ABSTRACT
We have developed a chimeric antigen receptor (CAR) against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in prostate cancer, Ewing sarcoma, and other malignancies. In the present study, we investigated the effect of substituting costimulatory domains and spacers in this STEAP1 CAR. We cloned four CAR constructs with either CD28 or 4-1BB costimulatory domains, combined with a CD8a-spacer (sp) or a mutated IgG-spacer. The CAR T-cells were evaluated in short- and long-term in vitro T-cell assays, measuring cytokine production, tumor cell killing, and CAR T-cell expansion and phenotype. A xenograft mouse model of prostate cancer was used for in vivo comparison. All four CAR constructs conferred CD4+ and CD8+ T cells with STEAP1-specific functionality. A CD8sp_41BBz construct and an IgGsp_CD28z construct were selected for a more extensive comparison. The IgGsp_CD28z CAR gave stronger cytokine responses and killing in overnight caspase assays. However, the 41BB-containing CAR mediated more killing (IncuCyte) over one week. Upon six repeated stimulations, the CD8sp_41BBz CAR T cells showed superior expansion and lower expression of exhaustion markers (PD1, LAG3, TIGIT, TIM3, and CD25). In vivo, both the CAR T variants had comparable anti-tumor activity, but persisting CAR T-cells in tumors were only detected for the 41BBz variant. In conclusion, the CD8sp_41BBz STEAP1 CAR T cells had superior expansion and survival in vitro and in vivo, compared to the IgGsp_CD28z counterpart, and a less exhausted phenotype upon repeated antigen exposure. Such persistence may be important for clinical efficacy.
Subject(s)
Prostatic Neoplasms , Receptors, Chimeric Antigen , Animals , Humans , Male , Mice , Antigens, Neoplasm/genetics , CD8-Positive T-Lymphocytes , Cytokines , Disease Models, Animal , Oxidoreductases , Prostate , Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Receptors, Chimeric Antigen/geneticsABSTRACT
In the field of sensing, finding high-performance amine molecular sensors has always been a challenging topic. Here, two highly stable 3D MOFs DUT-67(Hf) and DUT-67(Zr) with large specific surface areas and hierarchical pore structures were conveniently synthesized by solvothermal reaction of ZrCl4/HfCl4 with a simple organic ligand, 2,5-thiophene dicarboxylic acid (H2TDC) according to literature approach. By analyzing TGA data, it was found that the two MOFs have defects (unsaturated metal sites) that can interact with substrates (H2O and volatile amine gas), which is conducive to proton transfer and amine compound identification. Further experiments showed that at 100 °C and 98% relative humidity (RH), the optimized proton conductivities of DUT-67(Zr) and DUT-67(Hf) can reach the high values of 2.98 × 10-3 and 3.86 × 10-3 S cm-1, respectively. Moreover, the room temperature sensing characteristics of MOFs' to amine gases were evaluated at 68, 85 and 98% RHs, respectively. Impressively, the prepared MOFs-based sensors have the desired stability and higher sensitivity to amines. Under 68% RH, the detection limits of DUT-67(Zr) or DUT-67(Hf) for volatile amine gases were 0.5 (methylamine), 0.5 (dimethylamine) and 1 ppm (trimethylamine), and 0.5 (methylamine), 0.5 (dimethylamine) and 0.5 ppm (trimethylamine), respectively. As far as we know, this is the best performance of ammonia room temperature sensors in the past proton-conductive MOF sensors.
ABSTRACT
BACKGROUND: Radiotherapy-induced oral mucositis (RIOM) and chemotherapy-induced oral mucositis (CIOM) are common complications in cancer patients, leading to negative clinical manifestations, reduced quality of life, and unsatisfactory treatment outcomes. OBJECTIVE: The present study aimed to identify potential molecular mechanisms and candidate drugs by data mining. METHODS: We obtained a preliminary list of genes associated with RIOM and CIOM. In-depth information on these genes was explored by functional and enrichment analyses. Then, the drug-gene interaction database was used to determine the interaction of the final enriched gene list with known drugs and analyze the drug candidates. RESULTS AND CONCLUSION: This study identified 21 hub genes that may play an important role in RIOM and CIOM, respectively. Through our data mining, bioinformatics survey, and candidate drug selection, TNF, IL-6, and TLR9 could play an important role in disease progression and treatment. In addition, eight candidate drugs (olokizumab, chloroquine, hydroxychloroquine, adalimumab, etanercept, golimumab, infliximab, and thalidomide) were selected by the drug-gene interaction literature search additionally, as candidates for treating RIOM and CIOM.
