NK-derived exosome miR-1249-3p inhibits Mycobacterium tuberculosis survival in macrophages by targeting SKOR1.

Murine Natural Killer cells were cultivated in vitro to isolate NK-derived exosomes. Subsequent quantification via qPCR confirmed enrichment of miR-1249-3p. Ana-1 murine macrophages were cultured in vitro and subsequently inoculated with Mycobacterium tuberculosis (MTB) strain H37Rv. NK-exo and NK-exo miR-1249-3p were separately applied to the infection model, followed by immunological assays conducted post-48-hour co-culture. Western blot analyses corroborated that NK-exo exhibited exosomal marker proteins Granzyme A (GzmA), Granzyme B (GzmB), and Perforin (PFN), alongside a notable enrichment of miR-1249-3p. Functionally, NK-exo augmented the expression levels of Caspase-9,-8, and -3, as well as PARP, while attenuating the expression of NLRP3, ASC, and Cleaved-Caspase-1. Furthermore, qPCR demonstrated an up-regulation of Caspase-9, -8, and -3, along with pro-apoptotic factors Bax and Bid, and a concomitant down-regulation of the anti-apoptotic factor Bcl-2. The expression levels of inflammatory markers ASC, NLRP3, Cleaved-Caspase-1, and IL-1ß were concomitantly decreased. ELISA findings indicated diminished levels of TNF-α and ROS secretion. NK-exo miR-1249-3p specifically targeted and attenuated the expression of SKOR-1, engendering up-regulation of apoptosis-associated proteins and down-regulation of inflammation-related proteins, consequently affecting cellular fate.Our empirical evidence substantiates that NK-exo induces macrophage apoptosis, thereby mitigating MTB survival. Furthermore, NK-exo miR-1249-3p directly targets and inhibits SKOR-1 expression, leading to macrophage apoptosis and consequently hampering the proliferation of MTB. The data implicate the potential therapeutic relevance of NK-exo and miR-1249-3p in managing drug-resistant tuberculosis.

Feasibility of [18F]FSPG PET for Early Response Assessment to Combined Blockade of EGFR and Glutamine Metabolism in Wild-Type KRAS Colorectal Cancer.

Early response assessment is critical for personalizing cancer therapy. Emerging therapeutic regimens with encouraging results in the wild-type (WT) KRAS colorectal cancer (CRC) setting include inhibitors of epidermal growth factor receptor (EGFR) and glutaminolysis. Towards predicting clinical outcome, this preclinical study evaluated non-invasive positron emission tomography (PET) with (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG) in treatment-sensitive and treatment-resistant WT KRAS CRC patient-derived xenografts (PDXs). Tumor-bearing mice were imaged with [18F]FSPG PET before and one week following the initiation of treatment with either EGFR-targeted monoclonal antibody (mAb) therapy, glutaminase inhibitor therapy, or the combination. Imaging was correlated with tumor volume and histology. In PDX that responded to therapy, [18F]FSPG PET was significantly decreased from baseline at 1-week post-therapy, prior to changes in tumor volume. In contrast, [18F]FSPG PET was not decreased in non-responding PDX. These data suggest that [18F]FSPG PET may serve as an early metric of response to EGFR and glutaminase inhibition in the WT KRAS CRC setting.

The altered immunological status of children conceived by assisted reproductive technology.

