male; Neoplasm Metastasis; Prodrugs; Prostatic Neoplasms; Sesquiterpenes; Thapsigargin

Development of therapeutic resistance is responsible for most prostate cancer (PCa) related mortality. Resistance has been attributed to an acquired or selected cancer stem cell phenotype. Here we report the histone deacetylase inhibitor apicidin (APC) or ER stressor thapsigargin (TG) potentiate paclitaxel (TXL)-induced apoptosis in PCa cells and limit accumulation of cancer stem cells. TXL-induced responses were modulated in the presence of TG with increased accumulation of cells at G1-phase, rearrangement of the cytoskeleton, and changes in cytokine release. Cytoskeletal rearrangement was associated with modulation of the cytoplasmic and mitochondrial unfolded protein response leading to mitochondrial dysfunction and release of proapoptotic proteins from mitochondria. TXL in combination with APC or TG enhanced caspase activation. Importantly, TXL in combination with TG induced caspase activation and apoptosis in X-ray resistant LNCaP cells. Increased release of transforming growth factor-beta (TGF-β) was observed while phosphorylated β-catenin level was suppressed with TXL combination treatments. This was accompanied by a decrease in the CD44(+)CD133(+) cancer stem cell-like population, suggesting treatment affects cancer stem cell properties. Taken together, combination treatment with TXL and either APC or TG induces efficient apoptosis in both proliferating and cancer stem cells, suggesting this therapeutic combination may overcome drug resistance and recurrence in PCa.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; beta Catenin; Caspases; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Cytoskeleton; Enzyme Activation; G1 Phase; G2 Phase; HSP70 Heat-Shock Proteins; Humans; Interferon-gamma; Interleukin-8; Male; Matrix Metalloproteinases; Membrane Potential, Mitochondrial; Mitochondria; Neoplastic Stem Cells; Paclitaxel; Peptides, Cyclic; Phosphorylation; Prostatic Neoplasms; Reactive Oxygen Species; Thapsigargin; Transforming Growth Factor beta; Unfolded Protein Response; X-Rays

Several cellular mechanisms contribute to the neuroendocrine differentiation of prostate cancer cells, including exposure to sodium butyrate (NaBu), a naturally occurring salt of the short chain fatty acid n-butyric acid. NaBu belongs to a class of histone deacetylase inhibitors with potential anticancer function. T-type calcium channel expression constitutes an important route for calcium influx in tumor cells that may trigger changes in cell proliferation and differentiation. In this work we investigated the role NaBu on the differentiation of lymph node carcinoma of the prostate (LNCaP) cells and its effect on T-type Ca(2+) channel expression. NaBu stimulates the morphological and molecular differentiation of LNCaP cells. Stimulation of LNCaP cells with NaBu evokes a significant increase in the expression of the Cav3.2 T-type channel subunits. Furthermore, the increased Cav3.2 expression promotes membrane insertion of T-type Ca(2+) channels capable of generating fast inactivating Ca(2+) currents, sensitive to 100μM Ni(2+) ions. Inhibition of T-type Ca(2+) channel function reduces the outgrowth of neurite-like processes in LNCaP cells. NaBu-evoked expression of T-type Ca(2+) channels is also involved in the regulation of cell viability. Inhibition of T-type Ca(2+) channels causes a significant reduction in the viability of LNCaP cells treated with 1mM NaBu, suggesting that Ca(2+) influx via T-type channels can promote cell proliferation. However, increased expression of T-type Ca(2+) channels enhanced the cytotoxic effect of thapsigargin and paclitaxel on cell proliferation. These findings demonstrate that NaBu stimulates T-type Ca(2+) channel expression, thereby regulating both the morphological differentiation and growth of prostate cancer cells.

Topics: Butyric Acid; Calcium Channels, T-Type; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Histone Deacetylase Inhibitors; Humans; Male; Paclitaxel; Prostatic Neoplasms; Thapsigargin

Peripheral corticotropin-releasing hormone receptors (CRHRs) are G protein-coupled receptors that play different roles depending on tissue types. Previously, we discovered the mechanism of CRHR-mediated apoptosis of mouse prostate cancer cell line (RM-1) to be a change of Bcl-2:Bax ratio, and CRH was found to inhibit transforming growth factor β migration of breast cancer cells via CRHRs. In the present study, we investigated cytosolic calcium-dependent phospholipase A2 (cPLA2) bridging CRHR activations and Bcl-2:Bax ratio and the effect of CRHR activation on cell migration. Silencing of cPLA2 attenuated a CRHR1 agonist, CRH-induced apoptosis, and the decrease of the Bcl-2:Bax ratio, whereas silencing of cPLA2 aggravated CRHR2 agonist, Urocortin 2 (Ucn2)-inhibited apoptosis, and the increase of the Bcl-2:Bax ratio. CRH in a time- and concentration-dependent manner increased cPLA2 expression mainly through interleukin 1β (IL1β) upregulation. Ucn2 decreased cPLA2 expression through neither tumor necrosis factor α nor IL1β. CRH-suppressed decay of cPLA2 mRNA and Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated cPLA2 expression after CRH or Ucn2 stimulation respectively. In addition, both CRH and Ucn2 induced migration of RM-1 cells. Our observation not only established a relationship between CRHRs and cell migration but also for the first time, to our knowledge, demonstrated that cPLA2 participates in CRHR1-induced apoptosis and CRHR2-inhibited apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Movement; Corticotropin-Releasing Hormone; Dactinomycin; Down-Regulation; HEK293 Cells; Humans; Interleukin-1beta; Male; Mice; Mitomycin; Peptide Fragments; Phospholipases A2; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Pyrroles; Receptors, Corticotropin-Releasing Hormone; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Tumor Necrosis Factor-alpha; Up-Regulation; Urocortins

The inhibition of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) by thapsigargin (Tg) and Tg-type analogues is considered to trigger cell death by activation of apoptotic pathways. Some of these analogues may be useful as antineoplastic agents after appropriate targeting as peptide conjugated prodrugs to cancer cells. With this in mind, this study evaluates the effect on LNCaP androgen-sensitive cancer cells of thapsigargin substituted with 12-aminododecanoyl linkers and Leu (Leu-8ADT), aspartate (Asp-8ADT) or Boc-8ADT. Our results show that both Leu-8ADT and Asp-8ADT result in rapid ER calcium depletion and an influx of calcium across the plasma membrane by activation of store-operated calcium entry. By contrast, ER Ca(2+) depletion by Boc-8ADT is a very slow process that does not perceptibly increase cytosolic Ca(2+) and activate store-operated calcium entry, because the inhibition of SERCA with this compound is very slow. Nevertheless, we find that Boc-8ADT is a more efficient inducer of apoptosis than both Tg and Leu-8ADT. Compared with Tg and the other analogues, apoptosis induced by Asp-8ADT is very modest, although this compound also activates store-operated calcium entry and at high concentrations (1 μm) causes severe morphological changes, reflecting decreased cell viability. We conclude that many factors need to be considered for optimization of these compounds in antineoplastic drug design. Among these ER stress induced by Ca(2+) endoplasmic reticulum mobilization seems particularly important, whereas the early cytosolic increase of Ca(2+) concentration preceding the executive phase of apoptosis appears to be of no, or little, consequence for a subsequent apoptotic effect.

Topics: Apoptosis; Calcium; Calcium Channels; Cell Membrane; Cell Proliferation; Cytosol; Enzyme Inhibitors; Humans; Ion Transport; Male; Prostatic Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Tumor Cells, Cultured

Expression of clusterin (CLU) closely correlates with the regulation of apoptosis in cancer. Although endoplasmic reticulum (ER) stress-induced upregulation and retrotranslocation of cytoplasmic CLU (presecretory (psCLU) and secreted (sCLU) forms) has been linked to its anti-apoptotic properties, mechanisms mediating these processes remain undefined. Here, we show using human prostate cancer cells that GRP78 (Bip) associates with CLU under ER stress conditions to facilitate its retrotranslocation and redistribution to the mitochondria. Many ER stress inducers, including thapsigargin, MG132 or paclitaxel, increased expression levels of GRP78 and CLU, as well as post-translationally modified hypoglycosylated CLU forms. ER stress increased association between GRP78 and CLU, which led to increased cytoplasmic CLU levels, while reducing sCLU levels secreted into the culture media. GRP78 stabilized CLU protein and its hypoglycosylated forms, in particular after paclitaxel treatment. Moreover, subcellular fractionation and confocal microscopy with CLUGFP indicated that GRP78 increased stress-induced CLU retrotranslocation from the ER with co-localized redistribution to the mitochondria, thereby reducing stress-induced apoptosis by cooperatively stabilizing mitochondrial membrane integrity. GRP78 silencing reduced CLU protein, but not mRNA levels, and enhanced paclitaxel-induced cell apoptosis. Taken together, these findings reveal novel dynamic interactions between GRP78 and CLU under ER stress conditions that govern CLU trafficking and redistribution to the mitochondria, elucidating how GRP78 and CLU cooperatively promote survival during treatment stress in prostate cancer.

Topics: Apoptosis; Cell Line, Tumor; Clusterin; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Leupeptins; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Paclitaxel; Prostatic Neoplasms; Protein Transport; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Up-Regulation

GRP78, a major endoplasmic reticulum chaperone and signaling regulator, is commonly overexpressed in cancer. Moreover, induction of GRP78 by a variety of anti-cancer drugs, including histone deacetylase inhibitors, confers chemoresistance to cancer, thereby contributing to tumorigenesis. Thus, therapies aimed at decreasing GRP78 levels, which results in the inhibition of tumor cell proliferation and resensitization of tumor cells to chemotherapeutic drugs may hold promise for cancer treatment. Despite advances in our understanding of GRP78 actions, little is known about endogenous inhibitors controlling its expression. As endogenous regulators, microRNAs (miRNAs) play important roles in modulating gene expression; therefore, we sought to identify miRNA(s) that target GRP78, under the hypothesis that these miRNAs may serve as therapeutic agents. Here, we report that three miRNAs (miR-30d, miR-181a, miR-199a-5p) predicted to target GRP78 are down-regulated in prostate, colon and bladder tumors, and human cancer cell lines. We show that in C42B prostate cancer cells, these miRNAs down-regulate GRP78 and induce apoptosis by directly targeting its 3' untranslated region. Importantly, we demonstrate that the three miRNAs act cooperatively to decrease GRP78 levels, suggesting that multiple miRNAs may be required to efficiently control the expression of some genes. In addition, delivery of multiple miRNAs by either transient transfection or lentivirus transduction increased the sensitivity of cancer cells to the histone deacetylase inhibitor, trichostatin A, in C42B, HCT116 and HL-60 cells. Together, our results indicate that the delivery of co-transcribed miRNAs can efficiently suppress GRP78 levels and GRP78-mediated chemoresistance, and suggest that this strategy holds therapeutic potential.

Topics: 3' Untranslated Regions; Adenocarcinoma; Animals; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Heat-Shock Proteins; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Lentivirus; Male; Mice; Mice, Nude; MicroRNAs; Neovascularization, Pathologic; Prostatic Neoplasms; RNA; RNA, Messenger; Thapsigargin; Transcription, Genetic; Transfection; Tumor Stem Cell Assay; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line.. Cultured PC-3 cells were used in studies utilizing pharmacologic interventions, immunoblotting, and confocal immunolocalization.. Focal adhesion kinase (FAK), a tyrosine kinase involved in cell adhesion, is rapidly proteolyzed in serum-starved PC-3 cells exposed to the calcium ionophore, ionomycin; Nck, p130CAS, PKCα, and Ras-GAP are also degraded. Thapsigargin, which causes more moderate increases in intracellular calcium, induces partial proteolysis of these proteins. Calpain inhibitors block the proteolytic responses to ionomycin and thapsigargin. Ionomycin does not induce proteolysis in cells maintained in serum, suggesting a protective role for growth factors contained in serum. LPA causes minor FAK proteolysis when added alone, but protects against ionomycin-induced proteolysis in a time-dependent manner. LPA also protects against the cell detachment that eventually follows ionomycin treatment. The response to LPA is blocked by an LPA receptor antagonist. A similar effect of LPA is observed in ionomycin-treated Rat-1 fibroblasts. In PC-3 cells, the protective effects of LPA and serum are correlated with phosphorylation and redistribution of paxillin, suggesting roles for phosphorylation-mediated protein-protein interactions.. The complex effects of LPA on calpain-mediated proteolysis of FAK and other adhesion proteins are likely to play a role in the ability of LPA to promote attachment, migration, and survival of prostate cancer cells.

Topics: Adenocarcinoma; Animals; Calpain; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Screening Assays, Antitumor; Fibroblasts; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ionomycin; Isoxazoles; Lysophospholipids; Male; Paxillin; Phosphorylation; Propionates; Prostatic Neoplasms; Proteolysis; Rats; Thapsigargin

The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 µM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.

Topics: Calcium; Calcium Signaling; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; Fura-2; Homeostasis; Humans; Indoles; Male; Prostatic Neoplasms; Thapsigargin; Type C Phospholipases

Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG.. The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9-11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided.. FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC(50) = 3.5 nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206 mm(3), 95% CI = 0.068 to 0.344 mm(3); control: mean = 0.580 mm(3), 95% CI = 0.267 to 0.893 mm(3)) and 0.24 (treated: mean = 0.131 mm(3), 95% CI = 0.09 to 0.180 mm(3); control: mean = 0.543 mm(3), 95% CI = 0.173 to 0.913 mm(3)), respectively, on day 21 after therapy.. This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.

Topics: Amino Acids; Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Calcium; Cell Line, Tumor; Endopeptidases; Female; Gelatinases; Gene Expression Regulation, Neoplastic; Humans; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Neoplasms; Prodrugs; Prostatic Neoplasms; Serine Endopeptidases; Thapsigargin; Transplantation, Heterologous; Urinary Bladder Neoplasms

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca²⁺](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca²⁺](i) in a concentration-dependent fashion. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺ indicating that Ca²⁺ entry and release both contributed to the [Ca²⁺](i) rise. This Ca²⁺ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca²⁺ channels or by modulation of protein kinase C activity. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca²⁺ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca²⁺](i) rise, suggesting that thapsigargin released Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca²⁺](i) rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca²⁺ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca²⁺](i) rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2-sensitive Ca²⁺ channels. Thapsigargin also induced cell death via Ca²⁺-dependent pathways and Ca²⁺-independent apoptotic pathways.

Topics: Apoptosis; Aristolochic Acids; Calcium; Calcium Signaling; Cell Line, Tumor; Cell Survival; Estrenes; Fluorescence; Fura-2; Humans; Intracellular Space; Male; Manganese; Prostatic Neoplasms; Pyrrolidinones; Thapsigargin; Type C Phospholipases

Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production.. We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways.. Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation.. We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.