Subject(s)
Antineoplastic Agents , Mucositis , Neoplasms , Stomatitis , Humans , Mucositis/chemically induced , Quality of Life , Stomatitis/chemically induced , Stomatitis/drug therapy , Neoplasms/drug therapy , Antineoplastic Agents/adverse effectsABSTRACT
Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/ß-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.
Subject(s)
Heart/embryology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , cdc42 GTP-Binding Protein/metabolism , Animals , Cadherins/metabolism , Cell Adhesion , Cell Communication , Cell Membrane/metabolism , Cell Proliferation , Cells, Cultured , Embryo Loss/pathology , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Gene Deletion , Gene Expression Regulation, Developmental , Heart Septal Defects, Ventricular/embryology , Heart Septal Defects, Ventricular/pathology , Mice, Knockout , Myocytes, Cardiac/ultrastructure , Organ Specificity , Protein Transport , beta Catenin/metabolism , cdc42 GTP-Binding Protein/geneticsABSTRACT
In this study, we interestingly found that peroxydisulfate (PDS) could be activated by a commercial multiwalled carbon nanotube (CNT) material via a nonradical pathway. Iodide (I-) was quickly and almost completely oxidized to hypoiodous acid (HOI) in the PDS/CNT system over the pH range of 5-9, but the further transformation to iodate (IO3-) was negligible. A kinetic model was proposed, which involved the formation of reactive PDS-CNT complexes, and then their decomposition into sulfate anion (SO42-) via inner electron transfer within the complexes or by competitively reacting with I-. Several influencing factors (e.g., PDS and CNT dosages, and solution pH) on I- oxidation kinetics by this system were evaluated. Humic acid (HA) decreased the oxidation kinetics of I-, probably resulting from its inhibitory effect on the interaction between PDS and CNT to form the reactive complexes. Moreover, adsordable organic iodine compounds (AOI) as well as specific iodoform and iodoacetic acid were appreciably produced in the PDS/CNT/I- system with HA. These results demonstrate the potential risk of producing toxic iodinated organic compounds in the novel PDS/CNT oxidation process developed very recently, which should be taken into consideration before its practical application in water treatment.
Subject(s)
Iodides/chemistry , Nanotubes, Carbon , Iodates/chemistry , Oxidation-Reduction , Water Pollutants, Chemical , Water PurificationABSTRACT
Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca(2+) mobilization by releasing Ca(2+) from the endoplasmic reticulum and eliciting Ca(2+) entry across the plasma membrane. Herein, we show that histamine-evoked Ca(2+) entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca(2+) release-activated Ca(2+) (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca(2+) entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca(2+) influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca(2+) mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.
Subject(s)
Calcium Channels/physiology , Human Umbilical Vein Endothelial Cells/metabolism , Membrane Proteins/physiology , NFATC Transcription Factors/physiology , Neoplasm Proteins/physiology , Calcium/metabolism , Gene Silencing , Histamine/chemistry , Humans , Inflammation , Interleukin-8/metabolism , Interleukins/metabolism , ORAI1 Protein , ORAI2 Protein , RNA Interference , Signal Transduction , Stromal Interaction Molecule 1ABSTRACT
Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnß1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnß1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnß1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.