BACKGROUND: With the increased use of assisted reproductive technology (ART), assessing the potential health risks of children conceived on ART important to public health. Most research in this area has focused on the effects of ART on perinatal, metabolic, and oncological risks in children. Although an increased risk of immune-related diseases has been reported in children born after ART, there are no studies on the immunological status of these children. This study aimed to evaluate the impact of different embryo transfer methods and fertilization strategies on the immune status of the offspring. METHODS: A total of 69 children born to women treated with ART and a matched control group of 17 naturally conceived (NC) children, all aged from 3 to 6 years, were recruited in the reproductive hospital affiliated to Shandong University. The frequency of immune cells in the peripheral blood was assayed using flow cytometry; plasma cytokine levels were determined by multiplex cytokine immunoassay with human cytokine magnetic beads. RESULTS: Compared to children born after natural conception, children born after ART had elevated interferon-γ (IFN-γ) levels, regardless of embryo transfer and fertilization strategies. Children in the fresh-embryo transfer group had significantly higher IL-4 levels and a lower ratio of IFN-γ to IL-4 than those in the NC group ((P = 0.004, 10.41 ± 5.76 pg/mL vs 18.40 ± 7.01 pg/mL, P = 0.023, 1.00 ± 0.48 vs 0.67 ± 0.32, respectively). Similar results were shown in either the in vitro fertilization (IVF) group or the intra-cytoplasmic sperm injection (ICSI) group (P < 0.05 and P = 0.08 for IVF; P < 0.05 and P < 0.05 for ICSI, respectively). These alterations in IL-4 concentrations and the ratio of IFN-γ to IL-4 were statistically significantly correlated with supra-physical E2 (estradiol) levels on the day of hCG administration (R = 0.502, P = 0.017; R = - 0.537, P = 0.010, respectively). Consistently, the frozen embryo transfer did not result in alterations of these immune indicators in the offspring. Overall, there were no significant differences between the ART group and NC group in the frequencies of T cells, B cells, natural killer (NK) cells, CD4+T cells, CD8+T cells, T helper (TH)1 cells, TH17 cells, and regulatory T (Treg) cells and cytokine levels of IL-10 and IL-17a (all P > 0.05). CONCLUSIONS: Immunological alterations existed in children born after the use of ART. The elevated E2 levels before embryo implantation contributed to the increased IL-4 levels in children conceived by fresh embryo transfer. The assessment of immunological alteration is of importance to children conceived by ART for early monitoring and intervention.

Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer.

Improving response to epidermal growth factor receptor (EGFR)-targeted therapies in patients with advanced wild-type (WT) RAS colorectal cancer (CRC) remains an unmet need. In this preclinical work, we evaluated a new therapeutic combination aimed at enhancing efficacy by targeting cancer cell metabolism in concert with EGFR. We hypothesized that combined blockade of glutamine metabolism and EGFR represents a promising treatment approach by targeting both the "fuel" and "signaling" components that these tumors need to survive. To explore this hypothesis, we combined CB-839, an inhibitor of glutaminase 1 (GLS1), the mitochondrial enzyme responsible for catalyzing conversion of glutamine to glutamate, with cetuximab, an EGFR-targeted monoclonal antibody in preclinical models of CRC. 2D and 3D in vitro assays were executed following treatment with either single agent or combination therapy. The combination of cetuximab with CB-839 resulted in reduced cell viability and demonstrated synergism in several cell lines. In vivo efficacy experiments were performed in cell-line xenograft models propagated in athymic nude mice. Tumor volumes were measured followed by immunohistochemical (IHC) analysis of proliferation (Ki67), mechanistic target of rapamycin (mTOR) signaling (pS6), and multiple mechanisms of cell death to annotate molecular determinants of response. In vivo, a significant reduction in tumor growth and reduced Ki67 and pS6 IHC staining were observed with combination therapy, which was accompanied by increased apoptosis and/or necrosis. The combination showed efficacy in cetuximab-sensitive as well as resistant models. In conclusion, this therapeutic combination represents a promising new precision medicine approach for patients with refractory metastatic WT RAS CRC.

Leptin and its receptor in glucose metabolism of T-cell lymphoma.

T-cell lymphoma (TCL) is a group of heterogeneous disorders with a poor response to conventional treatment. In order to identify novel therapeutic targets, the present study investigated the effect of leptin and its receptor on glucose metabolism in TCL. The expression of the leptin receptor (ObR), and glucose transporter (Glut)1 and 4 was detected in TCL and reactive lymphoid hyperplasia (RLH) tissues by immunohistochemical analysis. A higher level of ObR expression was observed in the TCL tissues than in the RLH tissues (58.3 vs. 22.2%; P=0.012), and ObR overexpression was associated with high expression of Glut1 (P=0.007). In vitro analysis using the human TCL MOLT-3 cell line demonstrated that leptin stimulated cell glucose uptake via promoting recruitment and expression of Glut1, effects which were abolished by ObR-specific small interfering RNA (siRNA). Additionally, MOLT-3 cell viability was also increased following leptin treatment. ObR-specific siRNA abolished these responses. In conclusion, these results suggested that leptin serves a critical role in TCL glucose uptake via the ObR.

