thapsigargin and Neoplasms

Mipsagargin (G-202; (8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin)-Asp-γ-Glu-γ-Glu-γ-GluGluOH)) is a novel thapsigargin-based targeted prodrug that is activated by PSMA-mediated cleavage of an inert masking peptide. The active moiety is an inhibitor of the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump protein that is necessary for cellular viability. We evaluated the safety of mipsagargin in patients with advanced solid tumours and established a recommended phase II dosing (RP2D) regimen.. Patients with advanced solid tumours received mipsagargin by intravenous infusion on days 1, 2 and 3 of 28-day cycles and were allowed to continue participation in the absence of disease progression or unacceptable toxicity. The dosing began at 1.2 mg m(-2) and was escalated using a modified Fibonacci schema to determine maximally tolerated dose (MTD) with an expansion cohort at the RP2D. Plasma was analysed for mipsagargin pharmacokinetics and response was assessed using RECIST criteria.. A total of 44 patients were treated at doses ranging from 1.2 to 88 mg m(-2), including 28 patients in the dose escalation phase and 16 patients in an expansion cohort. One dose-limiting toxicity (DLT; Grade 3 rash) was observed in the dose escalation portion of the study. At 88 mg m(-2), observations of Grade 2 infusion-related reaction (IRR, 2 patients) and Grade 2 creatinine elevation (1 patient) led to declaration of 66.8 mg m(-2) as the recommended phase II dose (RP2D). Across the study, the most common treatment-related adverse events (AEs) were fatigue, rash, nausea, pyrexia and IRR. Two patients developed treatment-related Grade 3 acute renal failure that was reversible during the treatment-free portion of the cycle. To help ameliorate the IRR and creatinine elevations, a RP2D of 40 mg m(-2) on day 1 and 66.8 mg m(-2) on days 2 and 3 with prophylactic premedications and hydration on each day of infusion was established. Clinical response was not observed, but prolonged disease stabilisation was observed in a subset of patients.. Mipsagargin demonstrated an acceptable tolerability and favourable pharmacokinetic profile in patients with solid tumours.

Topics: Aged; Aged, 80 and over; Enzyme Inhibitors; Humans; Male; Middle Aged; Neoplasms; Prodrugs; Thapsigargin

Naturally occurring terpenoid lactones and their synthetic derivatives have attracted increasing interest for their promising antitumor activity and potential utilization in the discovery and design of new antitumor agents. In the present perspective article, selected plant-derived five-membered γ-lactones and six-membered δ-lactones that occur with terpenoid scaffolds are reviewed, with their structures, cancer cell line cytotoxicity and in vivo antitumor activity, structure-activity relationships, mechanism of action, and the potential for developing cancer chemotherapeutic agents discussed in each case. The compounds presented include artemisinin (ART,

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Biological Products; Diterpenes; Humans; Neoplasms; Sesquiterpenes; Structure-Activity Relationship; Triterpenes

In spite of the impressing cytotoxicity of thapsigargin (

Topics: Antineoplastic Agents, Phytogenic; Apiaceae; Butyrates; Carbon Dioxide; Chemistry Techniques, Synthetic; Chromatography, Supercritical Fluid; Fruit; Furans; Humans; Molecular Structure; Neoplasms; Plant Extracts; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Interleukin-8 (IL-8) and heat shock protein 60 (Hsp60) play crucial roles in cell survival and maintenance of cellular homoeostasis. However, cross talks between these two proteins are not defined.. IL-8 expression in tumour tissue sections was analysed by immunohistochemistry. IL-8 expression and release in cancer cells was quantified using enzyme-linked immunosorbent assay (ELISA). Apoptosis was quantified using caspase activity and Annexin-V/PI staining.. We observed IL-8 release from cancer cells in response to histone deacetylase inhibitor, apicidin (Api), and non-competitive inhibitor of the sarco/endoplasmic reticulum Ca. This study describes the underlying mechanism associated with apoptosis resistance mediated via Hsp60-IL-8 axis in cancer.