Subject(s)
Active Transport, Cell Nucleus , Endoplasmic Reticulum/metabolism , Fibroblast Growth Factor 1/metabolism , Membrane Proteins/physiology , alpha Karyopherins/metabolism , beta Karyopherins/metabolism , Biological Transport , Cell Nucleus/metabolism , Cytosol/metabolism , Humans , Nuclear Envelope/metabolism , Nuclear Localization Signals , Phosphorylation , Protein Kinase C-delta/metabolism , RNA, Small Interfering/metabolism , Subcellular Fractions/metabolism , ran GTP-Binding Protein/metabolismABSTRACT
Neural crest cells (NCCs) are physically responsible for craniofacial skeleton formation, pharyngeal arch artery remodeling and cardiac outflow tract septation during vertebrate development. Cdc42 (cell division cycle 42) is a Rho family small GTP-binding protein that works as a molecular switch to regulate cytoskeleton remodeling and the establishment of cell polarity. To investigate the role of Cdc42 in NCCs during embryonic development, we deleted Cdc42 in NCCs by crossing Cdc42 flox mice with Wnt1-cre mice. We found that the inactivation of Cdc42 in NCCs caused embryonic lethality with craniofacial deformities and cardiovascular developmental defects. Specifically, Cdc42 NCC knockout embryos showed fully penetrant cleft lips and short snouts. Alcian Blue and Alizarin Red staining of the cranium exhibited an unfused nasal capsule and palatine in the mutant embryos. India ink intracardiac injection analysis displayed a spectrum of cardiovascular developmental defects, including persistent truncus arteriosus, hypomorphic pulmonary arteries, interrupted aortic arches, and right-sided aortic arches. To explore the underlying mechanisms of Cdc42 in the formation of the great blood vessels, we generated Wnt1Cre-Cdc42-Rosa26 reporter mice. By beta-galactosidase staining, a subpopulation of Cdc42-null NCCs was observed halting in their migration midway from the pharyngeal arches to the conotruncal cushions. Phalloidin staining revealed dispersed, shorter and disoriented stress fibers in Cdc42-null NCCs. Finally, we demonstrated that the inactivation of Cdc42 in NCCs impaired bone morphogenetic protein 2 (BMP2)-induced NCC cytoskeleton remodeling and migration. In summary, our results demonstrate that Cdc42 plays an essential role in NCC migration, and inactivation of Cdc42 in NCCs impairs craniofacial and cardiovascular development in mice.
Subject(s)
Cardiovascular Abnormalities/embryology , Cardiovascular Abnormalities/enzymology , Craniofacial Abnormalities/embryology , Craniofacial Abnormalities/enzymology , Morphogenesis , Neural Crest/pathology , cdc42 GTP-Binding Protein/metabolism , Actins/metabolism , Animals , Bone Morphogenetic Protein 2/pharmacology , Cardiovascular Abnormalities/pathology , Cell Differentiation/drug effects , Cell Movement/drug effects , Craniofacial Abnormalities/pathology , Crosses, Genetic , Cytoskeleton/metabolism , Embryo, Mammalian/abnormalities , Embryo, Mammalian/drug effects , Embryo, Mammalian/pathology , Enzyme Activation/drug effects , Female , Gene Deletion , Genotype , Male , Mice , Mice, Knockout , Morphogenesis/drug effects , Neural Crest/drug effects , Neural Crest/enzymology , Osteogenesis/drug effects , Phenotype , Pseudopodia/drug effects , Pseudopodia/metabolism , Thymus Gland/abnormalities , Thymus Gland/drug effects , Thymus Gland/pathologyABSTRACT
Indirect pulp therapy (IPT) is a common conservative treatment for deep dental caries. However, the potential risk factors for the prognosis of IPT have not been well studied. This study retrospectively investigated the success rate of IPT in treating primary molars with deep caries and the factors potentially affecting the two-year success rate. A total of 303 primary molars in 202 children (106 boys and 96 girls) were included in this study. These primary molars were identified as having deep caries by clinical and radiographic examinations and were treated with IPT. The factors potentially affecting the IPT success rate were analyzed after two years of follow-up. The results indicated that the two-year IPT success rate was 86% (262/303). The success rate of primary molars with and without stainless steel crowns was 96% (120/125) and 80% (142/178), respectively. Primary molars treated with stainless steel crowns showed a significantly lower risk of failure (hazard ratio (HR) = 0.18, 95% confidence interval (CI): (0.10, 0.34), p = 0.01). There were no significant differences in other factors, including gender (male vs. female), age (preschool vs. school age), cooperation level (Frankl 2 vs. 3 or 4 scales), arch type (maxillary vs. mandibular), tooth type (first vs. second primary molar), or pulp capping material (calcium hydroxide vs. glass ionomer cement). IPT is an effective, conservative treatment modality for primary molars with deep caries. Stainless steel crowns could significantly improve the IPT success rate.