Topics: Acute-Phase Proteins; Adenocarcinoma; Animals; Cell Line, Tumor; Endoplasmic Reticulum; Gene Expression Regulation, Neoplastic; Glucose; Humans; Lipocalin-2; Lipocalins; Male; Mice; Neoplasm Proteins; NF-kappa B; Nitriles; Oncogene Proteins; Phenylbutyrates; Prostatic Neoplasms; Protein Biosynthesis; Proto-Oncogene Proteins; Sulfones; Thapsigargin; Transcription, Genetic; Tunicamycin; Unfolded Protein Response; Up-Regulation

The balance between proliferation and cell death is often disrupted in cancer leading to tumor growth. In prostate cancer, these events are regulated, at least in part, through androgen signaling. Prostate cancer is dependent on androgens for growth in the initial stages where apoptosis is simultaneously inhibited. Androgen signaling remains important in later stages of prostate cancer as well. Here, we provide methods to study apoptosis in prostate cancer cells and its regulation by androgens. In prostate cancer cells grown in vitro, apoptosis can be induced by different stimuli, such as the endoplasmic reticulum Ca2+ ATPase inhibitor Thapsigargin (TG) through the intrinsic apoptosis pathway, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plus the inhibition of PI3K, through the extrinsic signaling pathway; both of these apoptotic events can be blocked by androgens. Here, we provide protocols to assess apoptosis triggered by TG or TRAIL plus PI3K inhibitor LY294002, in prostate cancer cells in vitro using nuclear fragmentation and TUNEL assays aided by fluorescence microscopy or flow cytometry.

Topics: Androgens; Apoptosis; Cell Line, Tumor; Chromones; DNA Fragmentation; Flow Cytometry; Humans; In Situ Nick-End Labeling; Male; Microscopy, Fluorescence; Morpholines; Prostatic Neoplasms; Protein Kinase Inhibitors; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca(2+) pump that causes Ca(2+) efflux from ER stores, increased cytosolic [Ca(2+)] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu(98)-Leu(115)) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca(2+)] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca(2+), or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca(2+) from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.

Topics: Antibodies, Neoplasm; Autoantibodies; Calcium; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Epitopes; Heat-Shock Proteins; Humans; Male; Phosphatidylserines; Prostatic Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin; Thromboplastin; Type C Phospholipases; Venous Thromboembolism

The molecular nature of calcium (Ca(2+))-dependent mechanisms and the ion channels having a major role in the apoptosis of cancer cells remain a subject of debate. Here, we show that the recently identified Orai1 protein represents the major molecular component of endogenous store-operated Ca(2+) entry (SOCE) in human prostate cancer (PCa) cells, and constitutes the principal source of Ca(2+) influx used by the cell to trigger apoptosis. The downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways, such as those induced by thapsigargin (Tg), tumor necrosis factor α, and cisplatin/oxaliplatin. The transfection of functional Orai1 mutants, such as R91W, a selectivity mutant, and L273S, a coiled-coil mutant, into the cells significantly decreased both SOCE and the rate of Tg-induced apoptosis. This suggests that the functional coupling of STIM1 to Orai1, as well as Orai1 Ca(2+)-selectivity as a channel, is required for its pro-apoptotic effects. We have also shown that the apoptosis resistance of androgen-independent PCa cells is associated with the downregulation of Orai1 expression as well as SOCE. Orai1 rescue, following Orai1 transfection of steroid-deprived cells, re-established the store-operated channel current and restored the normal rate of apoptosis. Thus, Orai1 has a pivotal role in the triggering of apoptosis, irrespective of apoptosis-inducing stimuli, and in the establishment of an apoptosis-resistant phenotype in PCa cells.

Topics: Amino Acid Substitution; Antineoplastic Agents; Apoptosis; Calcium; Calcium Channels; Cell Line, Tumor; Cisplatin; Humans; Male; Membrane Proteins; Mutation; Neoplasm Proteins; ORAI1 Protein; Phenotype; Prostatic Neoplasms; Stromal Interaction Molecule 1; Thapsigargin; Tumor Necrosis Factor-alpha

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) in PC3 human prostate cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca²⁺]i levels in suspended PC3 cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-50 microM increased [Ca²⁺]i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 60% by removing extracellular Ca²⁺. M-3M3FBS-induced Ca²⁺ influx was inhibited by the store-operated Ca²⁺ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca²⁺-free medium, 30 microM m-3M3FBS pretreatment greatly inhibited the [Ca²⁺]i rise induced by the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or BHQ. Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid reduced the major part of m-3M3FBS-induced [Ca²⁺]i rise. Inhibition of phospholipase C with U73122 did not much alter m-3M3FBS-induced [Ca²⁺]i rise. Collectively, in PC3 cells, m-3M3FBS induced [Ca²⁺]i rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via store-operated Ca²⁺ channels.

Topics: Adenocarcinoma; Aristolochic Acids; Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Line, Tumor; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Humans; Indoles; Male; Models, Animal; Phospholipase A2 Inhibitors; Prostatic Neoplasms; Sulfonamides; Thapsigargin; Type C Phospholipases

During apoptosis, proteolytic cleavage of Bax at the amino terminus generates a truncated Bax of approximately 18 kDa (p18Bax) and an amino-terminal peptide of approximately 3 kDa (p3Bax). Whereas extensive studies have shown that p18Bax behaves like a BH3 protein with enhanced pro-apoptotic function over that of the full-length Bax (p21Bax), little is known about the function of p3Bax in apoptosis. We have previously shown that Bax and Ca2+ play a synergistic role in amplifying apoptosis signaling and that store-operated Ca2+ entry (SOCE) contributes to Bax-mediated apoptosis in prostate cancer cells. Here we test whether p3Bax can contribute to regulation of Ca2+ signaling during apoptosis through use of a membrane-penetrating peptide to facilitate delivery of recombinant p3Bax into NRP-154 cells, a prostate epithelial cell line with tumorigenic capacity. We find that human immunodefficiency virus transactivator of transcription protein (TAT)-p3Bax fusion peptide can enhance thapsigargin-induced apoptosis in NRP-154 cells, elevate SOCE activity, and increase inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores. Our data indicates that p3Bax can modulate the entry of extracellular Ca2+ and thus regulate the amplification of apoptosis in prostate cancer cells.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; HeLa Cells; Homeostasis; Humans; Inositol 1,4,5-Trisphosphate; Male; Mice; Peptide Fragments; Prostatic Neoplasms; Rats; Recombinant Fusion Proteins; tat Gene Products, Human Immunodeficiency Virus; Thapsigargin; Time Factors; Transfection

Taxanes are first line drugs for treating prostate cancer recurrence after the failure of anti-androgen therapy. There is a need to make taxanes more effective since they only provide palliative benefit. Exploiting endoplasmic reticulum (ER) stress death signaling to enhance drug efficacy has not been delineated. Human PC-3 cells were used as a model of hormone refractory prostate cancer. Thapsigargin and methylseleninic acid (MSA) were examined as sensitizers. Thapsigargin is a classic ER stress inducer. The activity of MSA in inducing ER stress has recently been studied by our group. The efficacy of single drug and the various combinations was evaluated by measuring apoptosis with a cell death ELISA kit. Thapsigargin increased the cell killing potency of paclitaxel or docetaxel by 10- to 12-fold, while MSA caused a 5- to 8-fold increase. Since thapsigargin is not used clinically because of its toxicity, the follow-up experiments were done with MSA. To test the hypothesis that a threshold level of ER stress is crucial to chemotherapeutic sensitization, three different approaches designed to dampen the severity of ER stress induced by MSA were examined. Lowering ER stress consistently attenuated the efficacy of MSA/taxane. GADD153 is a pro-apoptotic transcription factor which is upregulated during ER stress. Knocking down GADD153 by siRNA also reduced the cell killing effect of MSA/taxane. Both the intrinsic and extrinsic apoptotic pathways were involved in the sensitization mechanism. Our study supports the idea that marshalling ER stress apoptotic response is conducive to chemotherapeutic sensitization.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Death; Cell Line, Tumor; Docetaxel; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme-Linked Immunosorbent Assay; Formazans; Heat-Shock Proteins; Humans; Male; Molecular Chaperones; Organoselenium Compounds; Paclitaxel; Prostatic Neoplasms; RNA, Small Interfering; Taxoids; Tetrazolium Salts; Thapsigargin; Time Factors; Transcription Factor CHOP; Transfection

3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) is widely used for inhibition of leucotriene synthesis in in vitro studies, however, many of its other effects have been reported. The present study investigated the effect of MK-886 on cytosolic-free Ca(2+) concentrations ([Ca(2+)](i)) and viability in human PC3 prostate cancer cells. [Ca(2+)](i) in suspended cells was measured by using fura-2. MK-886 at concentrations of 1 microM and above increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 20 microM. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). MK-886 evoked Mn(2+) quenching of fura-2 fluorescence, implicating Ca(2+) entry. MK-886-induced Ca(2+) influx was inhibited by store-operated Ca(2+) entry inhibitors nifedipine, econazole and SKF96365. In Ca(2+)-free medium, after pre-treatment with 10 microM MK-886, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor)-induced [Ca(2+)](i) rises were abolished; and conversely, thapsigargin pre-treatment abolished MK-886-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with U73122 did not alter MK-886-induced [Ca(2+)](i) rises. MK-886 at concentrations of 1-100 microM concentration-dependently decreased cell viability with an IC(50) value of 60 microM. The cytotoxic effect of MK-886 was not inhibited by pre-chelating cytosolic Ca(2+) with BAPTA/AM. Together, in PC3 cells, MK-886 induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum; and Ca(2+) influx via store-operated Ca(2+) channels. Independently, MK-886 was cytotoxic to cells in a Ca(2+)-independent manner.

Topics: Arachidonate 5-Lipoxygenase; Calcium; Calcium Channel Blockers; Cations, Divalent; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Estrenes; Humans; Indoles; Lipoxygenase Inhibitors; Male; Prostatic Neoplasms; Pyrrolidinones; Thapsigargin; Type C Phospholipases

There are quantitative and/or qualitative mechanisms allowing androgen receptor (AR) growth signaling in androgen ablation refractory prostate cancer cells. Regardless of the mechanism, agents that deplete AR protein expression prevent such AR growth signaling. Thapsigargin (TG) is a highly cell-penetrant sequiterpene-lactone that once inside cells inhibits (IC(50), ∼ 10 nmol/L) critically important housekeeping SERCA 2b calcium pumps in the endoplasmic reticulum. Using a series of five genetically diverse androgen ablation refractory human prostate cancer lines (LNCaP, LAPC-4, VCaP, MDA-PCa-2b, and CWR22Rv1), TG inhibition of SERCA pumps consistently results in depletion of the endoplasmic reticulum Ca(+2) coupled with μmol/L elevation in the intracellular free Ca(+2) initiating a molecular cascade that: (a) inhibits Cap-dependent AR protein synthesis resulting in 90% depletion of AR protein by 24 hours of TG exposure, (b) arrests the cells in G(0), and (c) induces their apoptotic death. Unfortunately, due to its highly lipophilic nature, TG is not deliverable as a systemic agent without host toxicity. Therefore, TG analogues containing amino acids were developed, which retain ability to deplete AR protein and induce cell death and which can be covalently linked to peptide carriers producing water soluble prodrugs for systemic delivery. Specific amino acid sequences are used to restrict the liberation of cytotoxic amino acid containing TG analogues from the peptide prodrug by prostate-specific proteases, such as prostate-specific antigen and prostate-specific membrane antigen, or cancer-specific proteases, such as fibroblast activation protein, so that toxicity of these prodrugs is selectively targeted to metastatic sites of prostate cancer. Based on these results, these prodrugs are undergoing clinical development.

Topics: Adaptor Proteins, Signal Transducing; Animals; Calcium; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; DNA-Binding Proteins; Endoplasmic Reticulum; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Phosphoproteins; Phosphorylation; Prostatic Neoplasms; Rabbits; Receptors, Androgen; Signal Transduction; Thapsigargin; Transcription Factors

Binding of activated α(2)-macroglobulin to GRP78 on the surface of human prostate cancer cells promotes proliferation by activating signaling cascades. Autoantibodies directed against the activated α(2)-macroglobulin binding site in the NH(2)-terminal domain of GRP78 are receptor agonists, and their presence in the sera of cancer patients is a poor prognostic indicator. We now show that antibodies directed against the GRP78 COOH-terminal domain inhibit [(3)H]thymidine uptake and cellular proliferation while promoting apoptosis as measured by DNA fragmentation, Annexin V assay, and clonogenic assay. These antibodies are receptor antagonists blocking autophosphorylation and activation of GRP78. Using 1-LN and DU145 prostate cancer cell lines and A375 melanoma cells, which express GRP78 on their cell surface, we show that antibodies directed against the COOH-terminal domain of GRP78 up-regulate the tumor suppressor protein p53. By contrast, antibody directed against the NH(2)-terminal domain of GRP78 shows negligible effects on p53 expression. PC-3 prostate cancer cells, which do not express GRP78 on their cell surface, are refractory to the effects of anti-GRP78 antibodies directed against either the COOH- or NH(2)-terminal domains. However, overexpression of GRP78 in PC-3 cells causes translocation of GRP78 to the cell surface and promotes apoptosis when these cells are treated with antibody directed against its COOH-terminal domain. Silencing GRP78 or p53 expression by RNA interference significantly blocked the increase in p53 induced by antibodies. Antibodies directed against the COOH-terminal domain may play a therapeutic role in cancer patients whose tumors trigger the production of autoantibodies directed against the NH(2)-terminal domain of GRP78.

Topics: Antibodies; Apoptosis; Cell Line, Tumor; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Male; Phosphorylation; Prostatic Neoplasms; Protein Binding; RNA Interference; Signal Transduction; Thapsigargin; Tumor Suppressor Protein p53

During androgen ablation prostate cancer cells' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of alpha(1A)-adrenoceptors in prostate cancer epithelial cells, their exact functional role in androgen-independent cells has yet to be established. Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells.. In order to analyze the membrane topology of the alpha(1A)-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalization of the alpha(1A)-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the alpha(1A)-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of alpha(1A)-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK).. In conclusion, we propose that alpha(1A)-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.