Topics: Animals; Apoptosis; Caspase 8; Caspase 9; Chaperonin 60; Gene Knockdown Techniques; HCT116 Cells; Heterografts; Humans; Interleukin-8; Male; Mice; Mice, SCID; Mitochondrial Proteins; Neoplasms; PC-3 Cells; Peptides, Cyclic; Signal Transduction; Thapsigargin

CCAAT/enhancer binding protein beta (C/EBPβ), a transcription factor expressed in muscle satellite cells (SCs), inhibits the myogenic program and is downregulated early in differentiation. In a conditional null model in which C/EBPβ expression is knocked down in paired box protein 7+ (Pax7+) SCs, cardiotoxin (CTX) injury is poorly repaired, although muscle regeneration is efficient in control littermates. While myoblasts lacking C/EBPβ can differentiate efficiently in culture, after CTX injury poor regeneration was attributed to a smaller than normal Pax7+ population, which was not due to a failure of SCs to proliferate. Rather, the percentage of apoptotic SCs was increased in muscle lacking C/EBPβ. Given that an injury induced by BaCl2 is repaired with greater efficiency than controls in the absence of C/EBPβ, we investigated the inflammatory response following BaCl2 and CTX injury and found that the levels of interleukin-1β (IL-1β), a proinflammatory cytokine, were robustly elevated following CTX injury and could induce C/EBPβ expression in myoblasts. High levels of C/EBPβ expression in myoblasts correlated with resistance to apoptotic stimuli, while its loss increased sensitivity to thapsigargin-induced cell death. Using cancer cachexia as a model for chronic inflammation, we found that C/EBPβ expression was increased in SCs and myoblasts of tumor-bearing cachectic animals. Further, in cachectic conditional knockout animals lacking C/EBPβ in Pax7+ cells, the SC compartment was reduced because of increased apoptosis, and regeneration was impaired. Our findings indicate that the stimulation of C/EBPβ expression by IL-1β following muscle injury and in cancer cachexia acts to promote SC survival, and is therefore a protective mechanism for SCs and myoblasts in the face of inflammation.

Topics: Animals; Apoptosis; Barium Compounds; Cardiotoxins; CCAAT-Enhancer-Binding Protein-beta; Cell Line; Chlorides; Immunohistochemistry; Interleukin-1beta; Mice; Mice, Knockout; Mice, Transgenic; Myoblasts; Neoplasms; PAX7 Transcription Factor; RNA, Messenger; Thapsigargin; Up-Regulation

Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment because of its role in stabilizing the active form of a wide range of client oncoproteins. This study investigated the mechanism of apoptosis induced by the purine-scaffold Hsp90 inhibitor PU-H71 in different human cancer cell lines and examined the role of Bcl-2 and Bax in this process. We demonstrated that Hsp90 inhibition by PU-H71 generated endoplasmic reticulum (ER) stress and activated the Unfolded Protein Response (UPR) as evidenced by XBP1 mRNA splicing and up-regulation of Grp94, Grp78, ATF4 and CHOP. In response to PU-H71-induced ER stress, apoptosis was triggered in melanoma, cervix, colon, liver and lung cancer cells, but not in normal human fibroblasts. Apoptosis was executed through the mitochondrial pathway as shown by down-regulation of Bcl-2, up-regulation and activation of Bax, permeabilization of mitochondrial membranes, release of cytochrome c and activation of caspases. We also found that, in contrast to the ER stressor thapsigargin, PU-H71 induced apoptosis in cells overexpressing Bcl-2 and thus overcame the resistance conferred by this anti-apoptotic protein. In addition, although Bax deficiency rendered cells resistant to PU-H71, combined treatment with the anticancer drugs cisplatin or melphalan greatly sensitized these cells to PU-H71. Taken together, these data suggest that inhibition of Hsp90 by PU-H71 is a promising strategy for cancer treatment, particularly in the case of tumors resistant to conventional chemotherapy.