Subject(s)
Crowns , Dental Caries , Molar , Tooth, Deciduous , Humans , Male , Retrospective Studies , Female , Dental Caries/therapy , Child, Preschool , Child , Stainless Steel , Treatment Outcome , Dental Pulp Capping/methods , Risk Factors , Follow-Up StudiesABSTRACT
The elimination of antimony pollution has attracted increasing concerns because of its high toxicity to human health and the natural environment. In this work, biomimetic δ-MnO2 was synthesized by using waste tobacco stem-silks as biotemplate (Bio-δ-MnO2) and used in the capture of Sb(III)from aqueous solution. The tobacco stem-silks not only provided unique wrinkled morphologies but also contained carbon element self-doped into the resulting samples. The maximum Sb(III) adsorption capacity reached 763.4 mgâg -1, which is 2.06 times higher than δ-MnO2 without template (370.0 mgâg -1), 4.53 times than tobacco stem-silks carbon (168.5 mgâg -1), and 10.39 times than commercial MnO2 (73.5 mgâg -1), respectively. The isotherm and kinetic studies indicated that the adsorption behavior was consistent with the Langmuir isotherm model and the pseudo-second-order kinetic equation. As far as we are aware, the adsorption capacity of Bio-δ-MnO2 is much higher than that of most Sb(III) adsorbents. FT-IR, XPS, SEM, XRD, and Zeta potential analyses showed that the main mechanism for the adsorption of Sb(III) by Bio-δ-MnO2 includes electrostatic attraction, surface complexation, and redox. Overall, this study provides a new sustainable way to convert agricultural wastes to more valuable products such as biomimetic adsorbent for Sb(III) removal in addition to conventional activated carbon and biochar.
Subject(s)
Oxides , Water Pollutants, Chemical , Humans , Kinetics , Manganese Compounds , Spectroscopy, Fourier Transform Infrared , Water Pollutants, Chemical/analysis , AdsorptionABSTRACT
Therapy employing T cells modified with chimeric antigen receptors (CARs) is effective in hematological malignancies but not yet in solid cancers. CAR T cell activity in solid tumors is limited by immunosuppressive factors, including transforming growth factor ß (TGFß). Here, we describe the development of a switch receptor (SwR), in which the extracellular domains of the TGFß receptor are fused to the intracellular domains from the IL-2/15 receptor. We evaluated the SwR in tandem with two variants of a CAR that we have developed against STEAP1, a protein highly expressed in prostate cancer. The SwR-CAR T cell activity was assessed against a panel of STEAP1+/- prostate cancer cell lines with or without over-expression of TGFß, or with added TGFß, by use of flow cytometry cytokine and killing assays, Luminex cytokine profiling, cell counts, and flow cytometry phenotyping. The results showed that the SwR-CAR constructs improved the functionality of CAR T cells in TGFß-rich environments, as measured by T cell proliferation and survival, cytokine response, and cytotoxicity. In assays with four repeated target-cell stimulations, the SwR-CAR T cells developed an activated effector memory phenotype with retained STEAP1-specific activity. In conclusion, the SwR confers CAR T cells with potent and durable in vitro functionality in TGFß-rich environments. The SwR may be used as an add-on construct for CAR T cells or other forms of adoptive cell therapy.
ABSTRACT
Fibroblast growth factor 1 (FGF1) has the property to become translocated from the extracellular space into the cell cytosol and nucleus. Membrane translocation of FGF1 occurs subsequent to endocytic uptake and is strictly FGF-receptor (FGFR) dependent. Here we have investigated the timing of FGF1 translocation in relation to FGFR1 signalling. We found that the translocation of FGF1 is a periodic event that occurs with 24h intervals. Serum-starved cells translocated the growth factor with peak occurrences ~6 h, ~30 h, and ~54 h after the addition of FGF1. The periodic FGF1 translocation was totally independent of the FGFR1 tyrosine kinase activity as it proceeded unchanged when the kinase activity was chemically inhibited or the kinase domain was deleted. Furthermore, FGF1 translocation was not restricted to a particular phase of the cell cycle or dependent on cell cycle progression. The results demonstrate that the FGF1/FGFR1 complex constitutes a signalling module that independently of the receptor tyrosine kinase can convey a signal that initiates a strictly timed and periodic release of endocytosed FGF1 into the cytosol/nucleus.