Topics: Apoptosis; Caveolae; Cell Line, Tumor; Cell Survival; Cholesterol; Drug Resistance, Neoplasm; Humans; Male; Membrane Microdomains; Models, Biological; Neurotransmitter Agents; Prostatic Neoplasms; Receptors, Adrenergic, alpha-1; Signal Transduction; Sphingomyelins; Thapsigargin

Antrodia camphorata (AC) has been used as a health supplement in Asia to control different cancers; however, the cellular mechanisms of its effects are unclear. The effect of AC on cultured human prostate cancer cells (PC3) has not been explored. This study examined the effect of AC on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ handling in PC3 cells. AC at concentrations of 5-50 microg/ml did not affect cell viability, but at 100-200 microg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25-200 microg/ml did not alter basal [Ca2+]i, but at a concentration of 25 microg/ml decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment inhibited ATP-, bradykinin-, and histamine-induced enhancement on viability, but reversed thapsigargin-induced cytotoxicity. Immunoblotting showed that AC (200 microg/ml) did not induce the phosphorylation of ERK, JNK, and p38 MAPKs. Collectively, in PC3 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis via pathways unrelated to [Ca2+]i signal and phosphorylation of ERK, JNK and p38 MAPKs.

Topics: Adenosine Triphosphate; Agaricales; Antineoplastic Agents; Apoptosis; Bradykinin; Calcium; Cell Survival; Histamine Antagonists; Humans; Male; Mitogen-Activated Protein Kinases; Phosphorylation; Polyporales; Prostatic Neoplasms; Thapsigargin; Tumor Cells, Cultured

Inhibitors of the enzyme 17alpha-hydroxylase/17,20 lyase are a new class of anti-prostate cancer agents currently undergoing preclinical and clinical development. We have previously reported the superior anticancer activity of our novel 17alpha-hydroxylase/17,20 lyase inhibitor, VN/124-1, against androgen-dependent cancer models. Here, we examined the effect of VN/124-1 on the growth of the androgen-independent cell lines PC-3 and DU-145 and found that the compound inhibits their growth in a dose-dependent manner in vitro (GI50, 7.82 micromol/L and 7.55 micromol/L, respectively). We explored the mechanism of action of VN/124-1 in PC-3 cells through microarray analysis and found that VN/124-1 up-regulated genes involved in stress response and protein metabolism, as well as down-regulated genes involved in cell cycle progression. Follow-up real-time PCR and Western blot analyses revealed that VN/124-1 induces the endoplasmic reticulum stress response resulting in down-regulation of cyclin D1 protein expression and cyclin E2 mRNA. Cell cycle analysis confirmed G1-G0 phase arrest. Measurements of intracellular calcium levels ([Ca2+]i) showed that 20 micromol/L VN/124-1 caused a release of Ca2+ from endoplasmic reticulum stores resulting in a sustained increase in [Ca2+]i. Finally, cotreatment of PC-3 cells with 5, 10, and 20 micromol/L VN/124-1 with 10 nmol/L thapsigargin revealed a synergistic relationship between the compounds in inhibiting PC-3 cell growth. Taken together, these findings show VN/124-1 is endowed with multiple anticancer properties that may contribute to its utility as a prostate cancer therapeutic.

Topics: Androgens; Androstadienes; Animals; Benzimidazoles; Calcium; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Synergism; Endoplasmic Reticulum; Eukaryotic Initiation Factor-2; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Male; Oligonucleotide Array Sequence Analysis; Phosphorylation; Prostatic Neoplasms; Resting Phase, Cell Cycle; Steroid 17-alpha-Hydroxylase; Thapsigargin; Up-Regulation

Androgen ablation during the initial stages of prostate cancer causes regression of the tumor due to an increase in apoptosis and reduced cellular proliferation. However, prostate cancer invariably progresses to an androgen-independent state for poorly understood reasons. Previous studies showed that c-Jun NH(2) terminal kinase (JNK) is required for 12-O-tetradecanoylphorbol-13-acetate (TPA)- and thapsigargin (TG)-induced apoptosis in the androgen-responsive prostate cancer cell line LNCaP. Androgens protect LNCaP cells from TPA-induced or TG-induced apoptosis via down-regulation of JNK activation. However, the molecular mechanisms of this inhibition are not clear. Here, we systematically investigated the possible regulation of mitogen-activated protein kinase phosphatases/dual-specificity phosphatases during apoptosis of LNCaP cells and found that Vaccinia H1-related protein (VHR/DUSP3) is up-regulated by androgens during inhibition of apoptosis in LNCaP cells, but not in androgen-independent DU145 cells. Ectopic expression of wild-type VHR, but not a catalytically inactive mutant, interfered with TPA- and TG-induced apoptosis. Consistently, small interfering RNA-mediated knockdown of endogenous VHR increased apoptosis in response to TPA or TG in the presence of androgens. Furthermore, COS7 cells stably expressing wild-type VHR, but not a mutant, had a decrease in JNK phosphorylation. In vivo, VHR expression decreased in the androgen-dependent human prostate cancer xenograft CWR22 upon androgen withdrawal and was inversely correlated to JNK phosphorylation. Expression analysis in human prostate cancer specimens showed that VHR is increased in prostate cancer compared with normal prostate. These data show that VHR has a direct role in the inhibition of JNK-dependent apoptosis in LNCaP cells and may therefore have a role in prostate cancer progression.

Topics: Androgens; Apoptosis; Cell Line, Tumor; Dual Specificity Phosphatase 1; Dual Specificity Phosphatase 3; Humans; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Metribolone; Phosphorylation; Prostatic Neoplasms; RNA, Messenger; Tetradecanoylphorbol Acetate; Thapsigargin

Androgen deprivation induces the regression of prostate tumors mainly due to an increase in the apoptosis rate; however, the molecular mechanisms underlying the antiapoptotic actions of androgens are not completely understood. We have studied the antiapoptotic effects of androgens in prostate cancer cells exposed to different proapoptotic stimuli. Terminal deoxynucleotidyl transferase-mediated nick-end labeling and nuclear fragmentation analyses demonstrated that androgens protect LNCaP prostate cancer cells from apoptosis induced by thapsigargin, the phorbol ester 12-O-tetradecanoyl-13-phorbol-acetate, or UV irradiation. These three stimuli require the activation of the c-Jun N-terminal kinase (JNK) pathway to induce apoptosis and in all three cases, androgen treatment blocks JNK activation. Interestingly, okadaic acid, a phosphatase inhibitor that causes apoptosis in LNCaP cells, induces JNK activation that is also inhibited by androgens. Actinomycin D, the antiandrogen bicalutamide or specific androgen receptor (AR) knockdown by small interfering RNA all blocked the inhibition of JNK activation mediated by androgens indicating that this activity requires AR-dependent transcriptional activation. These data suggest that the crosstalk between AR and JNK pathways may have important implications in prostate cancer progression and may provide targets for the development of new therapies.

Topics: Androgen Antagonists; Androgen Receptor Antagonists; Androgens; Anilides; Apoptosis; Down-Regulation; Enzyme Activation; Humans; In Situ Nick-End Labeling; JNK Mitogen-Activated Protein Kinases; Male; Microscopy, Fluorescence; Neoplasms, Hormone-Dependent; Nitriles; Prostatic Neoplasms; Receptors, Androgen; RNA, Small Interfering; Tetradecanoylphorbol Acetate; Thapsigargin; Tosyl Compounds; Tumor Cells, Cultured; Ultraviolet Rays

The effect of the antidepressant desipramine on intracellular Ca(2+) movement and viability in prostate cancer cells has not been explored previously. The present study examined whether desipramine could alter Ca(2+) handling and viability in human prostate PC3 cancer cells. Cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of cells were measured using fura-2 as a probe. Desipramine at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner. The responses saturated at 300 microM desipramine. The Ca(2+) signal was reduced by half by removing extracellular Ca(2+), but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem or verapamil. In Ca(2+)-free medium, after treatment with 300 microM desipramine, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) failed to release Ca(2+) from endoplasmic reticulum. Conversely, desipramine failed to release more Ca(2+) after thapsigargin treatment. Inhibition of phospholipase C with U73122 did not affect desipramine-induced Ca(2+) release. Overnight incubation with 10-800 microM desipramine decreased viability in a concentration-dependent manner. Chelation of cytosolic Ca(2+) with BAPTA did not reverse the decreased cell viability. Collectively, the data suggest that in PC3 cells, desipramine induced a [Ca(2+)](i) increase by causing Ca(2+) release from endoplasmic reticulum in a phospholipase C-independent fashion and by inducing Ca(2+) influx. Desipramine decreased cell viability in a concentration-dependent, Ca(2+)-independent manner.

Topics: Antidepressive Agents; Calcium; Calcium Signaling; Cell Survival; Chelating Agents; Desipramine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fura-2; Humans; Male; Prostatic Neoplasms; Thapsigargin

Fatty acid synthase (FAS), the cellular enzyme that synthesizes palmitate, is expressed at high levels in tumor cells and is vital for their survival. Through the synthesis of palmitate, FAS primarily drives the synthesis of phospholipids in tumor cells. In this study, we tested the hypothesis that the FAS inhibitors induce endoplasmic reticulum (ER) stress in tumor cells. Treatment of tumor cells with FAS inhibitors induces robust PERK-dependent phosphorylation of the translation initiation factor eIF2alpha and concomitant inhibition of protein synthesis. PERK-deficient transformed mouse embryonic fibroblasts and HT-29 colon carcinoma cells that express a dominant negative PERK (DeltaC-PERK) are hypersensitive to FAS inhibitor-induced cell death. Pharmacologic inhibition of FAS also induces the processing of X-box binding protein-1, indicating that the IRE1 arm of the ER stress response is activated when FAS is inhibited. Induction of ER stress is further confirmed by the increased expression of the ER stress-regulated genes CHOP, ATF4, and GRP78. FAS inhibitor-induced ER stress is activated prior to the detection of caspase 3 and PARP cleavage, primary indicators of cell death, whereas orlistat-induced cell death is rescued by coincubation with the global translation inhibitor cycloheximide. Lastly, FAS inhibitors cooperate with the ER stress inducer thapsigargin to enhance tumor cell killing. These results provide the first evidence that FAS inhibitors induce ER stress and establish an important mechanistic link between FAS activity and ER function.

Topics: Activating Transcription Factor 4; Animals; Cell Line, Tumor; DNA-Binding Proteins; Drug Interactions; eIF-2 Kinase; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Eukaryotic Initiation Factor-2B; Fatty Acid Synthases; Heat-Shock Proteins; HeLa Cells; HT29 Cells; Humans; Lactones; Male; Mice; Molecular Chaperones; Nuclear Proteins; Orlistat; Phosphorylation; Prostatic Neoplasms; Regulatory Factor X Transcription Factors; Thapsigargin; Transcription Factor CHOP; Transcription Factors

It has recently been shown that the androgen receptor (AR) is the main factor that required for prostate cancer cells survival. We show that knocking down AR expression by siRNA induces PI3K-independent activation of Akt, which was mediated by calcium/calmodulin-dependent kinase II (CaMKII). We further show, for the first time, that prostate cancer cells express beta,gamma and delta CaMKII genes, and the expression of these genes is under the control of AR activity: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in elevated level of kinase activity and in enhanced expression of CaMKII-beta and -gamma genes. Overexpression of CaMKII genes results in resistance to apoptosis induced by KN-93, a CaMKII inhibitor, or wortmanninn, a PI3K/Akt inhibitor, in combination with doxorubicin, thapsigargin and TRAIL. Moreover, overexpression of CaMKII increases secretion of prostate specific antigen and promotes cell growth of LNCaP in steroid-free condition. Our data show that there is cross-talk between AR- and CaMKII-mediated pathways. The results of this study suggest that CaMKII is an important player in prostate cancer cells ability to escape apoptosis under androgen ablation and facilitate the progression of prostate cancer cells to an androgen independent state.

Topics: Androgen Receptor Antagonists; Androgens; Androstadienes; Antibiotics, Antineoplastic; Apoptosis; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Doxorubicin; Drug Therapy, Combination; Enzyme Inhibitors; Humans; Immunosuppressive Agents; Isoenzymes; Luciferases; Male; Phosphatidylinositol 3-Kinases; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sulfonamides; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Wortmannin

Cells with increasing resistance to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), ranging from 60-fold (PC3/TG(10) cells) to 1350-fold (PC3/TG(2000) cells), were derived from PC3 cells. SERCA2 is overexpressed in all PC3/TG cells but retains sensitivity to TG. siRNA-mediated downregulation of SERCA completely or partially reverses TG resistance in PC3/TG(10) or PC3/TG(2000) cells, respectively; thus SERCA overexpression mediates resistance in PC3/TG(10) cells but is not the only resistance mechanism in PC3/TG(2000) cells. By contrast, SERCA is not overexpressed in TG-resistant DU145/TG cells derived from DU145 cells. DU145/TG cells retain resistance while in PC3/TG cells resistance decreases upon removal of TG selection. The transport proteins PGP/BCRP/MRP1 and anti-apoptotic proteins Bcl2/Bcl(XL) are not involved in mediating resistance in either cell line. PARP and caspase 3 cleavage in response to other drugs demonstrate that the apoptotic pathways tested remain intact in these cells. Further, no cross-resistance occurs to other drugs. Thus, novel TG-specific resistance mechanisms are recruited by these cancer cells.

Topics: Androgens; Apoptosis; Calcium; Cell Line, Tumor; Cytosol; Drug Resistance; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Silencing; Humans; Male; Prostatic Neoplasms; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

The transient receptor potential channel, subfamily V, member 6 (TRPV6), is strongly expressed in advanced prostate cancer and significantly correlates with the Gleason >7 grading, being undetectable in healthy and benign prostate tissues. However, the role of TRPV6 as a highly Ca(2+)-selective channel in prostate carcinogenesis remains poorly understood. Here, we report that TRPV6 is directly involved in the control of prostate cancer cell (LNCaP cell line) proliferation by decreasing: (i) proliferation rate; (ii) cell accumulation in the S-phase of cell cycle and (iii) proliferating cell nuclear antigen (PCNA) expression. We demonstrate that the Ca(2+) uptake into LNCaP cells is mediated by TRPV6, with the subsequent downstream activation of the nuclear factor of activated T-cell transcription factor (NFAT). TRPV6-mediated Ca(2+) entry is also involved in apoptosis resistance of LNCaP cells. Our results suggest that TRPV6 expression in LNCaP cells is regulated by androgen receptor, however, in a ligand-independent manner. We conclude that the upregulation of TRPV6 Ca(2+) channel in prostate cancer cells may represent a mechanism for maintaining a higher proliferation rate, increasing cell survival and apoptosis resistance as well.