Topics: Antineoplastic Agents; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Benzodioxoles; Blotting, Western; Caspases; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Flow Cytometry; Fluorescent Antibody Technique; HSP90 Heat-Shock Proteins; Humans; Melphalan; Mitochondria; Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Purines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thapsigargin; Tumor Cells, Cultured; Unfolded Protein Response

Tasquinimod is an orally active antiangiogenic drug that is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer. However, the target of this drug has remained unclear. In this study, we applied diverse strategies to identify the histone deacetylase HDAC4 as a target for the antiangiogenic activity of tasquinimod. Our comprehensive analysis revealed allosteric binding (Kd 10-30 nmol/L) to the regulatory Zn(2+) binding domain of HDAC4 that locks the protein in a conformation preventing HDAC4/N-CoR/HDAC3 complex formation. This binding inhibited colocalization of N-CoR/HDAC3, thereby inhibiting deacetylation of histones and HDAC4 client transcription factors, such as HIF-1α, which are bound at promoter/enhancers where epigenetic reprogramming is required for cancer cell survival and angiogenic response. Through this mechanism, tasquinimod is effective as a monotherapeutic agent against human prostate, breast, bladder, and colon tumor xenografts, where its efficacy could be further enhanced in combination with a targeted thapsigargin prodrug (G202) that selectively kills tumor endothelial cells. Together, our findings define a mechanism of action of tasquinimod and offer a perspective on how its clinical activity might be leveraged in combination with other drugs that target the tumor microenvironment. Cancer Res; 73(4); 1386-99. ©2012 AACR.

Topics: Acetylation; Allosteric Regulation; Animals; Blotting, Western; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Histone Deacetylases; Histones; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Nude; Models, Molecular; Neoplasms; Prodrugs; Protein Binding; Protein Structure, Tertiary; Quinolines; Quinolones; Repressor Proteins; RNA Interference; Signal Transduction; Thapsigargin; Tumor Microenvironment; Xenograft Model Antitumor Assays

The family of ADAM (a disintegrin and metalloproteinase) proteins has been implicated in tumor initiation and progression. ADAM17/tumor necrosis factor-α (TNFα)-converting enzyme (TACE) has been initially recognized to release TNFα as well as its receptors (TNFRs) from the membrane. ADAM17, TNFα and TNFR have been found upregulated in cancer patients, although the underlying mechanisms remain largely unknown. As hypoxia is a hallmark of cancer that can lead to severe stress conditions accumulating in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), we investigated the role of these stress conditions in the regulation of ADAM17 and release of TNFR1.We found that severe hypoxia induced ADAM17 expression and activity. Although hypoxia-inducible factor 1α (HIF1α) was important to maintain basal ADAM17 mRNA levels during moderate hypoxia, it was not sufficient to induce ADAM17 levels under severe hypoxia. Instead, we found that ADAM17 induction by severe hypoxia can be mimicked by ER stressors such as Thapsigargin and occurs as a consequence of the activation of the PERK/eIF2α/ATF4 and activating transcription factor 6 (ATF6) arms of UPR in several tumor cell lines. ADAM17 expression was also increased in xenografts displaying ER stress because of treatment with the vascular endothelial growth factor (VEGF) inhibitory antibody Bevacizumab. Additionally, severe hypoxia and ER stress activated ADAM17 and ectodomain shedding of TNFR1 involving mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS). Collectively, these results show that ADAM17 is a novel UPR-regulated gene in response to severe hypoxia and ER stress, which is actively involved in the release of TNFR1 under these conditions. These data provide a novel link between severe hypoxic stress conditions and inflammation in the tumor environment.

Topics: Activating Transcription Factor 4; Activating Transcription Factor 6; ADAM Proteins; ADAM17 Protein; Animals; Antibodies, Monoclonal, Humanized; Bevacizumab; Cell Hypoxia; Cell Line, Tumor; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; MAP Kinase Signaling System; Mice; Mice, SCID; Neoplasms; Reactive Oxygen Species; Receptors, Tumor Necrosis Factor, Type I; Thapsigargin; Unfolded Protein Response