Pharmacological blockade of ASCT2-dependent glutamine transport leads to antitumor efficacy in preclinical models.

The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells, including biosynthesis, cell signaling, and oxidative protection. Herein we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux that selectively and potently targets the amino acid transporter ASCT2. Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively contributed to antitumor responses in vitro and in vivo. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, representing a new class of targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.

Krüppel-like factor 4 contributes to the pathogenesis of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma (NHL) with poor prognosis. Krüppel-like factor 4 (KLF4) has been reported as a bi-regulator in malignancies, but little is known about its role in MCL. Here, we showed that KLF4 was downregulated in three MCL cell lines and lymph nodes from MCL patients, which resulted in a negative prognosis. We also found that the regulation of KLF4 could inhibit the proliferation and induce apoptosis of Jeko-1 cells. The lentivirally over-expressed KLF4 protein was found bind to ß-catenin and could inhibit downstream molecules such as cyclinD1 and c-Myc. Furthermore, 5-azacytidine could decrease the expression of methyltransferase-1 (DNMT-1) and restore the KLF4 expression in MCL cell lines, indicating that methylation might play an important role in the downregulation of KLF4. KLF4 may be a potential therapeutic target as a tumor suppressor in MCL.

Bruton's tyrosine kinase inhibitor restrains Wnt signaling in chronic lymphocytic leukemia.

The B-cell receptor (BCR) signaling pathway serves an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and has been identified as a novel and effective therapeutic target of CLL, with particular focus its kinase factor, BTK. Previous studies have focused on combining the BTK inhibitor with additional chemotherapeutic agents to improve the prognosis of patients with CLL. Further investigation into the mechanism of the BTK inhibitor would promote an understanding of the pathogenesis of CLL. The current study investigated the association between ibrutinib and the Wnt signaling pathway, additionally focussing upon one of its regulators, metadherin (MTDH), which has been identified to be overexpressed in CLL and is considered a promoter of the Wnt pathway. The experiments in the current study were performed in the MEC-1 CLL cell line. Results indicated that MTDH, ß-catenin and lymphoid-enhancing factor-1 were inhibited subsequent to ibrutinib treatment. The results indicate that in CLL, ibrutinib is likely to possess an inhibitory role in Wnt signaling.

The STAT3 inhibitor WP1066 synergizes with vorinostat to induce apoptosis of mantle cell lymphoma cells.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL) characterized by the translocation t (11; 14) (q13; q32). Drug resistance remains a formidable obstacle to treatment and the median survival for MCL patients is between 3 and 5 years. Thus, there is an urgent need to discover novel approaches to MCL therapy. The signal transducer and activation of transcription 3 (STAT3) has been found to be constitutively activated in several subtypes of MCL cell lines and MCL tumors. WP1066, a small-molecule inhibitor of STAT3, exerted antitumor activity in hematological and solid malignancies by inhibiting key survival and growth signaling pathways. In the present study, we evaluated the antiproliferative and proapoptotic activity of WP1066 combined with pan-histone deacetylase (HDAC) inhibitor vorinostat (SAHA) in a panel of MCL cell lines. In addition, potential mechanisms involved were also explored. The outcome showed that combination of WP1066 with SAHA resulted in synergistic growth inhibition and apoptosis induction in MCL cell lines in vitro. Furthermore, combination of WP1066 with SAHA inhibited the constitutive STAT3 activation and modulated mRNA expressions of anti- and pro-apoptotic genes. Our findings suggest that agents targeting the STAT3 pathway such as WP1066 may be useful therapeutic drugs for MCL when combined with SAHA.

Targeting nucleophosmin 1 represents a rational strategy for radiation sensitization.

PURPOSE: To test the hypothesis that small molecule targeting of nucleophosmin 1 (NPM1) represents a rational approach for radiosensitization. METHODS AND MATERIALS: Wilde-type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine whether radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived non-small-cell lung cancer (NSCLC) tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. RESULTS: Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double- strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci that colocalized with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is overexpressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. CONCLUSIONS: These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity.