Subject(s)
Active Transport, Cell Nucleus/physiology , Cell Nucleus/metabolism , Cytosol/metabolism , Fibroblast Growth Factor 1/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , COS Cells , Chlorocebus aethiops , Fibroblast Growth Factor 1/genetics , Mice , NIH 3T3 Cells , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Signal Transduction/physiologyABSTRACT
Staphylococcus epidermidis (S. epidermidis), a human commensal, has been implicated in invasive infection in humans due to their ability to form biofilm. It is assumed that when a biofilm is dispersed it will subsequently cause a more severe infection. The clinical significance of S. epidermidis isolated from sterile body fluid (BF) remains unclear, and might be related to dispersal from catheter-associated biofilm infection. To evaluate this relationship, we evaluated S. epidermidis isolates from catheters (CA) or BF in hospitalized patients. Sequence type 2 (ST2) is the most prevalent type isolated from infection sites. Although the specific STs were also observed in isolates from different sites, we observed that the main sequence type was ST2, followed by ST59, among all the 114 isolates from different infection sites. Interestingly, ST2 strains isolated from BF exhibited significantly thicker biofilm than those from CA. The thicker biofilm was due to the higher expression of accumulation-associated protein (aap) but not intercellular adhesion (ica) operon. Moreover, the transcription of PSMδ and PSMε were significantly increased in ST2 strains isolated from BF. Although the bacterial loads on catheters were similar infected by CA- or BF-originated strains in mouse biofilm-associated infection model, we observed a higher CFU in peri-catheter tissues infected by ST2 clones isolated from BF, suggesting that S. epidermidis with thicker biofilm formation might be able to disperse. Taken together, our data suggested that S. epidermidis originated from diverse infection sites exhibited different biofilm forming capacity. The major ST2 clone isolated from BF exhibited thicker biofilm by increasing the expression of Aap. The higher expression of PSM of these strains may contribute to bacteria dispersal from biofilm and the following bacterial spread.
ABSTRACT
Chimeric antigen receptors (CARs) that retarget T cells against CD19 show clinical efficacy against B cell malignancies. Here, we describe the development of a CAR against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in â¼90% of prostate cancers, and subgroups of other malignancies. STEAP1 is an attractive target, as it is associated with tumor invasiveness and progression and only expressed at low levels in normal tissues, apart from the non-vital prostate gland. We identified the antibody coding sequences from a hybridoma and designed a CAR that is efficiently expressed in primary T cells. The T cells acquired the desired anti-STEAP1 specificity, with a polyfunctional response including production of multiple cytokines, proliferation, and the killing of cancer cells. The response was observed for both CD4+ and CD8+ T cells, and against all STEAP1+ target cell lines tested. We evaluated the in vivo CAR T activity in both subcutaneous and metastatic xenograft mouse models of prostate cancer. Here, the CAR T cells infiltrated tumors and significantly inhibited tumor growth and extended survival in a STEAP1-dependent manner. We conclude that the STEAP1 CAR exhibits potent in vitro and in vivo functionality and can be further developed toward potential clinical use.
ABSTRACT
One-carbon (1C) metabolism has a key role in metabolic programming with both mitochondrial (m1C) and cytoplasmic (c1C) components. Here we show that activating transcription factor 4 (ATF4) exclusively activates gene expression involved in m1C, but not the c1C cycle in prostate cancer cells. This includes activation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression, the central player in the m1C cycle. Consistent with the key role of m1C cycle in prostate cancer, MTHFD2 knockdown inhibited prostate cancer cell growth, prostatosphere formation, and growth of patient-derived xenograft organoids. In addition, therapeutic silencing of MTHFD2 by systemically administered nanoliposomal siRNA profoundly inhibited tumor growth in preclinical prostate cancer mouse models. Consistently, MTHFD2 expression is significantly increased in human prostate cancer, and a gene expression signature based on the m1C cycle has significant prognostic value. Furthermore, MTHFD2 expression is coordinately regulated by ATF4 and the oncoprotein c-MYC, which has been implicated in prostate cancer. These data suggest that the m1C cycle is essential for prostate cancer progression and may serve as a novel biomarker and therapeutic target. SIGNIFICANCE: These findings demonstrate that the mitochondrial, but not cytoplasmic, one-carbon cycle has a key role in prostate cancer cell growth and survival and may serve as a biomarker and/or therapeutic target.