Topics: Apoptosis; Calcium; Calcium Channels; Calcium Signaling; Cell Proliferation; Enzyme Inhibitors; Humans; Male; NFATC Transcription Factors; Prostatic Neoplasms; S Phase; Signal Transduction; Thapsigargin; TRPV Cation Channels; Tumor Cells, Cultured

The androgen-regulated protein androgen-induced bZIP (AIbZIP) is a bZIP transcription factor that localizes to the membrane of the endoplasmic reticulum (ER). The physiological role of AIbZIP is unknown, but other ER-bound transcription factors such as ATF6 and SREBPs play a crucial role in the regulation of protein processing and lipid synthesis, respectively. In response to alterations in the intracellular milieu, ATF6 and SREBPs are processed to their transcriptionally active forms by regulated intramembrane proteolysis. In humans, AIbZIP mRNA is expressed in several organs including the pancreas, liver, and gonads, but it is especially abundant in prostate epithelial cells. We therefore used LNCaP human prostate cancer cells as a model to identify stimuli that lead to AIbZIP activation and define the transcriptional targets of AIbZIP. In LNCaP cells, AIbZIP was processed to its transcriptionally active form by drugs that deplete ER calcium stores (i.e., A23187 and caffeine), but it was unaffected by an inhibitor of protein glycosylation (tunicamycin). To identify AIbZIP-regulated genes, we generated LNCaP cell lines that conditionally express the processed form of AIbZIP and used Affymetrix microarrays to screen for AIbZIP-regulated transcripts. Selected genes (n = 48) were validated by Northern blot hybridization. The results reveal that the downstream targets of AIbZIP include genes that are implicated in protein processing (e.g., BAG3, DNAJC12, KDELR3). Strikingly, a large number of AIbZIP-regulated transcripts encode proteins that are involved in transcriptional regulation, small molecule transport, signal transduction, and metabolism. These results suggest that AIbZIP plays a novel role in cell homeostasis.

Topics: Adenocarcinoma; Amino Acid Sequence; Basic-Leucine Zipper Transcription Factors; Brefeldin A; Caffeine; Calcimycin; Calcium Signaling; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Endoplasmic Reticulum; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Golgi Apparatus; Homeostasis; Humans; Male; Molecular Sequence Data; Neoplasm Proteins; Nuclear Proteins; Prostatic Neoplasms; Protein Processing, Post-Translational; Recombinant Fusion Proteins; RNA, Small Interfering; Thapsigargin; Transcription, Genetic; Tunicamycin

Prostate cancer targeted peptide prodrugs that are activated by the serine protease activity of prostate-specific antigen (PSA) are under development in our laboratory. To enhance delivery and solubility of these prodrugs, macromolecular carriers consisting of N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers were covalently coupled to a PSA-activated peptide prodrug. HPMA copolymers are water-soluble, nonimmunogenic synthetic carriers that exhibit promise for drug delivery applications. These macromolecular copolymers enter the interstitium of solid tumors by the enhanced permeability and retention effect. The PSA-activated peptide substrate imparts selectivity because it is specifically hydrolyzed to release a cytotoxin at the site of prostate tumor. Enzymatically active PSA is present in high amounts in the extracellular fluid of a tumor, but PSA is inactivated in blood by binding to serum protease inhibitors. As an initial proof of concept, the HPMA copolymer was synthesized with a peptide substrate (HSSKLQ) bound to a fluorophore, 7-amino-4-methylcoumarin (AMC). PSA cleavage of the HPMA-HSSKLQ-AMC copolymer was observed, which led to the synthesis of an HPMA-based copolymer with the prodrug SSKYQ-L12ADT [HPMA-morpholinocarbonyl-Ser-Ser-Lys-Tyr-Gln-Leu-12-aminododecanoyl thapsigargin (JHPD)]. L12ADT is a potent analogue of the highly cytotoxic natural product thapsigargin. HPMA-JHPD was hydrolyzed by PSA in vitro and was toxic to prostate cancer cells in the presence of active PSA. The HPMA-JHPD produced no systemic toxicity when given at a 500 micromol/L L12ADT equivalent dose. Analysis of tumor tissue from mice treated with a single or multiple dose of the HPMA-JHPD copolymer showed release and accumulation of the L12ADT toxin within the tumor tissue.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Coumarins; Drug Delivery Systems; Drug Screening Assays, Antitumor; Humans; Hydrolysis; Male; Mice; Mice, Nude; Micelles; Organ Specificity; Polymers; Polymethacrylic Acids; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Thapsigargin

Prostate cancer cells secrete unique proteases such as prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) that represent targets for the activation of prodrugs as systemic treatment of metastatic prostate cancer. Previously, a combinatorial peptide library was screened to identify a highly active peptide substrate for hK2. The peptide was coupled to an analog of the potent cytotoxin thapsigargin, L12ADT, to generate an hK2-activated prodrug that was efficiently hydrolyzed by purified hK2, stable to hydrolysis in human and mouse plasma in vitro and selectively toxic to hK2 producing prostate cancer cells in vitro.. In the current study, toxicology, pharmacokinetics, prodrug biodistribution, and antitumor efficacy studies were performed to evaluate the hK2-activated prodrug in vivo.. The single intravenous maximally tolerated dose of prodrug was 6 mg/kg (i.e., 3.67 micromole/kg) which produced peak serum concentration of approximately 36 microM and had a half-life of approximately 40 min. In addition, over a 24 hr period <0.5% of free L12ADT analog was observed in plasma. The prodrug demonstrated significant antitumor effect in vivo while it was being administered, but prolonged intravenous administration was not possible due to local toxicity to tail veins. Subcutaneous administration of equimolar doses produced lower plasma AUC compared to intravenous dosing but equivalent intratumoral levels of prodrug following multiple doses.. The hK2-activated prodrug was stable in vivo. The prodrug, however, was rapidly cleared and difficult to administer over prolonged dosing interval. Additional studies are underway to assess antitumor efficacy with prolonged administration of higher subcutaneous doses of prodrug. Second-generation hK2-activated thapsigargin prodrugs with increased half-lives and improved formulations are also under development.

Topics: Animals; Area Under Curve; Humans; Infusions, Intravenous; Male; Maximum Tolerated Dose; Mice; Mice, Inbred BALB C; Prodrugs; Prostatic Neoplasms; Thapsigargin; Tissue Kallikreins; Tumor Cells, Cultured

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.

Topics: Adrenergic alpha-Agonists; Blotting, Western; Calcineurin; Calcium; Calcium Signaling; Calmodulin; Cell Line, Tumor; Cytosol; Gene Expression Regulation; Humans; Ion Channels; Male; Models, Biological; NFATC Transcription Factors; Polymerase Chain Reaction; Prostatic Neoplasms; Protein Isoforms; Thapsigargin; Transcription, Genetic; TRPC Cation Channels; TRPV Cation Channels

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We asked whether elevated cytosolic free calcium [Ca2+]i is required for cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2 fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause apoptosis, measured by nucleosome release, under conditions that prevented elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER calcium. C33A and DU145 responded differently to perturbations in Ca2+ homeostasis, suggesting that DIM induces apoptosis by different mechanisms in these two cell lines and/or that calcium mobilization also activates different survival pathways in C33A and DU145. Apoptosis in C33A was independent of increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+ signaling is important for cell survival but the characteristics of this response also differed in the two cell lines. Our results underscore the complex and variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both calcium-dependent and calcium-independent mechanisms.

Topics: Apoptosis; Benzylamines; Calcium; Calcium Signaling; Chelating Agents; Cyclosporine; Cytosol; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Female; Humans; Indoles; Male; Prostatic Neoplasms; Sulfonamides; Thapsigargin; Uterine Cervical Neoplasms

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.

Topics: Adenoviridae; Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Down-Regulation; Electrophoretic Mobility Shift Assay; Endoplasmic Reticulum; Endoribonucleases; Enzyme Activation; Female; Fibroblasts; Gene Expression Regulation; Genes, Reporter; Humans; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; Kinetics; L Cells; Luciferases; Lung Neoplasms; Male; Membrane Proteins; Mice; Mice, Knockout; NF-kappa B; Precipitin Tests; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Thapsigargin; TNF Receptor-Associated Factor 2; Tumor Necrosis Factor-alpha; Tunicamycin

Neuroendocrine differentiation (NED) has been implicated in prostate cancer progression and hormone-therapy failure. Neuroendocrine cells are non-proliferating and escape apoptotic cell death, although their origin and the causes of their apoptotic resistance have as yet been poorly elucidated. This study demonstrates a new mechanism involved in controlling NED. We report that epidermal growth factor (5-50 ng/ml) promotes neuroendocrine-like differentiation of androgen-independent DU145 prostate cancer cells. This differentiation is associated with an increase in the expression of Neuron Specific Enolase (NSE) and a reduction in cell proliferation and is blocked by inhibiting tyrosine kinase activity with genistein and with compound 56 (C56). An increase in the cAMP level, using dibutryl cAMP (db-cAMP) (1 mM) and isobutylmethylxanthine (100 microM), does not promote NED by itself, but does increase the effect of EGF on NED. In addition, EGF-induced NED protects cells from apoptosis induced with thapsigargin (1 microM) by reducing the thapsigargin-induced cytosolic calcium overload. In order to describe how EGF-induced NED protects cells against thapigargin-induced calcium overload we investigated the spatiotemporal calcium signalling linked to apoptosis. By using thapsigargin in various conditions on DU145 cells and using micro-fluorimetric calcium measurements, we show that depletion of intracellular calcium store induces apoptosis and that the amplitude and duration of the capacitive calcium entry are two apoptosis-modulating parameters. We show that protection against thapsigargin-induced apoptosis conferred by NED is achieved by reducing the amount and the speed of calcium that can be released from calcium pools, as well as modulating the amplitude of the subsequent calcium entry.

Topics: Androgens; Antineoplastic Agents; Apoptosis; Calcium; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Male; Neoplasms, Hormone-Dependent; Neurosecretory Systems; Phosphopyruvate Hydratase; Prostatic Neoplasms; Protein-Tyrosine Kinases; Thapsigargin

Thapsigargin (TG) is a potent inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCAs). TG-based prodrugs are being developed for the treatment of prostate cancer (PC). To develop optimal TG-based therapeutics it is important to understand the mechanisms of resistance to TG that may potentially occur in cancer cells.. DU145/TG and PC3/TG cells were derived from human PC DU145 and PC3 cells, respectively, by incremental exposure to TG. Growth assays, Western blot analyses, cDNA microarrays, semiquantitative and real-time polymerase chain reaction (PCR), Northern blot analyses, and immunohistochemistry were used to study these cells.. DU145/TG cells are 1100-fold and PC3/TG cells are 1350-fold resistant to TG. Although expression of both SERCA and p-glycoprotein can mediate TG resistance in hamster cells, neither is modulated in DU145/TG cells. In contrast, in PC3/TG cells, SERCA, and not p-glycoprotein, is significantly overexpressed but cannot by itself account for the 1350-fold resistance to TG in these cells. Several genes not previously identified to be altered by TG selection are modulated in DU145/TG and PC3/TG cells. Furthermore, the spectrum of genes modulated in DU145/TG cells are distinct from that in PC3/TG cells, even though both cells are of prostate origin and share the same TG-resistant phenotype.. PC cells can adapt to SERCA inhibition by TG. However, they demonstrate cell type-specific plasticity with respect to gene expression upon TG selection. Further, previously not described mechanisms of resistance appear to be recruited in the TG-resistant PC cells, which provide a novel model to study mechanisms of resistance and adaptation in PC on TG-mediated dysregulation of Ca2+ homeostasis.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Cricetinae; Drug Resistance, Neoplasm; Enzyme Inhibitors; Fibroblasts; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Thapsigargin; Up-Regulation

Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca(2+) homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NE-differentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca(2+)-store content due to both SERCA 2b Ca(2+) ATPase and luminal Ca(2+) binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca(2+) current. NE-differentiated cells showed enhanced resistance to thapsigargin- and TNF-alpha-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca(2+) homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.

Topics: Apoptosis; Blotting, Western; Calcium; Calcium Channels; Calcium-Transporting ATPases; Calreticulin; Cell Differentiation; Cell Line, Tumor; Electric Capacitance; Electric Impedance; Electrophysiology; Endoplasmic Reticulum; Epithelial Cells; Fluorescent Dyes; Fura-2; Homeostasis; Humans; Kinetics; Male; Models, Biological; Neurosecretory Systems; Patch-Clamp Techniques; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Thapsigargin; Tumor Necrosis Factor-alpha

The one or more coupling mechanisms of store-operated channels (SOCs) to endoplasmic reticulum (ER) Ca2+ store depletion as well as the molecular identity of SOCs per se still remain a mystery. Here, we demonstrate the co-existence of two populations of molecular distinct endogenous SOCs in LNCaP prostate cancer epithelial cells, which are preferentially activated by either active inositol 1,4,5-trisphosphate (IP3)-mediated or passive thapsigargin-facilitated store depletion and have different ER store content sensitivity. The first population, called SOC(CC) (for "conformational coupling"), is characterized by preferential IP3 receptor-dependent mode of activation, as judged from sensitivity to cytoskeleton modifications, and dominant contribution of transient receptor potential (TRP) TRPC1 within it. The second one, called SOC(CIF) (for "calcium influx factor"), depends on Ca(2+)-independent phospholipase A2 for activation with probable CIF involvement and is mostly represented by TRPC4. The previously identified SOC constituent in LNCaP cells, TRPV6, seems to play equal role in both SOC populations. These results provide new insight into the nature of SOCs and their representation in the single cell type as well as permit reconciliation of current SOC activation hypotheses.

Topics: Actins; Blotting, Western; Calcium; Calcium Channels; Calcium Signaling; Cell Line, Tumor; Cytoskeleton; Dose-Response Relationship, Drug; Electrophysiology; Endoplasmic Reticulum; Epithelial Cells; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Microscopy, Fluorescence; Models, Biological; Oligonucleotides, Antisense; Prostatic Neoplasms; Protein Conformation; Protein Isoforms; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Thapsigargin; Time Factors; TRPV Cation Channels

In human prostate cancer PC3 cells, the effect of Y-24180, a presumed specific platelet activation factor (PAF) receptor antagonist, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2 as a Ca2+-sensitive fluorescent probe. Y-24180 (1-10 microM) caused a rapid and sustained [Ca2+]i rise in a concentration-dependent manner. The [Ca2+]i rise was prevented by 40% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of 10 microM Y-24180 on [Ca2+]i was reduced by 67%; conversely, depletion of Ca2+ stores with 10 microM Y-24180 abolished thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phospholipase C, inhibited ATP-, but not Y-24180-induced [Ca2+]i rise. Activation of protein kinase C with phorbol-12-myristate-13-acetate (PMA) enhanced Y-24180-induced [Ca2+]i rise by 70%. Overnight treatment with 0.1-10 microM Y-24180 inhibited cell proliferation in a concentration-dependent manner. Collectively, these results suggest that Y-24180 acts as a potent and cytotoxic Ca2+ mobilizer in prostate cancer cells, by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release. Since alterations in Ca2+ movement may interfere with many cellular signalling processes unrelated to modulation of PAF receptors, caution must be applied in using this reagent as a selective PAF receptor antagonist.