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

Topics: Animals; Antineoplastic Agents; Calcium; Calcium Signaling; Cell Death; Cell Survival; Cell-Penetrating Peptides; CHO Cells; Cricetinae; Cricetulus; Crotalid Venoms; Crotalus; Endoplasmic Reticulum; Flow Cytometry; HEK293 Cells; Humans; Injections, Intraperitoneal; Lysosomes; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Microscopy, Confocal; Neoplasms; Thapsigargin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Src, the canonical member of the non-receptor family of tyrosine kinases, is deregulated in numerous cancers, including colon and breast cancers. In addition to its effects on cell proliferation and motility, Src is often considered as an inhibitor of apoptosis, although this remains controversial. Thus, whether the ability of Src to generate malignancies relies on an intrinsic aptitude to inhibit apoptosis or requires preexistent resistance to apoptosis remains somewhat elusive. Here, using mouse fibroblasts transformed with v-Src as a model, we show that the observed Src-dependent resistance to cell death relies on Src ability to inhibit the mitochondrial pathway of apoptosis by specifically increasing the degradation rate of the BH3-only protein Bik. This effect relies on the activation of the Ras-Raf-Mek1/2-Erk1/2 pathway, and on the phosphorylation of Bik on Thr124, driving Bik ubiquitylation on Lys33 and subsequent degradation by the proteasome. Importantly, in a set of human cancer cells with Src-, Kras- or BRAF-dependent activation of Erk1/2, resistances to staurosporine or thapsigargin were also shown to depend on Bik degradation rate via a similar mechanism. These results suggest that Bik could be a rate-limiting factor for apoptosis induction of tumor cells exhibiting deregulated Erk1/2 signaling, which may provide new opportunities for cancer therapies.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Membrane Proteins; Mice; Mitochondrial Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasms; NIH 3T3 Cells; Oncogene Protein p21(ras); Proteolysis; raf Kinases; src-Family Kinases; Staurosporine; Thapsigargin

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

In contrast to other cytotoxic agents including anthracyclins and oxaliplatin (OXP), cisplatin (CDDP) fails to induce immunogenic tumor cell death that would allow to stimulate an anticancer immune response and hence to amplify its therapeutic efficacy. This failure to induce immunogenic cell death can be attributed to CDDP's incapacity to elicit the translocation of calreticulin (CRT) from the lumen of the endoplasmic reticulum (ER) to the cell surface. Here, we show that, in contrast to OXP, CDDP is unable to activate the protein kinase-like ER kinase (PERK)-dependent phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Accordingly, CDDP also failed to stimulate the formation of stress granules and macroautophagy, two processes that only occur after eIF2α phosphorylation. Using a screening method that monitors the voyage of CRT from the ER lumen to the cell surface, we identified thapsigargin (THAPS), an inhibitor of the sarco/ER Ca(2+)-ATPase as a molecule that on its own does not stimulate CRT exposure, yet endows CDDP with the capacity to do so. The combination of ER stress inducers (such as THAPS or tunicamycin) and CDDP effectively induced the translocation of CRT to the plasma membrane, as well as immunogenic cell death, although ER stress or CDDP alone was insufficient to induce CRT exposure and immunogenic cell death. Altogether, our results underscore the contribution of the ER stress response to the immunogenicity of cell death.

Topics: Antineoplastic Agents; Apoptosis; Calbindin 2; Cell Line, Tumor; Cell Separation; Cisplatin; Endoplasmic Reticulum; Flow Cytometry; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Microscopy, Confocal; Neoplasms; Organoplatinum Compounds; Oxaliplatin; Protein Transport; S100 Calcium Binding Protein G; Stress, Physiological; Thapsigargin

Autocrine motility factor/ phosphoglucose isomerase (AMF/PGI) promotes cell survival by the pAkt survival pathway. Its receptor, gp78/AMFR, is an E3 ubiquitin ligase implicated in endoplasmic reticulum (ER)-associated protein degradation. We demonstrate here that AMF/PGI also protects against thapsigargin (TG)- and tunicamycin (TUN)-induced ER stress and apoptosis. AMF/PGI protection against the ER stress response is receptor mediated as it is not observed in gp78/AMFR-knockdown HEK293 cells. However, AMF/PGI protection against the ER stress response by TG and TUN was mediated only partially through PI3K/Akt activation. AMF/PGI reduction of the elevation of cytosolic calcium in response to either TG or inositol 1,4,5-trisphosphate receptor activation with ATP was gp78/AMFR-dependent, independent of mitochondrial depolarization and not associated with changes in ER calcium content. These results implicate regulation of ER calcium release in AMF/PGI protection against ER stress and apoptosis. Indeed, sequestration of cytosolic calcium with BAPTA-AM limited the ER stress response. Importantly, elevation of cytosolic calcium upon treatment with the calcium ionophore ionomycin, while not inducing an ER stress response, did prevent AMF/PGI protection against ER stress. By regulating ER calcium release, AMF/PGI interaction with gp78/AMFR therefore protects against ER stress identifying novel roles for these cancer-associated proteins in promoting tumor cell survival.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Calcium; Calcium Signaling; Cell Survival; Chlorocebus aethiops; COS Cells; Endoplasmic Reticulum; Enzyme Inhibitors; Glucose-6-Phosphate Isomerase; HEK293 Cells; Humans; Neoplasms; Receptors, Autocrine Motility Factor; Receptors, Cytokine; Thapsigargin; Tunicamycin; Ubiquitin-Protein Ligases; Unfolded Protein Response