Subject(s)
Carbon Cycle/genetics , Prostatic Neoplasms/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Disease Progression , Humans , Male , Mice , Mice, NudeABSTRACT
Cancer cells exploit many of the cellular adaptive responses to support their survival needs. One such critical pathway in eukaryotic cells is the unfolded protein response (UPR) that is important in normal physiology as well as disease states, including cancer. Since UPR can serve as a lever between survival and death, regulated control of its activity is critical for tumor formation and growth although the underlying mechanisms are poorly understood. Here we show that one of the main transcriptional effectors of UPR, activating transcription factor 4 (ATF4), is essential for prostate cancer (PCa) growth and survival. Using systemic unbiased gene expression and proteomic analyses, we identified a novel direct ATF4 target gene, family with sequence similarity 129 member A (FAM129A), which is critical in mediating ATF4 effects on prostate tumorigenesis. Interestingly, FAM129A regulated both PERK and eIF2α in a feedback loop that differentially channeled the UPR output. ATF4 and FAM129A protein expression is increased in patient PCa samples compared with benign prostate. Importantly, in vivo therapeutic silencing of ATF4-FAM129A axis profoundly inhibited tumor growth in a preclinical PCa model. These data support that one of the canonical UPR branches, through ATF4 and its target gene FAM129A, is required for PCa growth and thus may serve as a novel therapeutic target.
Subject(s)
Activating Transcription Factor 4/physiology , Biomarkers, Tumor/physiology , Neoplasm Proteins/physiology , Prostatic Neoplasms/metabolism , Unfolded Protein Response/genetics , Animals , Cell Proliferation/genetics , Endoplasmic Reticulum Stress/genetics , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Male , Mice , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Signal Transduction/genetics , Tumor Cells, CulturedABSTRACT
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid mediators, which are known to play major roles in allergic reactions as well as in tumor pathogenesis. Here, the biological activities and signal pathways of these lysophospholipids (LPLs) in dendritic cells (DCs) were characterized further. Flow cytometric and immunoblot analyses indicate that immature as well as mature DCs express the LPL receptors S1P1, S1P3, S1P5, and LPA2, but not S1P2, S1P4, LPA1, or LPA3. Moreover, enzyme-linked immunosorbent assay experiments demonstrate that simultaneous addition of these LPLs to immature DCs in the presence of lipopolysaccharide enhanced the secretion of the inflammatory cytokines interleukin (IL)-6 and IL-8 in maturing DCs. In contrast, no modification of IL-6 or IL-8 release was observed after exposure of mature DCs to LPLs alone. In addition, studies with pertussis toxin and mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 suggested that Gi proteins and MAPK pathway are involved in these LPL-induced cell responses. Corroborating these findings, we observed that LPLs induce the phosphorylation of extracellular signal-regulated kinase 1/2 in immature DCs but not in mature DCs. Further analyses show that inhibitors of phosholipase D, Rho, and protein kinase C also inhibited the LPL-induced release of IL-6 and IL-8. Therefore, our findings suggest that lipopolysaccharide in DCs uncouples LPL receptors from the signal-transducing machinery during maturation and that exposure of LPLs at early time-points to maturing DCs modifies the proinflammatory capacity of mature DCs.
Subject(s)
Dendritic Cells/physiology , Interleukin-6/blood , Interleukin-8/blood , Lysophospholipids/pharmacology , Cells, Cultured , Dendritic Cells/metabolism , Dose-Response Relationship, Drug , GTP-Binding Proteins/physiology , Humans , Mitogen-Activated Protein Kinase Kinases/physiology , Protein Kinase C/physiology , Receptor Cross-Talk , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Time Factors , rho GTP-Binding Proteins/physiologyABSTRACT
The transformation efficiency and products of an odorous compound 2,4,6-trichloroanisole (TCA) at the wavelength of 254 nm in the presence of persulfate were investigated for the first time. The effects of water matrix (i.e., natural organic matter (NOM), pH, carbonate/bicarbonate (HCO3(-)/CO3(2-)), and chloride ions (Cl(-))) were evaluated. The second order rate constant of TCA reacting with sulfate radical (SO4(-)) was determined to be (3.72 ± 0.10) × 10(9) M(-1) s(-1). Increasing dosage of persulfate increased the observed pseudo-first-order rate constant for TCA degradation (kobs), and the contribution of SO4(-) to TCA degradation was much higher than that of HO at each experimental condition. Degradation rate of TCA decreased with pH increasing from 4.0 to 9.0, which could be explained by the lower radical scavenging effect of dihydrogen phosphate than hydrogen phosphate in acidic condition (pH < 6). NOM significantly decreased kobs due to the effects of radical scavenging and UV absorption with the former one being dominant. kobs decreased from 2.32 × 10(-3) s(-1) to 0.92 × 10(-3) s(-1) with the CO3(2-)/HCO3(-) concentration increased from 0.5 mM to 10 mM in the UV/persulfate process, while kobs slightly decreased from 2.54 × 10(-3) s(-1) in the absence of Cl(-) to 2.10 × 10(-3) s(-1) in the presence of 10 mM Cl(-). Most of these kinetic results could be described by a steady-state kinetic model. Furthermore, liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry at powerful precursor ion scan approach was used to selectively detect oxidation products of TCA. It was found that 2,4,6-trichorophenol (TCP) was the major oxidation product (i.e., the initial yield of TCP was above 90%). The second order rate constant between TCP and SO4(-) was estimated to be (4.16 ± 0.20) × 10(9) M(-1) s(-1). In addition, three products (i.e., 2,6-dichloro-1,4-benzoquinone and two aromatic ring-opening products) were detected in the reaction of TCP with SO4(-), which also appeared in the oxidation of TCA in the UV/persulfate process. A tentative pathway was proposed, where the initial one-electron oxidation of TCA by SO4(-) and further reactions (e.g., ipso-hydroxylation and aromatic ring-cleavage) of the formed cation intermediate TCA were involved.
Subject(s)
Sulfates/chemistry , Water Pollutants, Chemical/chemistry , Benzoquinones , Carbonates , Hydrogen-Ion Concentration , Kinetics , Oxidation-ReductionABSTRACT
We demonstrate the expression of S1P(1,3,4,5) the receptors for sphingosine 1-phosphate (S1P), and LPA(1,2,3) the receptors for lysophosphatidic acid (LPA) in T helper 1 (Th1) and T helper 2 (Th2) cells. S1P and LPA induce the chemotaxis of Th1 and Th2 cells, an activity that is resistant to pertussis toxin (PTX) pretreatment in Th1, but is sensitive in Th2 cells. Also, I-TAC-induced Th1 and eotaxin-induced Th2 cell chemotaxis are blocked by PTX pretreatment. LPA but not S1P induces calcium flux response in Th1 and Th2 cells, which is due to the influx of extracellular calcium and is mediated by receptor activation, since EGTA and suramin (SUR) completely abrogate LPA-induced the release of calcium. No cross-desensitization is observed between thapsigargin (TG) and LPA in both cell types. PTX and SUR but not EGTA inhibit I-TAC- or eotaxin-induced [Ca(2+)](i) release in Th1 and Th2 cells. Our results indicate that lysophospholipids and chemokines stimulate different signal transduction pathways.
Subject(s)
Chemokines/pharmacology , Lysophospholipids/pharmacology , Signal Transduction/drug effects , Th1 Cells/drug effects , Th2 Cells/drug effects , Calcium/metabolism , Cell Line , Chemokine CCL11 , Chemokine CXCL11 , Chemokines, CC/pharmacology , Chemokines, CXC/pharmacology , Chemotaxis/drug effects , Humans , Receptors, Lysophosphatidic Acid/metabolism , Receptors, Lysosphingolipid/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
Platelet factor four (PF4), an inhibitor of endothelial cell proliferation in vitro, inhibits angiogenesis and tumor growth in vivo in experimental animals. The present study was designed to determine whether gene therapy-mediated expression of a form of PF4 lacking 16 amino acids of N-terminus from tumor cells could inhibit angiogenesis and tumor growth in vivo. Two replication-defective recombinant retroviral vectors were constructed. One encodes human PF4 (rRV-PF4) and the other encodes the N-truncated peptide (rRVp17-70). These vectors were then used to transduce KB cells, a human head and neck squamous carcinoma cell line. Expression of PF4 and p17-70 transgenes was confirmed by Western blot analysis. In vitro, both rRV-PF4 and rRVp17-70 were able to inhibit selectively the proliferation of human umbilical vascular endothelial cells (HUVEC) but not KB cells. In vivo activity was assessed by injecting 10(7) KB cells subcutaneously into nude mice and by monitoring subsequent tumor growth, xenograft vascular histochemistry, and animal survival. Viral vector-mediated cDNA transfer of PF4 and p17-70 resulted in inhibiting solid tumors through an anti-angiogenic action in vivo. Our data indicate that targeting tumor angiogenesis using viral-mediated gene transfer of full-length and N-terminal truncated PF4 represents a promising strategy for cancer gene therapy.