Topics: Adenosine Triphosphate; Azepines; Calcium; Calcium Signaling; Cell Proliferation; Dihydropyridines; Diltiazem; Enzyme Inhibitors; Fura-2; Humans; Male; Phorbol Esters; Platelet Membrane Glycoproteins; Prostatic Neoplasms; Protein Kinase C; Receptors, G-Protein-Coupled; Signal Transduction; Thapsigargin; Triazoles; Tumor Cells, Cultured; Verapamil

An in vitro screen for identification of novel anti-cancer agents, which can induce proliferation-independent apoptosis of prostate cancer (PCA) cells, is required, since the proliferative growth fraction of human prostate cancers in patients is usually <10%. This is possible using the PCA cell line LNCaP, which can be permanently transdifferentiated into a quiescent neuroendocrine (NE) phenotype without undergoing apoptosis by the cytokine interleukine-6 (IL-6). Transdifferentiation of LNCaP cells into a NE phenotype was documented using western blot analysis and immunohistochemistry for the NE markers, neuron-specific enolase (NSE) and beta III tubulin. Accumulation of NE cells in the G0 phase of the cell cycle was demonstrated by Ki-67 immunohistochemistry. The effects of paclitaxel, vinblastine and thapsigargin (TG) on viability and apoptosis of NE and LNCaP cells were assessed by trypan blue exclusion and 4', 6-diamidino-2-phenylindole nuclear staining assays. In proliferating LNCaP cells, there was a significant decrease in viable cells after 48 h exposure to paclitaxel and vinblastine and a dramatic increase of apoptosis as compared with the controls. On the other hand, treatment with paclitaxel or vinblastine decreased the viability of NE cells only slightly without markedly increasing their rate of apoptosis compared to controls. In contrast, both LNCaP and NE cells showed a significant and comparable decrease in cell viability and similar high levels of apoptosis when treated with TG. These results demonstrate that terminally transdifferentiated NE cells represent a useful in vitro screening system for identification of novel anti-cancer agents, like TG, that can induce apoptosis without requiring proliferation.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Survival; Drug Evaluation, Preclinical; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Male; Neurosecretory Systems; Paclitaxel; Prostatic Neoplasms; Resting Phase, Cell Cycle; Thapsigargin; Tumor Cells, Cultured; Vinblastine

Variations in calcium concentration within the endoplasmic reticulum ([Ca(2+)](ER)) may play a role in cell growth. This study evaluates the regulation of calcium pools by growth modulators of prostate cancer (PC) cells, the insulin growth factor (IGF), and the tumor necrosis growth factor-alpha (TNFalpha) as well as evaluating the possible role of [Ca(2+)](ER) variations as signals for growth modulation. We show that IGF (5 ng/ml), which increases cell growth, induces an increase in [Ca(2+)](ER) whereas TNFalpha (1 ng/ml) which reduces cell proliferation and induces apoptosis, reduces [Ca(2+)](ER). IGF-induced [Ca(2+)](ER) increase is correlated to an overexpression of the sarcoendoplasmic calcium-ATPase 2B (SERCA2b), whereas TNFalpha-induced [Ca(2+)](ER) decrease is associated to a reduction in SERCA2b expression. Pretreatment with epidermal growth factors (EGF) or IGF does not prevent TNFalpha from affecting the induction of apoptosis, [Ca(2+)](ER) reduction and SERCA2b downregulation. Reduction in [Ca(2+)](ER) induced by thapsigargin (TG) (from 1 pM to 1 microM, 48 h) reduces LNCaP growth in a dose dependent manner and induces apoptosis when cells are treated with 1 microM TG. We also show that a transient TG application (1 pM, 1 nM, 1 microM 15 min) is insufficient to induce a long lasting decrease in [Ca(2+)](ER), since [Ca(2+)](ER) remains identical to the control for 48 h following TG application. These treatments (1 pM and 1 nM, 15 min) do not modify cell growth. However, TG (1 microM, 15 min) induces apoptosis. We thus identify [Ca(2+)](ER) and SERCA2b as a central targets for causing LNCaP PC cell life or death induced by growth modulators. Furthermore our results indicate that calcium pool contents can regulate cell growth.

Topics: Apoptosis; Blotting, Western; Calcium; Calcium-Transporting ATPases; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Flow Cytometry; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Insulin-Like Growth Factor I; Male; Prostatic Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Tumor Necrosis Factor-alpha

Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation-independent apoptosis through dysregulation of intracellular calcium levels.. To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity.. Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 micromol/L, kcat of 1.09 s(-1), and a kcat/Km ratio of 41,132 s(-1) mol/L(-1). The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2.. The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Cathepsin B; Cell Line, Tumor; Drug Evaluation, Preclinical; Humans; Hydrolysis; Male; Mice; Molecular Structure; Peptide Library; Peptides; Prodrugs; Prostatic Neoplasms; Substrate Specificity; Thapsigargin; Tissue Kallikreins; Trypsin

This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.

Topics: Adenosine Triphosphate; Calcium; Calcium Channels; Calcium Signaling; Carcinoma; Cell Division; Cell Line, Tumor; Cytoplasm; Down-Regulation; Endoplasmic Reticulum; Humans; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Fluid; Male; Prostatic Neoplasms; Protein Kinase C; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Type C Phospholipases

Standard anti-proliferative chemotherapy is relatively ineffective against slowly proliferating androgen-independent prostate cancer cells within metastatic sites. In contrast, the lipophilic cytotoxin thapsigargin, which causes apoptosis by disrupting intracellular free Ca2+ levels, is effective against both proliferative and quiescent (i.e., G0-arrested) cells. However, thapsigargin's mechanism of action indicates that it is unlikely to be selective for cancer cells or prostate cells.. We coupled a chemically modified form of thapsigargin, L12ADT, to a peptide carrier that is a substrate for the prostate-specific antigen (PSA) protease to produce a soluble, cell-impermeant latent prodrug that is specifically activated extracellularly within metastatic prostate cancer sites by PSA. We analyzed the kinetics of PSA hydrolysis of the prodrug, the in vitro cytotoxicity of the prodrug against PSA-producing LNCaP human prostate cancer and PSA non-producing HCT-116 human colon cancer cells, and the in vivo pharmacokinetics of the prodrug in mice. We also analyzed antitumor efficacy of the prodrug in nude mice xenograft models of prostate cancer (using LNCaP cells) and renal carcinoma (using human SN12C cells).. The L12ADT peptide prodrug was hydrolyzed efficiently by PSA, was selectively toxic to PSA-producing prostate cancer cells in vitro, and was stable in human plasma. A single dose of 7 mg/kg resulted in a peak serum prodrug concentration of 15.4 +/- 1.1 microM and a half-life of approximately 2.8 hours. Over 24 hours, less than 0.5% of free L12ADT was observed in plasma. Levels of prodrug and liberated L12ADT in prostate cancer xenograft tumors were approximately eightfold and sixfold, respectively, higher than the in vitro LD50s. Prostate cancer xenograft tumors in mice treated with prodrug by intravenous administration were growth-inhibited without substantial host toxicity. Continuous subcutaneous prodrug administration in mice produced complete growth inhibition of established PSA-producing prostate cancer xenograft tumors but had no effect on PSA non-producing renal carcinoma xenograft tumors.. Further development of PSA-activated thapsigargin prodrugs as therapy for metastatic prostate cancer is warranted.

Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Humans; Hydrolysis; Male; Mice; Mice, Nude; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Thapsigargin; Transplantation, Heterologous; Treatment Outcome

Thapsigargin treatment of cultured cells leads to an increase in the intracellular calcium concentration, activation of calpain, and, in some cell types, apoptosis. Using a human prostate epithelial cell line that undergoes apoptosis in the presence of thapsigargin, we find decreased levels of IRS-1 protein levels during apoptosis. Inhibition of calpain prevents this decrease in IRS-1 protein; however, inhibitors of caspases or the proteasome are ineffective in maintaining IRS-1 levels. In terms of IGF-I-related second messenger proteins, the effect of thapsigargin is specific for IRS-1 since the protein levels of IGF-I receptor beta-subunit, Akt, Erk, and Shc are not affected. In addition to preventing the reduction in IRS-1, treatment of cells with calpain inhibitor II prevents apoptosis in response to thapsigargin. Finally, IRS-1 and calpain can be identified in protein complexes isolated using IRS-1-specific antibodies, indicating that calpain can associate with either IRS-1 or one of the proteins present in protein complexes that contain IRS-1. In total, these results suggest that IRS-1 may be targeted for degradation by calpain during apoptosis.

Topics: Apoptosis; Calcium; Calcium Signaling; Calpain; Carcinoma; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Insulin Receptor Substrate Proteins; Intracellular Fluid; Macromolecular Substances; Male; Phosphoproteins; Prostatic Neoplasms; Second Messenger Systems; Thapsigargin; Tumor Cells, Cultured

Previously it has been reported that caveolin-1 (cav-1) has antiapoptotic activities in prostate cancer cells and functions downstream of androgenic stimulation. In this study, we demonstrate that cav-1 overexpression significantly reduced thapsigargin (Tg)-stimulated apoptosis. Examination of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling cascade revealed higher activities of PDK1 and Akt but not PI3-K in cav-1-stimulated cells compared to control cells. We subsequently found that cav-1 interacts with and inhibits serine/threonine protein phosphatases PP1 and PP2A through scaffolding domain binding site interactions. Deletion of the cav-1 scaffolding domain significantly reduces phosphorylated Akt and cell viability compared with wild-type cav-1. Analysis of potential substrates for PP1 and PP2A revealed that cav-1-mediated inhibition of PP1 and PP2A leads to increased PDK1, Akt, and ERK1/2 activities. We demonstrate that increased Akt activities are largely responsible for cav-1-mediated cell survival using dominant-negative Akt mutants and specific inhibitors to MEK1/MEK and show that cav-1 increases the half-life of phosphorylated PDK1 and Akt after inhibition of PI3-K by LY294002. We further demonstrate that cav-1-stimulated Akt activities lead to increased phosphorylation of multiple Akt substrates, including GSK3, FKHR, and MDM2. In addition, overexpression of cav-1 significantly increases translocation of phosphorylated androgen receptor to nucleus. Our studies therefore reveal a novel mechanism of Akt activation in prostate cancer and potentially other malignancies.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Active Transport, Cell Nucleus; Binding Sites; Caveolin 1; Caveolins; Cell Death; Cell Line, Tumor; Enzyme Activation; Humans; In Vitro Techniques; Male; Phosphoprotein Phosphatases; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Thapsigargin; Transfection

In early, androgen dependent stages of prostate cancer, androgen withdrawal, the major course of therapy in prostate cancer, leads to a rapid regression of the tumor as a result of apoptosis. However, prostate cancer invariably progresses to an androgen independent and apoptosis resistant stage for which no curative treatment is available. The molecular details of regression upon androgen withdrawal and progression to a resistant state are largely unknown. Here we show that c-Jun N-terminal Kinase (JNK) is activated strongly and in a sustained fashion by 12-O-tetradecanoylphorbol 13-acetate (TPA) and thapsigargin (TG), two agents which were previously shown to lead to apoptosis in the androgen responsive prostate cancer cell line LNCaP. The time course of JNK induction by both compounds correlated very well with the onset and progression of apoptosis in LNCaP cells. Inhibition of either ERK or p38 pathways did not affect TPA-induced cell death. In the androgen-independent prostate cancer cell lines DU-145 and PC-3, and in the cervical carcinoma cell line HeLaS3, TPA did not lead to apoptosis and there were no significant changes in JNK activity upon TPA treatment. The failure of TPA to induce JNK activity in PC-3, DU-145, and HelaS3 cells was not due to a general defect in JNK signaling since ultraviolet (UV) irradiation dramatically increased JNK activity in all four cell lines. Specific inhibition of JNK by expression of the JNK Inhibitory Protein (JIP) dramatically inhibited both TPA- and TG-induced apoptosis. Furthermore, apoptosis induced by both agents was completely blocked by ectopic expression of the baculovirus caspase-inhibitor P35. Surprisingly, ZVAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases, had no effect on TPA-induced apoptosis, whereas it completely inhibited TG-induced cell death; JNK activity was not affected in either case. This indicates that ZVAD-fmk does not inhibit some of the caspases involved in TPA-induced apoptosis, and that despite the common requirement of JNK activation, TPA- and TG-induced cell death are mechanistically different. Furthermore, it also suggests that JNK is either upstream or independent of caspases in LNCaP cells. Collectively, these results indicate that apoptosis in LNCaP cells requires a sustained increase in JNK activity and caspase activation; components of these signaling pathways may be defective in the androgen independent prostate cancer cell lines.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Androgens; Apoptosis; Carrier Proteins; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Humans; Inhibitor of Apoptosis Proteins; JNK Mitogen-Activated Protein Kinases; Kinetics; Male; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Tetradecanoylphorbol Acetate; Thapsigargin; Transfection; Tumor Cells, Cultured; Viral Proteins

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.

Topics: Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Drug Antagonism; Fura-2; Humans; Male; Nicardipine; Nifedipine; Nimodipine; Prostatic Neoplasms; Thapsigargin; Triethyltin Compounds; Tumor Cells, Cultured

Loss of contact with the extracellular matrix (ECM) triggers a specialized form of apoptosis known as "anoikis" in normal epithelial cells. Dependence on adhesion to ECM is often lost in transformed cells, and the degree of anchorage independence may vary in non-metastatic and metastatic cancer cells. BCL-2 oncoprotein overexpression correlates with the progression and metastases of prostate cancer. Materials and Methods We studied anoikis in suspension cultures of PC-3 and LNCaP prostate carcinoma cells selected for enhanced metastatic potential in vivo and in PC-3 and LNCaP cells stably transfected with BCL-2. Apoptosis-associated DNA fragmentation was measured by agarose gel electrophoresis and propidium iodide staining and flow cytometry. Expression of BCL-2 family polypeptides was determined by immunoblotting.. Non-metastatic PC-3P cells were significantly more sensitive to anoikis than the metastatic PC-3 variants (PC-3M, PC-3M-PRO-4, and PC-3M-LN-4), but anoikis resistance did not correlate with metastatic potential in LNCaP-derived cell lines. Expression of BCL-2 was higher in metastatic PC-3 and LNCaP subclones compared to isogenic non-metastatic cells, but these levels were not affected by anoikis. Enforced overexpression of BCL-2 did not protect either PC-3P or LNCaP-PRO-5 cells from anoikis, even though it rendered them resistant to thapsigargin and inhibited cytochrome c release. Strikingly, cells that died of anoikis maintained their pretreatment levels of BCL-2, whereas the cells that survived anoikis expressed much lower levels of the protein.. Sensitivity to anoikis is regulated by BCL-2 independent mechanisms in LNCaP and PC-3 prostate cancer cells.