Rapid growth of tumor cells coupled with inadequate vascularization leads to shortage of oxygen and nutrients. The unfolded protein response (UPR), a defense cellular mechanism activated during such stress conditions, is a complex process that includes upregulation of the endoplasmic reticulum chaperones, such as glucose-regulated protein 78 (GRP78). Due to its central role in UPR, GRP78 is overexpressed in many cancers; it is implicated in cancer cell survival through supporting of drug- and radioresistance as well as metastatic dissemination, and is generally associated with poor outcome. This is the reason why selective destruction of GRP78 could become a novel anticancer strategy. GRP78 is the only known substrate of the proteolytic A subunit (SubA) of a bacterial AB(5) toxin, and the selective SubA-induced cleavage of GRP78 leads to massive cell death. Targeted delivery of SubA into cancer cells via specific receptor-mediated endocytosis could be a suitable strategy for assaulting tumor cells. We fused SubA to epidermal growth factor (EGF), whose receptor (EGFR) is frequently overexpressed in tumor cells, and demonstrated that the resulting EGF-SubA immunotoxin is an effective killer of EGFR-positive tumor cells. Furthermore, because of its unique mechanism of action, EGF-SubA synergizes with UPR-inducing drugs, which opens a possibility for the development of mechanism-based combination regimens for effective anticancer therapy. In this chapter, we provide experimental protocols for the assessment of the effects of EGF-SubA on EGFR-positive cancer cells, either alone or in combination with UPR-inducing drugs.

Topics: Animals; Antineoplastic Agents; Catechin; Cell Line; Drug Screening Assays, Antitumor; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Heat-Shock Proteins; Humans; Neoplasms; Thapsigargin; Unfolded Protein Response

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

Topics: Crystallography, X-Ray; Drug Delivery Systems; Enzyme Inhibitors; Humans; Hydrophobic and Hydrophilic Interactions; Neoplasms; Protein Binding; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Cadmium; Cell Line, Tumor; Cisplatin; Etoposide; Flow Cytometry; Fluorescent Antibody Technique; Mice; Mitomycin; Mitoxantrone; Neoplasms; Organoplatinum Compounds; Oxaliplatin; Sphingosine; Thapsigargin

Tumor cells deficient in the proapoptotic proteins Bak and Bax are resistant to chemotherapeutic drugs. Here, we demonstrate that murine embryonic fibroblasts deficient for both Bak and Bax are, however, efficiently killed by thapsigargin, a specific inhibitor of ER Ca(2+) pumps that induces ER stress by depleting ER Ca(2+) stores. In the presence of Bak and Bax, thapsigargin eliminates cells by release of mitochondrial cytochrome c and subsequent caspase activation, which leads to the proteolytic inactivation of the molecular necrosis switch PARP-1 and results in apoptosis. By contrast, in the absence of Bak and Bax, a failure to activate caspases results in PARP-1-mediated ATP depletion. The subsequent necrosis is not prevented by autophagy as an alternative energy source. Moreover, in cells deficient for both Bak and Bax, thapsigargin induces permanent mitochondrial damage by Ca(2+) overload, permeability transition and membrane rupture. Thus, even though deficiency in Bak and Bax protects these cells against apoptosis, it does not compromise necrosis induced by SERCA inhibitors. Importantly, thapsigargin induces caspase-independent cell death also in colon and prostate carcinoma cells deficient in Bak and Bax expression. Therefore, targeted application of ER stressors such as thapsigargin might be a promising approach for the treatment of Bak- and Bax-deficient, drug-resistant tumors.