Mitigation of oxygen-induced retinopathy in α2ß1 integrin-deficient mice.

PURPOSE: The α2ß1 integrin plays an important but complex role in angiogenesis and vasculopathies. Published GWAS studies established a correlation between genetic polymorphisms of the α2ß1 integrin gene and incidence of diabetic retinopathy. Recent studies indicated that α2-null mice demonstrate superior vascularization in both the wound and diabetic microenvironments. The goal of this study was to determine whether the vasculoprotective effects of α2-integrin deficiency extended to the retina, using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP). METHODS: In the OIR model, wild-type (WT) and α2-null mice were exposed to 75% oxygen for 5 days (postnatal day [P] 7 to P12) and subsequently returned to room air for 6 days (P12-P18). Retinas were collected at postnatal day 7, day 13, and day 18 and examined via hematoxylin and eosin and Lectin staining. Retinas were analyzed for retinal vascular area, neovascularization, VEGF expression, and Müller cell activation. Primary Müller cell cultures from WT and α2-null mice were isolated and analyzed for hypoxia-induced VEGF-A expression. RESULTS: In the retina, the α2ß1 integrin was minimally expressed in endothelial cells and strongly expressed in activated Müller cells. Isolated α2-null primary Müller cells demonstrated decreased hypoxia-induced VEGF-A expression. In the OIR model, α2-null mice displayed reduced hyperoxia-induced vaso-attenuation, reduced pathological retinal neovascularization, and decreased VEGF expression as compared to WT counterparts. CONCLUSIONS: Our data suggest that the α2ß1 integrin contributes to the pathogenesis of retinopathy. We describe a newly identified role for α2ß1 integrin in mediating hypoxia-induced Müller cell VEGF-A production.

[Research progress of microRNA in chronic lymphocytic leukemia].

MicroRNA (miRNA) is a small endogenous noncoding single-stranded RNA molecule with about 22 nucleotides, and the high conservation in a variety of species exists in most eukaryotes. miRNA can recognize and bind to the 3'untranslated region (3'UTR) of target mRNA via complementary base pairing, then degrading or inhibiting translation of the target mRNA. The miRNA participates in the growth and progression development of individual organism and of the disease by regulating the gene expression after transcription. Chronic lymphocytic leukemia (CLL) is a malignant proliferative disease of B lymphocytes with insidious onset, showing significant heterogeneity in the incidence of individual, disease progression, treatment response, clinical prognosis and so on. Now, more and more studies have shown that mutation or abnormal expression of miRNA are closely related to CLL occurrence, progression, prognosis and curative efficacy. Complex and diverse miRNA in CLL may play a role of oncogenes or tumor suppressor genes. In the near future, some miRNAs may even become fresh biomarkers or therapeutic targets of CLL. This review will focus on miRNA molecules related to the pathogenesis, prognosis and treatment of CLL.

Genome-wide association study identifies eight new risk loci for polycystic ovary syndrome.

Following a previous genome-wide association study (GWAS 1) including 744 cases and 895 controls, we analyzed genome-wide association data from a new cohort of Han Chinese (GWAS 2) with 1,510 polycystic ovary syndrome (PCOS) cases and 2,016 controls. We followed up significantly associated signals identified in the combined results of GWAS 1 and 2 in a total of 8,226 cases and 7,578 controls. In addition to confirming the three loci we previously reported, we identify eight new PCOS association signals at P < 5 × 10(-8): 9q22.32, 11q22.1, 12q13.2, 12q14.3, 16q12.1, 19p13.3, 20q13.2 and a second independent signal at 2p16.3 (the FSHR gene). These PCOS association signals show evidence of enrichment for candidate genes related to insulin signaling, sexual hormone function and type 2 diabetes (T2D). Other candidate genes were related to calcium signaling and endocytosis. Our findings provide new insight and direction for discovering the biological mechanisms of PCOS.

The novel chemical entity YTR107 inhibits recruitment of nucleophosmin to sites of DNA damage, suppressing repair of DNA double-strand breaks and enhancing radiosensitization.