Topics: Adenocarcinoma; Anoikis; Blotting, Western; Carcinogens; Cell Adhesion; DNA Fragmentation; Electrophoresis, Agar Gel; Flow Cytometry; Humans; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Thapsigargin; Transfection; Tumor Cells, Cultured

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca(2+) indicator. Clomiphene at concentrations between 10-50 microM increased [Ca(2+)](i) in a concentration-dependent manner. The [Ca(2+)](i) signal was biphasic with an initial rise and a slow decay. Ca(2+) removal inhibited the Ca(2+) signal by 41%. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with clomiphene in Ca(2+)-free medium, confirming that clomiphene induced Ca(2+) entry. In Ca(2+)-free medium, pretreatment with 50 microM brefeldin A (to permeabilize the Golgi complex), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca(2+) pump), and 2 microM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 microM clomiphene-induced store Ca(2+) release. Conversely, pretreatment with 50 microM clomiphene in Ca(2+)-free medium abolished the [Ca(2+)](i) increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 microM clomiphene-induced Ca(2+)release was unaltered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 microM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca(2+)](i) increases in PC3 cells by releasing store Ca(2+) from multiple stores in an phospholipase C-independent manner, and by activating Ca(2+) influx; and clomiphene was of mild cytotoxicity.

Topics: Brefeldin A; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Clomiphene; Dose-Response Relationship, Drug; Drug Interactions; Estrenes; Fertility Agents, Female; Humans; Male; Prostatic Neoplasms; Pyrrolidinones; Thapsigargin; Tumor Cells, Cultured

A series of thapsigargin (TG) analogues, containing an amino acid applicable for conjugation to a peptide specifically cleaved by prostate-specific antigen (PSA), has been prepared to develop the drug-moiety of prodrugs for treatment of prostatic cancer. The analogues were synthesized by converting TG into O-8-debutanoylthapsigargin (DBTG) and esterifying O-8 of DBTG with various amino acid linkers. The compounds were evaluated for their ability to elevate the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in TSU-Pr1 cells, their ability to inhibit the rabbit skeletal muscle SERCA pump, and their ability to induce apoptosis in TSU-Pr1 human prostatic cancer cells. The activity of analogues, in which DBTG were esterified with omega-amino acids [HOOC(CH(2))(n)()NH(2), n = 5-7, 10, 11], increased with the linker length. Analogues with 3-[4-(L-leucinoylamino)phenyl]propanoyl, 6-(L-leucinoylamino)hexanoyl, and 12-(L-serinoylamino)dodecanoyl were considerably less active than TG, and analogues with 12-(L-alaninoylamino)dodecanoyl and 12-(L-phenylalaninoylamino)dodecanoyl were almost as active as TG. The 12-(L-leucinoylamino)dodecanoyl gave an analogue equipotent with TG, making this compound promising as the drug-moiety of a PSA sensitive prodrug of TG.

Topics: Animals; Antineoplastic Agents; Apoptosis; Calcium; Calcium-Transporting ATPases; Drug Design; Drug Screening Assays, Antitumor; Humans; Male; Muscle, Skeletal; Prostatic Neoplasms; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Structure-Activity Relationship; Thapsigargin; Tumor Cells, Cultured

Prostate cancer is the second leading cause of cancer death in men. The most common treatment of prostate cancer is androgen ablation therapy which leads to regression of the tumor due to increased cell death. However, at later stages, the tumor becomes resistant to androgen ablation. Ceramide is a lipid second messenger that mediates cell death in prostate cancer cells. Previous studies suggested that ceramide may cause either apoptosis or growth arrest in the androgen-responsive prostate cancer cell line LNCaP. However, the molecular details of ceramide-induced cell death in LNCaP cells remain to be elucidated.. To investigate the mechanisms of cell death in LNCaP cells, we used various methods, including cell viability assays, fluorescence image analysis, internucleosomal DNA fragmentation analysis, Western blotting, and protein kinase assays.. Ceramide caused LNCaP cell death without exhibiting typical signs of apoptosis, such as internucleosomal DNA fragmentation and poly(ADP)-ribose-polymerase (PARP) proteolysis. In addition, the general caspase inhibitor z-VAD-fmk did not alter ceramide-induced cell death in LNCaP cells, whereas it efficiently inhibited thapsigargin-induced apoptosis under similar conditions. However, ceramide treatment of LNCaP cells resulted in nuclear fragmentation, which is characteristic of apoptosis. Ceramide induced a strong and prolonged activation of c-Jun N-terminal Kinase (JNK) that correlated very well with the time course of cell death. Whereas the PKC inhibitor bisindolylmaleimide prevented phorbol ester-induced apoptosis in LNCaP cells, it did not affect ceramide-induced cell death. These results suggest that LNCaP cell death induced by ceramide progresses through a novel pathway that is more necrotic than apoptotic.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase Inhibitors; Cell Death; Ceramides; DNA, Neoplasm; Enzyme Inhibitors; Fluorescence; Humans; Indoles; Male; Maleimides; Necrosis; Prostatic Neoplasms; Protein Kinase C; Thapsigargin; Time Factors; Tumor Cells, Cultured

Functional overexpression of Bcl-2 has been reported to confer an anti-apoptotic potential in a variety of cell types. The role of Bcl-2 in epithelial cell-cycle control and in interactions with other cell-cycle regulators is not clearly understood. Its expression has been correlated with the hormono- and chemo-resistant phenotype in advanced prostate cancer. The aim of this study was to investigate the mechanisms through which Bcl-2 mediates increased cytotoxic chemoresistance by assessing alterations in the expression of cell death regulatory molecules. The DU145 human prostatic adenocarcinoma cell line was stably transfected with a Bcl-2 encoding expression plasmid. Two Bcl-2 transfectants, DKC9 and DKC11, were expanded for further study. The effects of Bcl-2 expression on cellular proliferation, cell death (+/- adriamycin or thapsigargin), and expression of cell-cycle/death regulators (p53, PCNA, Bax, Bak, Bcl-X(L)) were evaluated. Compared with controls, Bcl-2 transfectants showed no difference in the rate of proliferation, a decrease in p53 (approximately two-fold), an increase in Bax (approximately two-fold) and PCNA (approximately three-fold), and no change in the levels of Bcl-X(L) and Bak proteins. DKC9 and DKC11 also exhibited a significantly increased chemoresistance to adriamycin (0.0025-5 microM) and thapsigargin (0.0025-5 microM) compared with controls. In the presence of thapsigargin or adriamycin, levels of Bcl-2 and its heterodimeric partner Bax were elevated approximately two-fold with no change in Bak in Bcl-2 transfectants in contrast to controls, where Bak was increased (two-fold). This is the first study to demonstrate that Bcl-2 transfection modulates the expression of mutant p53, Bax, and PCNA in prostate cancer cells. Moreover, Bcl-2 overexpression conferred a significant cytotoxic chemoresistance and altered the balance of expression of death promoters (from Bak, a dominant death promoter in controls, to Bax) in response to thapsigargin and adriamycin.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Cycle Proteins; Cell Death; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Male; Prostatic Neoplasms; Thapsigargin; Transfection; Tumor Cells, Cultured

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.

Topics: Alternative Splicing; Apoptosis; bcl-X Protein; Breast Neoplasms; Cell Death; Female; Flow Cytometry; Genes, Regulator; Genetic Variation; Humans; Male; Prostatic Neoplasms; Protein Isoforms; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Precursors; Thapsigargin; Transfection; Tumor Cells, Cultured

The effect of estrogens (diethylstilbestrol [DES], 17 beta-estradiol) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in hormone-insensitive PC3 human prostate cancer cells was examined.. [Ca(2+)](i) changes in suspended cells were measured by using the Ca(2+)-sensitive fluorescent dye fura-2.. Estrogens (1--20 microM) increased [Ca(2+)](i) concentration-dependently with DES being more potent. Ca(2+) removal inhibited 50 +/- 10% of the signal. In Ca(2+)-free medium, pretreatment with 20 microM estrogens abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), but pretreatment with CCCP and thapsigargin did not alter DES-induced Ca(2+) release and partly inhibited 17 beta-estradiol-induced Ca(2+) release. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 1- 20 microM estrogens in Ca(2+)-free medium. Pretreatment with 1 microM U73122 to block phospholipase C-coupled inositol 1,4,5-trisphosphate formation did not alter estrogens-induced Ca(2+) release. The effect of 20 microM estrogen on [Ca(2+)](i) was not affected by pretreatment with 0.1 microM estrogens.. Estrogen induced significant Ca(2+) release and Ca(2+) influx in an inositol 1,4,5-trisphosphate-independent manner in PC3 cells. These effects of estrogens on Ca(2+) signaling appear to be nongenomic. Prostate 47:141-148, 2001.

Topics: Antineoplastic Agents, Hormonal; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Diethylstilbestrol; Enzyme Inhibitors; Estradiol; Estrenes; Fluorescent Dyes; Fura-2; Humans; Male; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Pyrrolidinones; Thapsigargin; Tumor Cells, Cultured; Uncoupling Agents

Osteoblastic metastases are common in lethal prostate cancer. Effective therapy for bone metastases is lacking. Thus, developing an appropriate in vitro screening system is critical to prioritize which of the newly developed agents should undergo additional expensive and time-consuming in vivo evaluation in bone metastases animal models. In the past, such in vitro screening evaluated the response of prostate cancer cells to chemotherapeutic agents in monoculture without the presence of osteoblasts. In such monoculture, prostate cancer cells have a high (i.e., >90%) proliferative growth fraction. In contrast, the growth fraction (i.e., mean: 7.1 +/- 0.8%; median: 3.1%) in 117 metastatic sites of prostate cancer obtained from 11 androgen ablation failing patients at "warm" autopsy was found to be >10-fold lower. To better mimic the lower growth fraction observed clinically, LNCaP human prostate cancer cells were cocultured with membrane-separated hFOB human osteoblasts. Such coculturing significantly lowered the growth fraction of the LNCaP cells (i.e., from >90 to <30%) without enhancing their low rate (i.e., <5%) of apoptosis. This lowering of the growth fraction was documented using flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using RNase protection assays, it was documented that coculture with osteoblasts causes enhanced p53, p27, and p21 expression leading to a decrease in the number of LNCaP cells entering the cell cycle (i.e., enhanced number of LNCaP cells in G(0)-G(1) and a decrease in S and G(2)-M and thus the growth fraction). This osteoblast-induced enhanced G(0)-G(1) checkpoint control affected the chemosensitivity of LNCaP cells. This was documented by coculturing LNCaP cells with hFOB cells to condition the medium for 3 days to lower the growth fraction to <30% before exposing the LNCaP cells for 48 h to various concentrations of Taxol, doxorubicin, or thapsigargin (TG). In standard high (i.e., >90%) growth fraction cultures (i.e., cultures in the absence of osteoblast-conditioned medium), there was a dose-dependent and significant (P < 0.05) increase in apoptosis of LNCaP cells exposed to Taxol or doxorubicin. In contrast, even the highest dose of Taxol (1 microM) did not enhance apoptosis of lower growth fraction LNCaP cells cultured in osteoblast-conditioned medium. Similarly, only the highest concentration of doxorubicin (1 microM) enhanced apoptosis in lower growth fraction cells. In contrast, 100

Topics: Antineoplastic Agents; Apoptosis; Cell Communication; Cell Cycle; Cell Division; Coculture Techniques; Culture Media, Conditioned; Doxorubicin; G1 Phase; Humans; Male; Neoplasms, Hormone-Dependent; Osteoblasts; Paclitaxel; Prostatic Neoplasms; Resting Phase, Cell Cycle; Thapsigargin; Tumor Cells, Cultured

The present study demonstrates for the first time that intracellular calcium-ATPases and calcium pool content are closely associated with prostate cancer LNCaP cell growth. Cell growth was modulated by changing the amount of epidermal growth factor, serum, and androgene in culture media. Using the microspectrofluorimetric method with Fura-2 and Mag Fura-2 as probes, we show that in these cells, the growth rate is correlated with intracellular calcium pool content. Indeed, an increased growth rate is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load. Using Western blotting and immunocytochemistry, we show that endoplasmic reticulum calcium pump expression is closely linked to LNCaP cell growth, and are a common target of physiological stimuli that control cell growth. Moreover, we clearly demonstrate that inhibition of these pumps, using thapsigargin, inhibits LNCaP cell growth and prevents growth factor from stimulating cell proliferation. Our results thus provide evidence for the essential role of functional endoplasmic reticulum calcium pumps and calcium pool in control of prostate cancer LNCaP cell growth, raising the prospect of new targets for the treatment of prostate cancer.

Topics: Blotting, Western; Calcium; Calcium-Transporting ATPases; Cell Division; Chelating Agents; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Fura-2; Humans; Immunohistochemistry; Intracellular Membranes; Male; Microscopy, Fluorescence; Microsomes; Prostatic Neoplasms; Protein Binding; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrophotometry; Thapsigargin; Time Factors; Tumor Cells, Cultured

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.