Topics: Animals; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biological Transport; Calcium; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Humans; Mice; Mice, Knockout; Mitochondria; Necrosis; Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the 'ground state', remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation, we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.

Topics: Animals; Cell Survival; Cells, Cultured; Mice; Molecular Structure; Neoplasms; Neurons; Pharmaceutical Preparations; Sensitivity and Specificity; Stem Cells

To establish the effects of Na(+)/Ca(2+) exchanger (NCX) blockers on 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) and (11)C-choline accumulation in different cancer cells.. The tumor cells were incubated with NCX inhibitors, and the uptakes of (18)FDG and (11)C-choline were measured. Flow cytometric measurements of intracellular Ca(2+) and Na(+) concentrations were carried out. The presence of the NCX antigen in the cancer cells was proved by Western blotting, flow cytometry and confocal laser scanning microscopy.. The NCX is expressed at a noteworthy level in the cytosol and on the cytoplasmic membrane of the examined cells. Incubation of the cells with three chemically unrelated NCX blockers (bepridil, KB-R7943 or 3',4'-dichlorobenzamil hydrochloride) resulted in an increase in the intracellular Ca(2+) concentration, with a simultaneous decrease in the intracellular Na(+) concentration. The treatment with the NCX inhibitors increased the energy consumption of the tumor cells by 50-100%. Thapsigargin abolished the NCX-induced (18)FDG accumulation in the cells. The NCX blockers applied decreased the (11)C-choline accumulation of all the investigated cancer cells by 60-80% relative to the control.. A possible masking effect of NCX medication must be taken into consideration during the diagnostic interpretation of PET scans.

Topics: Bepridil; Calcium; Carbon Radioisotopes; Cell Line, Tumor; Choline; Flow Cytometry; Fluorodeoxyglucose F18; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Neoplasms; Positron-Emission Tomography; Radioactive Tracers; Sodium; Sodium-Calcium Exchanger; Thapsigargin

Nonsteroidal anti-inflammatory drugs (NSAID) have shown chemopreventive effects in both preclinical and clinical studies; however, the precise molecular mechanism governing this response remains unclear. We used DNA microarray techniques to search for genes whose expression is induced by the NSAID indomethacin in human gastric carcinoma (AGS) cells. Among identified genes, we focused on those related to tight junction function (claudin-4, claudin-1, and occludin), particularly claudin-4. Induction of claudin-4 by indomethacin was confirmed at both mRNA and protein levels. NSAIDs, other than indomethacin (diclofenac and celecoxib), also induced claudin-4. All of the tested NSAIDs increased the intracellular Ca2+ concentration. Other drugs that increased the intracellular Ca2+ concentration (thapsigargin and ionomycin) also induced claudin-4. Furthermore, an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] inhibited the indomethacin-dependent induction of claudin-4. These results strongly suggest that induction of claudin-4 by indomethacin is mediated through an increase in the intracellular Ca2+ concentration. Overexpression of claudin-4 in AGS cells did not affect cell growth or the induction of apoptosis by indomethacin. On the other hand, addition of indomethacin or overexpression of claudin-4 inhibited cell migration. Colony formation in soft agar was also inhibited. Suppression of claudin-4 expression by small interfering RNA restored the migration activity of AGS cells in the presence of indomethacin. Based on these results, we consider that the induction of claudin-4 and other tight junction-related genes by NSAIDs may be involved in the chemopreventive effect of NSAIDs through the suppression of anchorage-independent growth and cell migration.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Biomarkers, Tumor; Calcium; Cell Movement; Chemoprevention; Claudin-4; Colony-Forming Units Assay; Enzyme Inhibitors; Gene Expression Profiling; Humans; Indomethacin; Ionomycin; Ionophores; Membrane Proteins; Neoplasms; Oligonucleotide Array Sequence Analysis; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Tight Junctions; Tumor Cells, Cultured

We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4, 5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.

Topics: Caffeine; Calcium; Culture Media; Endothelium; Humans; Inositol 1,4,5-Trisphosphate; Neoplasms; Ryanodine; Thapsigargin; Tumor Cells, Cultured; Uridine Triphosphate