PURPOSE: Radiation therapy continues to be an important therapeutic strategy for providing definitive local/regional control of human cancer. However, oncogenes that harbor driver mutations and/or amplifications can compromise therapeutic efficacy. Thus, there is a need for novel approaches that enhance the DNA damage produced by ionizing radiation. EXPERIMENTAL DESIGN: A forward chemical genetic approach coupled with cell-based phenotypic screening of several tumor cell lines was used to identify a novel chemical entity (NCE) that functioned as a radiation sensitizer. Proteomics, comet assays, confocal microscopy, and immunoblotting were used to identify the biological target. RESULTS: The screening process identified a 5-((N-benzyl-1H-indol-3-yl)-methylene)pyrimidine-2,4,6(1H,3H,5H)trione as an NCE that radiosensitized cancer cells expressing amplified and/or mutated RAS, ErbB, PIK3CA, and/or BRAF oncogenes. Affinity-based solid-phase resin capture followed by liquid chromatography/tandem mass spectrometry identified the chaperone nucleophosmin (NPM) as the NCE target. SiRNA suppression of NPM abrogated radiosensitization by the NCE. Confocal microscopy showed that the NCE inhibited NPM shuttling to radiation-induced DNA damage repair foci, and the analysis of comet assays indicated a diminished rate of DNA double-strand break repair. CONCLUSION: These data support the hypothesis that inhibition of DNA repair due to inhibition of NPM shuttling increases the efficacy of DNA-damaging therapeutic strategies.

Evaluation of the association between GHR exon 3 polymorphism and polycystic ovary syndrome among Han Chinese women.

BACKGROUND: Polycystic ovary syndrome (PCOS) and type 2 diabetes (T2D) are two common metabolic disorders in reproductive-aged women, and both are associated with insulin resistance. Evidence has indicated that the growth hormone receptor (GHR) exon 3 polymorphism is associated with T2D and the GHRd3 allele may have the preventive effect on the disease. However, the genetic effect of this polymorphism on PCOS is unknown. The present study thus aims to evaluate the association between the GHR exon 3 polymorphism and PCOS. DESIGN: A total of 432 patients with PCOS and 441 healthy control subjects were included. All of them were Han Chinese women and well characterized. Genotyping experiments of GHR exon 3 polymorphism were performed with a standard protocol of PCR and gel electrophoresis. RESULTS: GHRd3 allele frequency in PCOS patients was significantly higher compared to the control subjects (19.1% vs. 14.3%; P=0.007, OR=1.416; 95% CI=1.099-1.825). Further analyses indicated that the GHRd3 allele was associated with increased waist and hip circumstance in healthy women (P=0.016; 0.003), and also with 1-h, 2-h and area under the curve (AUC) plasma glucose levels among PCOS patients (all P<0.05). But, no association of GHR exon 3 polymorphism with insulin resistance in the patients was observed. CONCLUSIONS: The present study provides the first evidence that GHR exon 3 polymorphism is associated with PCOS in Han Chinese women. The GHRd3 allele may contribute to an impact of glucose metabolism but not insulin resistance.

Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3, 2p21 and 9q33.3.

Polycystic ovary syndrome (PCOS) is a common metabolic disorder in women. To identify causative genes, we conducted a genome-wide association study (GWAS) of PCOS in Han Chinese. The discovery set included 744 PCOS cases and 895 controls; subsequent replications involved two independent cohorts (2,840 PCOS cases and 5,012 controls from northern Han Chinese; 498 cases and 780 controls from southern and central Han Chinese). We identified strong evidence of associations between PCOS and three loci: 2p16.3 (rs13405728; combined P-value by meta-analysis P(meta) = 7.55 × 10⁻²¹, odds ratio (OR) 0.71); 2p21 (rs13429458, P(meta) = 1.73 × 10⁻²³, OR 0.67); and 9q33.3 (rs2479106, P(meta) = 8.12 × 10⁻¹9, OR 1.34). These findings provide new insight into the pathogenesis of PCOS. Follow-up studies of the candidate genes in these regions are recommended.

A new class of molecular targeted radioprotectors: GSK-3beta inhibitors.

PURPOSE: Development of new treatments is critical to effective protection against radiation-induced injury. We investigate the potential of developing small-molecule inhibitors of glycogen synthase kinase 3beta (GSK-3beta)-SB216763 or SB415286-as radioprotective agents to attenuate intestinal injury. METHODS AND MATERIALS: A survival study was done by use of C57BL/6J mice to evaluate the radioprotective effect of GSK-3beta inhibitors. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemical staining for Bax and Bcl-2 were used to assess apoptosis in the small intestines of the treated mice. A clonogenic survival study, apoptosis assays (staining with annexin V or 4',6-diamidino-2-phenylindole), and immunoblot analysis of beta-catenin, Bcl-2, Bax, and caspase 3 were done by use of Rat intestinal epithelial cell line IEC-6 cells. RESULTS: Pretreatment with SB415286 significantly improved survival of mice irradiated with 8 and 12 Gy. Mice pretreated with SB216763 or SB415286 showed a significant reduction in TUNEL- and Bax-positive cells and an increase in Bcl-2-positive cells in intestinal crypts at 4 and/or 12 h after radiation with 4 and/or 8 Gy compared with radiation alone. Pretreatment of irradiated IEC-6 cells with GSK-3beta inhibitors significantly increased clonogenic survival compared with cells treated with radiation alone. This increase was due to the attenuation of radiation-induced apoptosis, as shown by annexin V and 4',6-diamidino-2-phenylindole assays, as well as immunoblot analysis of Bcl-2, Bax, and caspase 3. CONCLUSIONS: Glycogen synthase kinase 3beta small-molecule inhibitors protect mouse intestine from radiation-induced damage in cell culture and in vivo and improve survival of mice. Molecular mechanisms of this protection involve attenuated radiation-induced apoptosis regulated by Bcl-2, Bax, and caspase 3. Therefore GSK-3beta inhibitors reduce deleterious consequences of intestinal irradiation and thereby improve quality of life during radiation therapy.

Radiation induces an antitumour immune response to mouse melanoma.

PURPOSE: Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response. MATERIALS AND METHODS: For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels. RESULTS: Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC). CONCLUSION: The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.

Indolyl-quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation-mediated control of tumor vasculature.

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.

Cytosolic phospholipase A2: targeting cancer through the tumor vasculature.

PURPOSE: In vascular endothelial cells, low doses of ionizing radiation trigger the immediate activation of cytosolic phospholipase A2 (cPLA2). This event initiates prosurvival signaling that could be responsible for radioresistance of tumor vasculature. Thus, the development of radiosensitizers targeting these survival pathways may enhance tumor response to radiation therapy. Arachidonyltrifluoromethyl Ketone (AACOCF3), a specific cPLA2 inhibitor, was studied as a potential radiosensitizer. EXPERIMENTAL DESIGN: Vascular endothelial cells (3B11 and MPMEC) and lung tumor cells (LLC and H460) were treated with 1 micromol/L AACOCF3 for 30 minutes prior to irradiation. Treatment response was evaluated by clonogenic survival, activation of extracellular signal-regulated kinase 1/2 (ERK1/2), tubule formation, and migration assays. For in vivo experiments, mice with LLC or H460 tumors in the hind limbs were treated for 5 consecutive days with 10 mg/kg AACOCF3 administered daily 30 minutes prior to irradiation. Treatment response was assessed by tumor growth delay, Power Doppler Sonography, and immunohistochemistry. RESULTS: In cell culture experiments, inhibition of cPLA2 with AACOCF3 prevented radiation-induced activation of ERK1/2 and decreased clonogenic survival of irradiated vascular endothelial cells but not the lung tumor cells. Treatment with AACOCF3 also attenuated tubule formation and migration in irradiated vascular endothelial cells. In both tumor mouse models, treatment with AACOCF3 prior to irradiation significantly suppressed tumor growth and decreased overall tumor blood flow and vascularity. Increased apoptosis in both tumor cells and tumor vascular endothelium was determined as a possible mechanism of the observed effect. CONCLUSION: These findings identify cPLA2 as a novel molecular target for tumor sensitization to radiation therapy through the tumor vasculature.