Topics: Calcium; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Estrenes; Fluorescent Dyes; Fura-2; Histamine; Histamine H1 Antagonists; Histamine H2 Antagonists; Humans; Inositol 1,4,5-Trisphosphate; Ion Transport; Male; Phosphodiesterase Inhibitors; Prostatic Neoplasms; Pyrilamine; Pyrrolidinones; Thapsigargin; Tumor Cells, Cultured; Type C Phospholipases

Nuclear envelope (NE) cisternal Ca2+ and cytosolic ATP are required for nuclear-pore-complex-(NPC-) mediated transport of DNAs, RNAs, transcription factors and other large molecules. Isolated cardiomyocyte nuclei, capable of macromolecular transport (MMT), have intrinsic NPC ion channel behavior. The large ion conductance (gamma) activity of the NPC channel (NPCC) is blocked by the NPC monoclonal antibody mAb414, known to block MMT, and is also silenced during periods of MMT. In cardiomyocytes, neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. To test the role of Ca2+ and ATP in NPCC activity, we carried out the present patch-clamp study with the pipette attached to the outer NE membrane of nuclei isolated from cultured Dunning G prostate cancer cells. Our investigations demonstrate that in these isolated nuclei neither cytosolic Ca2+ nor ATP alone directly affects NPCC gating. However, when simultaneously applied to the bath and pipette, they transiently silence NPCC activity through stimulation of MMT by raising the Ca2+ concentration in the NE cisterna ([Ca2+]NE). Our fluorescence microscopy observations with nuclear-targeted macromolecular fluorochromes (B-phycoerythrin and plasmid for the enhanced green fluorescence protein EGFP, pEGFP-C1) and with FITC-labeled RNA support the view that channel silence accompanies MMT. Repeated Ca2+ loading of the NE with Ca2+ and ATP, after unloading with 1-5 microM inositol 1,4,5-trisphosphate (IP3), thapsigargin (TSG) or 5 mM BAPTA or EGTA, failed to affect channel gating. This result indicates that other factors are involved in this phenomenon and that they are exhausted during the first cycle of NE Ca2+ loading/unloading--in agreement with current theories of NPC-mediated MMT. The results explain how Ca2+ and IP3 waves may convert the NE into an effective Ca2+ barrier and, consequently, affect the regulation of gene activity and expression through their feedback on MMT and NPCC gating. Thus, [Ca2+]NE regulation by intracellular messengers is an effective mechanism for synchronizing gene activity and expression to the cellular rhythm.

Topics: Adenosine Triphosphate; Animals; Antibodies, Monoclonal; Biological Transport; Calcium; Calcium Channels; Chelating Agents; Cytosol; Dextrans; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Inositol 1,4,5-Trisphosphate; Ion Channel Gating; Male; Nuclear Envelope; Oocytes; Patch-Clamp Techniques; Prostatic Neoplasms; Thapsigargin; Tumor Cells, Cultured; Xenopus laevis

Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca(2+) stores are released and plasma membrane "store-operated" Ca(2+) entry channels subsequently open, cytosolic [Ca(2+)] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca(2+) stores are resistant to apoptotic stimuli, indicating that Ca(2+) store release is also important. We investigated whether release of intracellular Ca(2+) stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca(2+) without elevating cytosolic [Ca(2+)]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca(2+) store release with the dual insults of store release and cytosolic [Ca(2+)] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca(2+) perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins Bclx(L) and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca(2+) store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca(2+)] elevation and intracellular store release.

Topics: Apoptosis; bcl-X Protein; Calcium; Caspase 7; Caspase 9; Caspases; Endoplasmic Reticulum; Humans; Male; Nickel; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Thapsigargin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The proinflammatory protein endothelial monocyte-activating polypeptide II (EMAP-II) was first detected in supernatants of murine tumor cells by virtue of its ability to stimulate endothelial-dependent coagulation in vitro. The purified protein has pleiotropic effects on endothelial cells, monocytes, and neutrophils; however, its function in vivo is unknown, and the mechanism whereby it is released from cells is poorly understood. We investigated the expression of EMAP-II in human prostate adenocarcinoma specimens by immunohistochemistry and in LNCaP and DU-145 human prostate adenocarcinoma cells by reverse transcription-PCR, flow cytometry, and Western blotting. We then examined the effects of chemical and physiological stress on release and processing of EMAP-II by LNCaP and DU-145 cells. These cells constitutively express a Mr 34,000 form of EMAP-II that is retained intracellularly. Exposure to agents that induce apoptosis or, in some cases, necrosis induces the release of the Mr 34,000 form and further processing to the Mr 27,000 and Mr 22,000 forms. Hypoxia, but not heat shock, is a potent inducer of release and processing of biologically active EMAP-II by LNCaP and DU-145 cells. We suggest that release of EMAP-II by prostate adenocarcinoma cells as a consequence of treatment with anticancer agents or as a result of constitutive hypoxia may potentiate the effects of those agents through the localized activation of host effector mechanisms.

Topics: Adenocarcinoma; Anti-Bacterial Agents; Antimycin A; Apoptosis; Blotting, Western; Cell Hypoxia; Cytokines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Glucose; Humans; Immunohistochemistry; Ionomycin; Ionophores; Male; Necrosis; Neoplasm Proteins; Peptides; Prostate; Prostatic Neoplasms; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Stress, Physiological; Thapsigargin; Tumor Cells, Cultured

New agents are required for the treatment of androgen-independent prostate cancer. Due to the low rate of proliferation of these malignant cells, agents which can activate the apoptotic death of these cells without requiring the cells being in the proliferative cell cycle are critically required. Thapsigargin (TG), via its ability to perturb intracellular free calcium [Ca(2+)](i), is such a cell proliferation-independent cytotoxic agent. The present study focuses on more completely describing the biochemical cascade during the apoptotic death of androgen-independent prostate cancer cells induced by TG and on the mechanistic requirements for this death.. A variety of cell and molecular biology techniques (e.g., time-lapse video, fluorescence image analysis, Northern and Western blotting) were used to examine the temporal relationship between changes in [Ca(2+)](i), GADD 153 transcription, translocation of the NFATc transcription factor to the nucleus, translocation of BAD from the cytosol to the mitochondria, caspase 9 activation, DNA fragmentation, and the loss of clonogenic survival induced by TG treatment of both human TSU-prl and rat AT3.1 prostate cancer cells in vitro. Additional studies using both microinjection of inhibitors of calmodulin and DNA transfections to induce expression of Ca(2+) binding proteins, e.g., calbindin, were performed to evaluate the causal relationship between [Ca(2+)](i) elevation, calmodulin/calcineurin activation, and apoptosis of prostate cancer cells.. Using simultaneous fluorescence ratiometric and phase contrast image analysis in individual cells followed longitudinally for several days, it was documented that TG induced early (1-12 hr) moderate (i.e., <500 nM) elevation in [Ca(2+)](i). During this early rise in [Ca(2+)](i), genes like GADD 153 are induced at the transcriptional level. This early rise is followed by a return of [Ca(2+)](i) to baseline (i.e., approximately 50 nM) before the induction of a delayed (i.e., >12 hr) secondary rise ( approximately 10 microM) in [Ca(2+)](i). During the secondary rise in [Ca(2+)](i), Ca(2+) binds to calcineurin and calmodulin, allowing these proteins to form a complex which activates calcineurin's latent phosphatase activity. Once activated, calcineurin dephosphorylates NFATc and BAD, allowing translocation of these proteins to the nucleus and mitochondria, respectively. BAD translocation induces the release of cytochrome C from the mitochondria into the cytoplasm, which results in activation of caspase 9 and DNA fragmentation. If the TG-induced rise in [Ca(2+)](i) is blocked by overexpressing calbindin, or if calmodulin function is inhibited, these apoptotic events are prevented.. TG induces the apoptotic death of prostate cancer cells via the activation of a reversible signaling phase induced by a transient nanomolar rise in [Ca(2+)](i), which involves new gene transcription and translation. This reversible signaling phase is followed by an irreversible commitment to undergo the execution phase which is induced by a secondary micromolar rise in [Ca(2+)](i). This secondary [Ca(2+)](i) rise irreversibly commits the cell to a calmodulin/calcineurin-dependent cascade, which results in DNA and cellular fragmentation into apoptotic bodies.

Topics: Animals; Apoptosis; bcl-Associated Death Protein; Calcineurin; Calcium; Calmodulin; Carrier Proteins; Cytochrome c Group; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Male; NFATC Transcription Factors; Nuclear Proteins; Prostatic Neoplasms; Rats; Signal Transduction; Thapsigargin; Transcription Factors; Tumor Cells, Cultured

1. In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+-ATPase inhibitor thapsigargin (TG), in androgen-sensitive human prostate cancer LNCaP cells. 2. Exposure of fura-2-loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store-operated Ca2+ channels (SOCs). 3. For the first time we have identified and characterized the SOC-mediated membrane current (Istore) in prostate cells using whole-cell, cell-attached, and perforated patch-clamp techniques, combined with fura-2 microspectrofluorimetric and Ca2+-imaging measurements. 4. Istore in LNCaP cells lacked voltage-dependent gating and displayed an inwardly rectifying current-voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 +/- 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5-3 mM) and La3+ (1 microM). 5. Treatment of LNCaP cells with TG (0.1 microM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG-induced apoptosis. 6. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA-AM). 7. These results indicate that in androgen-sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store-activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.

Topics: Androgens; Apoptosis; Calcium; Calcium Channels; Calcium-Transporting ATPases; Electric Conductivity; Humans; Lanthanum; Male; Microscopy, Fluorescence; Nickel; Patch-Clamp Techniques; Prostatic Neoplasms; Thapsigargin; Tumor Cells, Cultured

The effects of mitochondrial uncoupling on the calcium homeostasis of prostatic cells were investigated using the prostatic cancer cell line LNCaP and indo-1 spectrofluorimetry. Carbonyl cyanide m-chloro-phenylhydrazone (CCCP) was used as uncoupler. Resting LNCaP cells responded to CCCP by a biphasic increase in [Ca2+]i. The first phase of increase which corresponded to the release of a mitochondrial CCCP-sensitive Ca2+ store was followed by a second increase phase consisting of Ca2+ influx through the plasma membrane. The relationship between the CCCP- and the InsP3-sensitive stores was investigated using thapsigargin (TG). The release part of the Ca2+ response to TG was reduced in a time-dependent manner by previous exposure of the cells to CCCP, suggesting that CCCP also acts on non-mitochondrial stores. Our results show that CCCP releases Ca2+ from both mitochondrial and non-mitochondrial stores in prostatic cells. The possible mechanisms of these effects are discussed.

Topics: Adenosine Triphosphate; Antimycin A; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Enzyme Inhibitors; Fluorescent Dyes; Humans; Indoles; Male; Mitochondria; Oligomycins; Prostatic Neoplasms; Spectrometry, Fluorescence; Thapsigargin; Time Factors; Tumor Cells, Cultured; Uncoupling Agents

Caspases are a family of cysteine proteases capable of characteristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical initiators in the signaling phase, while others (e.g., caspases 3, 6, and 7) have been implicated in the effector or execution phase of apoptosis. Thapsigargin (TG) is capable of inducing cell proliferation-independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspase inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apoptosis in prostate cancer cells.. Caspase activity was evaluated by Western blot analysis of the cleavage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specific fluorescent peptide substrates was assayed in lysates from TG-treated cells. Clonogenic survival assays were performed following treatment of rat AT3 and human TSU-Pr1 prostate cancer cell lines with TG and 5-FrdU in the presence and absence of peptide caspase inhibitors. AT3.1 cells transfected with the crmA gene, encoding a viral protein with caspase-inhibitory activity, were also tested for clonogenic survival following TG and 5-FrdU exposure.. During treatment with TG, Rb is first dephosphorylated and then proteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hydrolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspase inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect TSU and AT3 cells from apoptosis induced by TG or 5-FrdU.. Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest that caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A strategy for targeting TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; Caspase Inhibitors; Cell Survival; Clone Cells; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Floxuridine; Humans; Male; Oligopeptides; Prostatic Neoplasms; Rats; Retinoblastoma Protein; Signal Transduction; Thapsigargin; Tumor Cells, Cultured

Prostate carcinoma (PCA) is the most frequently diagnosed malignancy in American men. PCA at advanced stages can both proliferate abnormally and resist apoptosis. Among the many known signal transduction pathways, phosphatidylinositide-3'OH kinase (PI3-kinase) has been shown to play an important role in cell survival and resistance to apoptosis. In this study, we investigate the involvement of Etk/Bmx, a newly discovered tyrosine kinase that is a substrate of PI3-kinase, in protection of prostate cancer cells from apoptosis. Parental LNCaP cells and two derivative cell lines, one overexpressing wild type Etk (Etkwt) and the other expressing a dominant negative Etk (EtkDN), were used to study the function of Etk. The cells were treated with photodynamic therapy (PDT), a newly approved cancer treatment which employs a photosensitizer and visible light to produce an oxidative stress in cells, often leading to apoptosis. Our results indicate that PDT induces apoptosis in LNCaP cells, as measured by DNA fragmentation and by cleavage of poly(ADP-ribose) polymerase (PARP), and moreover, the extent of apoptosis was much reduced in Etkwt cells as compared to LNCaP or EtkDN cells. Assay of overall cell viability confirmed that Etkwt cells were considerably less sensitive to PDT than were the parental LNCaP or EtkDN cells. Similar results were found in response to thapsigargin (TG). A specific inhibitor of PI3-kinase, LY294002, abolished Etk activity and markedly increased TG-induced PARP cleavage. The results suggest that Etk/Bmx is an efficient effector of PI3-kinase and that the newly described PI3-kinase/Etk pathway is involved in the protection of prostate carcinoma cells from apoptosis in response to PDT or TG.

Topics: Apoptosis; Carcinoma; Cell Survival; Chromones; DNA Fragmentation; Enzyme Inhibitors; Humans; Male; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Photochemotherapy; Photosensitizing Agents; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein-Tyrosine Kinases; Thapsigargin; Tumor Cells, Cultured

Calcium seems to be a major second messenger involved in the regulation of prostatic cell functions, but the mechanisms underlying its control are poorly understood. We investigated spatiotemporal aspects of Ca2+ signals in the LNCaP cell line, a model of androgen-dependent prostatic cells, by using non-invasive external electric field pulses that hyperpolarize the anode facing membrane and depolarize the membrane facing the cathode. Using high-speed fluo-3 confocal imaging, we found that an electric field pulse (10-15 V/cm, 1-5 mA, 5 ms) initiated rapidly, at the hyperpolarized end of the cell, a propagated [Ca2+]i wave which spread through the cell with a constant amplitude and an average velocity of about 20 microns/s. As evidenced by the total wave inhibition either by the block of Ca2+ entry or the depletion of Ca2+ stores by thapsigargin, a specific Ca(2+)-ATPase inhibitor, the [Ca2+]i wave initiation may imply a localized Ca2+ influx linked to a focal auto-regenerative process of Ca2+ release. Using different external Ca2+ and Ca2+ entry blockers concentrations, Mn2+ quenching of fluo-3 and fura-2 fluorescence and inhibitors of InsP3 production, we found evidence that the [Ca2+]i wave progression required, in the presence of basal levels of InsP3, an interplay between Ca2+ release from InsP3-sensitive Ca2+ stores and Ca2+ influx through channels possibly activated by the [Ca2+]i rise.

Topics: Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Polarity; Electric Stimulation; Enzyme Inhibitors; Fura-2; Gadolinium; Humans; Lanthanum; Male; Manganese; Microscopy, Confocal; Nimodipine; Prostatic Neoplasms; Ryanodine; Thapsigargin; Tumor Cells, Cultured

A number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPases have been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an aromatic amine. The amine can be used as an anchoring point for attaching a peptide group sensitive to the proteolytic enzyme, prostate specific antigen, secreted by prostate cancer cells. Like thapsigargin, the analogues are capable of elevating the cytoplasmic Ca2+ concentration approximately sevenfold when tested at effective cytotoxic doses. The analogues in which the 8-O-butanoyl group has been replaced with 3-(4-aminophenyl)propanoyl or 4-aminocinnamoyl were found potently to induce programmed cell death of the prostate cancer cells.

Topics: Androgens; Animals; Apoptosis; Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Male; Muscle, Skeletal; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Rabbits; Sarcoplasmic Reticulum; Structure-Activity Relationship; Thapsigargin; Tumor Cells, Cultured

A human prostate cancer cell line (PC3) with abundant neurotensin (NT) receptors was used to demonstrate that NT potentiated 3',5'-cyclic adenosine monophate (cAMP) accumulation in response to a variety of stimuli, including both direct forskolin (F) and indirect (prostaglandin, (PGE2), isoproterenol (ISO) and cholera toxin (CTx)) activators of adenylyl cyclase. Several mechanisms were investigated and our results indicated an effect on the rate of cAMP formation and not on degradation or extrusion. For each stimulus, NT enhanced efficacy without altering EC50. The effect of NT did not involve stimulatory G-protein (Gs)-activation or interference with a tonic inhibitory G-protein (Gi)-mediated inhibition. A similar response was obtained when NT was added with the stimulus or given as a two minute pulse which was removed prior to addition of stimulus. The potentiating activity disappeared with a t1,2 of approximately 15 min. NT transiently elevated cellular [Ca2+]i and its effects on cAMP could be mimicked by [Ca2+]i-elevating agents (uridine triphosphate (UTP), thapsigargin and ionomycin). Buffering cellular [Ca2+]i with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) inhibited cAMP responses to ISO and F in presence and absence of NT. These data support the idea that NT potentiated cAMP formation in response to a variety of stimuli by facilitating the activation of Ca2+ -dependent adenylyl cyclases.

Topics: Adenylyl Cyclases; Calcium; Cholera Toxin; Colforsin; Cyclic AMP; Dinoprostone; Drug Synergism; Egtazic Acid; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Humans; Ionomycin; Isoproterenol; Male; Neurotensin; Prostatic Neoplasms; Thapsigargin; Tumor Cells, Cultured; Uridine Triphosphate

Prostate-specific antigen (PSA) is a serine protease secreted by both normal prostate glandular cells and prostate cancer cells. The major proteolytic substrates for PSA are the gel-forming proteins in semen, semenogelin (Sg) I and II. On the basis of the PSA cleavage map for Sg I and II, a series of small peptides (i.e., < or = 7 amino acids) was synthesized and coupled at the COOH terminus to 7-amino-4-methyl coumarin. Using these fluorescently tagged substrates, K(m)s and k(cat)s were determined for PSA hydrolysis, and the substrates were also tested for activity against a panel of purified proteases. Previously, a variety of chymotrypsin substrates have been used to assay the enzymatic activity of PSA. The present studies have identified a peptide sequence with a high degree of specificity for PSA (ie., no detectable hydrolysis by chymotrypsin) and improved K(m)s and k(cat)s over previously used substrates. On the basis of these parameters, the best peptide substrate for PSA has the amino acid sequence HSSKLQ. Using PC-82 human prostate cancer xenografts and human prostate tissues, this PSA substrate was used to document that prostate cancer cells secrete enzymatically active PSA into the extracellular fluid but that once in the blood, PSA is not enzymatically active. On the basis of this information, it should be possible to use the HSSKLQ peptide as a carrier to target peptide-coupled prodrugs for selective activation within sites of PSA-secreting, metastatic prostate cancer cells and not within the blood or other nonprostatic normal tissues.

Topics: alpha 1-Antichymotrypsin; Animals; Enzyme Inhibitors; Extracellular Space; Gonadal Steroid Hormones; Humans; Hydrolysis; Kinetics; Male; Mice; Mice, Nude; Molecular Sequence Data; Peptide Fragments; Peptide Mapping; Precipitin Tests; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Seminal Vesicle Secretory Proteins; Substrate Specificity; Thapsigargin; Transplantation, Heterologous; Tumor Cells, Cultured

More than 95% of metastatic androgen independent prostatic cancer cells per day are in a proliferatively quiescent G0 state [Berges et al.: Clin Cancer Res 1:473-480, 1995] limiting their responsiveness to anti-proliferative chemotherapeutic agents. Novel therapeutics capable of activating the programmed (apoptotic) death pathway in these cells without requiring entrance into the proliferative cell cycle are urgently needed. Thapsigargin (TG) treatment of rapidly growing androgen independent prostatic cancer cells arrests such cells in G0 and induces their programmed death. This raises not only the issue of the mechanism for such growth arrest, but also whether this programmed death is simply a response of rapidly growing cells to growth arrest making cytotoxicity still dependent upon the initial rate of cell proliferation.. To resolve the mechanism of TG induced growth arrest, rat AT3.1 prostatic cancer cells were analyzed for RNA and protein expression of the growth arrest gene, gadd153, intracellular free Ca2+ levels (Cai), and cell cycle distribution on exposure to TG alone and in combination with Ca2+ chelation induced by BAPTA-AM or BAPTA-AM/EGTA. To resolve whether growth arrest is required for TG cytotoxicity, primary cultures of proliferatively quiescent, human prostatic cancer cells were exposed to TG.. Co-treatment of androgen independent AT-3 rat prostatic cancer cells with the Cai chelator BAPTA plus TG prevented growth arrest, as monitored by DNA flow cytometry, and failure to induce mRNA and protein for gadd153, demonstrating that growth arrest is due to Cai elevation, not depletion of intracellular Ca2+ pools. In addition, proliferatively quiescent G0 primary cultures of human prostatic cancer cells were resistant to anti-proliferative agents, but could be induced to undergo programmed death by TG as documented by morphological criteria and 14C-labeled DNA fragmentation assays.. These results demonstrate that TG with its ability to elevate Cai induces proliferating prostate cancer cells to growth arrest. Such Cai dependent growth arrest is not required, however, since TG can induce the programmed death of proliferatively quiescent G0 prostatic cancer cells without requiring either growth arrest or progression through the proliferative cell cycle.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Northern; Blotting, Western; Calcium; Cell Division; Chelating Agents; DNA Damage; DNA, Neoplasm; Egtazic Acid; Flow Cytometry; Floxuridine; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Neoplasms; Rats; RNA, Messenger; RNA, Neoplasm; Thapsigargin; Time Factors; Tumor Cells, Cultured

Bcl-2 expression is associated with the progression of prostate cancer from androgen-dependence to androgen-independence. Bcl-2 is an integral membrane protein which localizes to mitochondria, endoplasmic reticulum, and the nuclear envelope. Using spectrofluorometry and laser confocal microscopy, the ability of bcl-2 to modulate intracellular Ca2+ was examined in the Dunning G prostate carcinoma cell line following apoptosis induction by adriamycin. Adriamycin and thapsigargin, an endoplasmic reticulum Ca2+-pump inhibitor, were effective inducers of apoptosis in control, but not bcl-2 transfected, cells. Treatment with adriamycin was accompanied by a sustained rise in cytoplasmic Ca2+ in control and bcl-2 transfected cells. An increase in intranuclear Ca2+ was observed in control cells only. Apoptosis induction by thapsigargin was associated with an increase in cytoplasmic Ca2+ in control cells that was not detected in the resistant bcl-2 transfectants. Ca2+ was excluded from nuclei isolated from bcl-2 expressing cells, but was sequestered in control nuclei, following the addition of ATP. These findings suggest that bcl-2 may regulate levels of intranuclear Ca2+ independently of cytosolic Ca2+ levels. The ability of bcl-2 to modulate, directly or indirectly, sustained increases in both cytosolic and intranuclear Ca2+ may provide a common basis for bcl-2 function in different subcellular compartments.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Calcium; Cell Nucleus; Cytosol; DNA, Neoplasm; Doxorubicin; Enzyme Inhibitors; Male; Microscopy, Confocal; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

The aim of this study was to determine whether stable differences in apoptosis sensitivity were selected for in nonmetastatic and metastatic variants of the LNCaP human prostate carcinoma line that had been isolated from tumors grown orthotopically in the prostate glands and regional lymph nodes of nude mice. The nonmetastatic LNCaP-Pro5 cells were significantly more sensitive to thapsigargin-induced apoptosis than were the metastatic LNCaP-LN3 cells, as measured by viability, DNA fragmentation, and interleukin 1beta-converting enzyme family-mediated cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase. Apoptosis resistance in the metastatic cells was associated with higher levels of expression of the cell death suppressor BCL-2 and lower levels of the death promoters BAX and BAK than were detected in the nonmetastatic LNCaP-Pro5 cells, whereas levels of two other BCL-2 family members (BCL-X(L) and BAD) were indistinguishable. Our data support the hypothesis that apoptosis resistance contributes to prostate cancer metastasis and that elevated expression of BCL-2 is involved.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinogens; Doxorubicin; Drug Resistance; Humans; Male; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; Thapsigargin; Tumor Cells, Cultured

Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of interest to determine whether Ca2+ would affect the expression of androgen receptor messenger RNA (mRNA) and protein, thus affecting the ability of androgens to control prostate function. AR-positive human prostate cancer cells, LNCaP, were incubated with either the calcium ionophore A23187 or the intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. Subsequently, AR mRNA and protein levels were assessed by Northern and Western blot analysis. Both A23187 and thapsigargin were found to down-regulate steady state AR mRNA levels in a time- and dose-dependent manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of incubation, respectively. In contrast, control mRNA (glyceraldehyde 3-phosphate dehydrogenase) did not change significantly during the treatments with either A23187 or thapsigargin. AR protein levels were found to be decreased after 12 h of incubation with either 10(-6) M A23187 or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was found to alter cell morphology within the treatment time. Cycloheximide and actinomycin D were unable to change the calcium-mediated decrease in AR mRNA, ruling out the necessity for de novo protein synthesis or a change in mRNA stability. Moreover, the decrease in AR mRNA induced by calcium does not seem to involve protein kinase C- or calmodulin-dependent pathways, since inhibitors of these cellular components had no effect. Nuclear run-on assays demonstrated little or no effects of either A23187 or thapsigargin treatment on AR gene transcription (8 h and 10 h). In conclusion, these studies show that intracellular calcium seems to be a potent regulator of AR gene expression in LNCaP cells.

Topics: Alkaloids; Apoptosis; Blotting, Northern; Blotting, Western; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Line; Cell Nucleus; Dactinomycin; Dose-Response Relationship, Drug; Gene Expression; Humans; Kinetics; Male; Prostatic Neoplasms; Protein Kinase C; Receptors, Androgen; RNA, Messenger; RNA, Neoplasm; Staurosporine; Terpenes; Thapsigargin; Tumor Cells, Cultured

Calcium (Ca2+) accumulates within the endoplasmic reticulum of cells through function of the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase family of intracellular Ca(2+)-pumping ATPases. The resulting pools have important signaling functions. Thapsigargin (TG) is a sesquiterpene gamma-lactone which selectively inhibits the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase pumps with a 50% inhibitory concentration of approximately 30 nM. Treatment of androgen-independent prostate cancer cells of both rat and human origin with TG inhibits their endoplasmic reticulum Ca(2+)-dependent ATPase activity, resulting in a 3-4-fold elevation in the level of intracellular free Ca2+ (Cai) within minutes of exposure. Due to a secondary influx of extracellular Ca2+, this increase in Cai is sustained, resulting in morphological (cell rounding) and biochemical changes within 6-12 h (enhanced calmodulin, glucose regulated protein, and tissue transglutaminase expression, and decreased expression of the G1 cyclins). Within 24 h of exposure, androgen-independent prostatic cancer cells stop progression through the cell cycle, arrest out of cycle in G0, and irreversibly lose their ability to proliferate with a median effective concentration value of 31 nM TG. During the next 24-48 h, the genomic DNA of the G0-arrested cells undergoes double-strand fragmentation. This is followed by the loss of plasma membrane integrity and fragmentation of the cell into apoptotic bodies. During this process, there is no acidification in the intracellular pH. Using cells transfected with the avian M(r) 28,000 calbindin D Ca(2+)-buffering protein, it was demonstrated that the programmed death initiated by TG is critically dependent upon an adequate (i.e., 3-4-fold) sustained (> 1 h) elevation in Cai and not depletion of the endoplasmic reticulum pools of Ca2+. These results demonstrate that TG induces programmed cell death in androgen-independent prostatic cancer cells in a dose-dependent manner and that this death does not require proliferation or intracellular acidification but is critically dependent upon an adequate, sustained (i.e., > 1 h) elevation in Cai.

Topics: Androgens; Animals; Apoptosis; Calcium; Calcium-Transporting ATPases; Cell Cycle; Cell Division; DNA; Humans; Hydrogen-Ion Concentration; Male; Prostatic Neoplasms; Protein Biosynthesis; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

Androgen-independent Dunning R-3327 AT-3 rat prostatic cancer cells can be induced to undergo programmed cell death in either a proliferation-dependent or independent manner depending upon the therapeutic agent used. In the present study, 5-fluorodeoxyuridine (5-FrdU) was used to induce proliferation-dependent death of the AT-3 cells via its ability to inhibit thymidylate synthetase. Ionomycin and thapsigargin were used to induce proliferation-independent death of these cells via their ability to sustain an elevation in intracellular free Ca2+. Based upon the temporal sequence of DNA fragmentation, morphologic changes, and loss of cell viability, each of the three test agents, at the doses used, induces the programmed death of AT-3 cells with essentially identical kinetics. Based upon these similarities, comparisons of the pattern of gene expression during the proliferation-dependent (i.e., 5-FrdU-induced) vs. proliferation-independent (i.e., ionomycin and thapsigargin-induced) programmed death of AT-3 cells allow identification of genes whose enhanced expression is involved in the initiation vs. completion of programmed cell death. Based upon this approach, enhanced H-ras and TRPM-2 expression is associated with initiation of proliferation-dependent programmed death of AT-3 cells while enhanced c-myc, calmodulin, and alpha-prothymosin expression is associated with initiation of proliferation-independent programmed death of these cells. In contrast, enhanced expression of glucose-regulated 78 kilodalton and tissue transglutaminase genes are associated with the completion of programmed cell death, since their expression is enhanced in both proliferation-dependent and independent programmed cell death of AT-3 cells.

Topics: Animals; Apoptosis; Cell Division; DNA, Neoplasm; Floxuridine; Gene Expression Regulation, Neoplastic; Ionomycin; Male; Prostatic Neoplasms; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured