Real-time PCR Array Research Articles

Existence of Circulating Mitochondria in Human and Animal Peripheral Blood.

Mitochondria are usually located in the cytoplasm of cells where they generate adenosine triphosphate (ATP) to empower cellular functions. However, we found circulating mitochondria in human and animal blood. Electron microscopy confirmed the presence of mitochondria in adult human blood plasma. Flow cytometry analyses demonstrated that circulating mitochondria from the plasma of human cord blood and adult peripheral blood displayed the immune tolerance-associated membrane molecules such as CD270 and PD-L1 (programmed cell death-ligand 1). Similar data were obtained from fetal bovine serum (FBS) and horse serum of different vendors. Mitochondria remained detectable even after 56 °C heat inactivation. A real-time PCR array revealed purified mitochondria from animal sera expressed several genes that contribute to human T- and B-cell activation. Transwell experiments confirmed the migration capability of mitochondria through their expression of the chemokine receptor CXCR4 in responses to its ligand stromal-derived factor-1α (SDF-1α). Functional analysis established that human plasma mitochondria stimulated the proliferation of anti-CD3/CD28 bead-activated PBMC, up-regulated the percentage of activated CD4+ T and CD8+ T cells, and reduced the production of inflammatory cytokines. These findings suggested that the existence of circulating mitochondria in blood may function as a novel mediator for cell-cell communications and maintenance of homeostasis. Plasma-related products should be cautiously utilized in cell cultures due to the mitochondrial contamination.

Targeting fatty acid synthase sensitizes human nasopharyngeal carcinoma cells to radiation via downregulating frizzled class receptor 10.

Objective: Our aim was to test the hypothesis that fatty acid synthase (FASN) expression contributes to radioresistance of nasopharyngeal carcinoma (NPC) cells and that inhibiting FASN enhances radiosensitivity.Methods: Targeting FASN using epigallocatechin gallate (EGCG) or RNA interference in NPC cell lines that overexpress endogenous FASN was performed to determine their effects on cellular response to radiation in vitro using MTT and colony formation assays, and in vivo using xenograft animal models. Western blot, immunohistochemistry, real-time PCR arrays, and real-time RT-PCR were used to determine the relationship between FASN and frizzled class receptor 10 (FZD10) expression. FZD10 knockdown and overexpression were used to determine its role in mediating FASN function in cellular response to radiation. Immunohistochemical staining was used to determine FASN and FZD10 expressions in human NPC tissues, followed by analysis of their association with the overall survival of patients.Results: FASN knockdown or inhibition significantly enhanced radiosensitivity of NPC cells, both in vitro and in vivo. There was a positive association between FASN and FZD10 expression in NPC cell lines grown as monolayers or xenografts, as well as human tissues. FASN knockdown reduced FZD10 expression, and rescue of FZD10 expression abolished FASN knockdown-induced enhancement of radiosensitivity. FASN and FZD10 were both negatively associated with overall survival of NPC patients.Conclusions: FASN contributes to radioresistance, possibly via FZD10 in NPC cells. Both FZD10 and FASN expressions were associated with poor outcomes of NPC patients. EGCG may sensitize radioresistance by inhibiting FASN and may possibly be developed as a radiosensitizer for better treatment of NPCs.

M6A RNA modification modulates PI3K/Akt/mTOR signal pathway in Gastrointestinal Cancer.

Rationale: Methylation at the N6 position of adenosine (m6A) is the most prevalent RNA modification within protein-coding mRNAs in mammals, and it is a reversible modification with various important biological functions. The formation and function of m6A are regulated by methyltransferases (writers), demethylases (erasers), and special binding proteins (readers) as key factors. However, the underlying modification mechanisms of m6A in gastrointestinal (GI) cancer remain unclear. Here, we performed comprehensive molecular profiling of the nine known m6A writer, eraser, and reader proteins in GI cancer.Methods: Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were used. Gene alteration and pathway analysis were done in cBioportal. The protein network of m6A regulators and its related pathway members was analyzed in STRING online platform. Phylogenetic tree was constructed in MEGA7. m6A modification sites were predicted by SRAMP. m6A related SNPs were analyzed by m6ASNP. The modulation of m6A on its related pathway members was validated by m6A-seq, real-time PCR and phosphor-MAPK array.Results: We found that m6A regulators were mostly upregulated in GI cancer and their differential expression significantly influenced the overall survival of patients with GI cancer. The phosphatidylinositol-3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) signaling pathways were found to be potentially affected by m6A modification in most human cancers, including GI cancer, which was further verified by m6A-Seq and phospho-MAPK array.Conclusions: Our findings suggest that m6A RNA modification has a fundamental role in the regulation of PI3K/Akt and mTOR signaling pathway function in cancer.

The dual role of BI 2536, a small-molecule inhibitor that targets PLK1, in induction of apoptosis and attenuation of autophagy in neuroblastoma cells.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood with the overall 5 years' survival less than 40%. Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase expressed during mitosis and over expressed in multiple cancers, including neuroblastoma. We found that higher PLK1 expression related to poor outcome of NB patients. BI2536, a small molecule inhibitor against PLK1, significantly reduced cell viability in a panel of NB cell lines, with IC50 less than 100 nM. PLK1 inhibition by BI 2536 treatment induced cell cycle arrest at G2/M phase and cell apoptosis in NB cells. Realtime PCR array revealed the PLK1 inhibition related genes, such as BIRC7, TNFSF10, LGALS1 and DAD1 et al. Moreover, autophagy activity was investigated in the NB cells treated with BI 2536. BI 2536 treatment in NB cells increased LC3-II puncta formation and LC3-II expression. Formation of autophagosome induced by BI 2536 was observed by transmission electron microscopy. However, BI2536 abrogated the autophagic flux in NB cells by reducing SQSTM1/p62 expression and AMPKαT172 phosphorylation. These results provide new clues for the molecular mechanism of cell death induced by BI 2536 and suggest that BI 2536 may act as new candidate drug for neuroblastoma.

Use of human neuroblastoma SH-SY5Y cells to evaluate glyphosate-induced effects on oxidative stress, neuronal development and cell death signaling pathways

Glyphosate-containing herbicides are the most used agrochemicals in the world. Their indiscriminate application raises some concerns regarding the possible health and environmental hazards. In this study, we investigated in human neuroblastoma cell line SH-SY5Y if oxidative stress, altered neurodevelopment and cell death pathways are involved in response to glyphosate and its metabolite aminomethylphosphonic acid (AMPA) exposures.MTT and LDH assays were carried out to assess the glyphosate and AMPA cytotoxicity. Lipid peroxides measured as malondialdehyde (MDA), nitric oxide (NO) and reactive oxygen species (ROS) production, and caspase-Glo 3/7 activity were evaluated. The neuroprotective role of melatonin (MEL), Trolox, N-acetylcysteine (NAC) and Sylibin against glyphosate- and AMPA-induced oxidative stress was examined. Glyphosate and AMPA effects on neuronal development related gene transcriptions, and gene expression profiling of cell death pathways by Real-Time PCR array were also investigated. Glyphosate (5 mM) and AMPA (10 mM) induced a significant increase in MDA levels, NO and ROS production and caspase 3/7 activity. Glyphosate exposure induced up-regulation of Wnt3a, Wnt5a, Wnt7a, CAMK2A, CAMK2B and down-regulation of GAP43 and TUBB3 mRNA expression involved in normal neural cell development. In relation to gene expression profiling of cell death pathways, of the 84 genes examined in cells a greater than 2-fold change was observed for APAF1, BAX, BCL2, CASP3, CASP7, CASP9, SYCP2, TNF, TP53, CTSB, NFκB1, PIK3C3, SNCA, SQSTMT, HSPBAP1 and KCNIPI mRNA expression for glyphosate and AMPA exposures. These gene expression data can help to define neurotoxic mechanisms of glyphosate and AMPA.Our results demonstrated that glyphosate and AMPA induced cytotoxic effects on neuronal development, oxidative stress and cell death via apoptotic, autophagy and necrotic pathways and confirmed that glyphosate environmental exposure becomes a concern.This study demonstrates that SH-SY5Y cell line could be considered an in vitro system for pesticide screening.

Long noncoding RNA cytoskeleton regulator RNA promotes cell invasion and metastasis by titrating miR-613 to regulate ANXA2 in nasopharyngeal carcinoma.

BackgroundNasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors. Long noncoding RNAs play critical roles in tumorigenesis.MethodsReal‐time quantitative PCR arrays were used to evaluate the expression levels of cytoskeleton regulator RNA (CYTOR) in NPC tissues and cells. Cell counting kit‐8 and colony formation analyses were used to test the NPC cell viability, while wound healing and transwell assays were employed to detect cell invasion and migration ability. Luciferase reporter assay and Western blot analyses were employed to explore the relationships among CYTOR, miR‐613, and ANXA2.ResultsWe found that CYTOR expression was elevated both in NPC tissues and cells. Functional assays revealed that CYTOR promoted the invasion and migration of NPC cells. The established spontaneous lymph node metastasis model also confirmed that CYTOR promoted NPC cell metastasis in vivo. Mechanically, we found that the subcellular localization of CYTOR mostly occurred in the cell cytoplasm. Luciferase reporter and RIP assays confirmed that CYTOR functioned as the molecular sponge of miR‐613. Subsequent experiments confirmed that ANXA2 was directly targeted by miR‐613. Gain‐ and loss‐of‐function studies further confirmed that CYTOR induced the upregulation of ANXA2 by competitively binding to miR‐613, thus leading to NPC metastasis.ConclusionOur results highlight the importance of CYTOR in NPC development and provide new insights into potential therapeutic targets for NPC.

The environmental hormone nonylphenol interferes with the therapeutic effects of G protein-coupled estrogen receptor specific agonist G-1 on murine allergic rhinitis

The G protein-coupled estrogen receptor (GPER) specific agonist G-1 has therapeutic effects in patients with allergic diseases, but any role for G-1 as a therapy for inflammation associated with allergic rhinitis (AR) remains unclear. The structure of the environmental hormone nonylphenol (NP) is very similar to that of estrogen; it binds to the estrogen receptor to produce estrogen-like effects and thus may also bind to the membrane GPER. We explored whether NP administration would reduce the effects of G-1 on AR, the interactions between the two materials, and their mechanisms of action using a murine model of AR. Mice were randomly assigned into control, AR, G-1, and G-1 + NP groups (n = 10/group). AR nasal symptoms were scored. Eosinophils in nasal mucosa were counted after staining with hematoxylin and eosin. Serum ovalbumin (OVA)-specific IgE was determined by ELISA. The proportions of splenic Th1, Th2, and Treg cells were determined by flow cytometry. The expression of transcription factors unique to Th1, Th2, Treg cells and cytokine levels in nasal mucosa were evaluated by real-time PCR and cytometric bead arrays. AR nasal symptoms, including sneezing, nasal scratching, eosinophil infiltration of nasal mucosa, and serum IgE, were reduced in G-1 group. After injection, Th2 cells proportions, Th2-immune response-related cytokines (IL-4, IL-5, and IL-13), and a Th2 cell-specific transcription factor (GATA-3) were significantly decreased in G-1 group. Treg immune response was enhanced (as reflected by Treg cell, IL-10, and Foxp3 levels). The levels of all of these were significantly increased after adding NP, and the Treg immune response was significantly decreased. These results indicate that G-1 attenuated the nasal symptoms, serum OVA-specific IgE, and Th2 cell immune response, whereas it enhanced Treg immune response, in mice with AR. Adding NP weakened these therapeutic effects.

LINC00459 sponging miR-218 to elevate DKK3 inhibits proliferation and invasion in melanoma

The lncRNA biomarkers in melanoma remain to be further explored. The lncRNAs with different expression levels in melanoma tissue were identified by microarray analysis. To investigate the biological functions of target lncRNA, several in-vivo and in-vitro studies were performed. Potential mechanisms of competitive endogenous RNAs (ceRNAs) were predicted by using bioinformatics analysis and explored by western blot assay, fluorescence in situ hybridization assay, real-time quantitative PCR (RT-qPCR) array, RNA pull-down analysis, AGO2-RIP assay, and dual-luciferase reporter assay. The results demonstrated decreased LINC00459 in melanoma cell lines and tissues. According to the in-vitro and in-vivo experiments, up-regulated LINC00459 had inhibitory effect on cell proliferation and invasion. Bioinformatics analyses suggested that miR-218 could be a direct target of LINC00459. In addition, miR-218 was proved to be able to directly target the dickkopf-related protein 3 (DKK3) gene. In conclusion, our analysis suggested that the LINC00459 could sponge miR-218 and increase the expression of DKK3 gene, thus inhibiting the invasion and proliferation of melanoma cells, which indicated that the LINC00459 could be an effective biomarker for melanoma and its potential as the therapeutic target.

Patterns of Gene Expression Variation across Body Parts of the Hydrothermal Vent Shrimp Nautilocaris saintlaurentae

Since deep-sea hydrothermal vents have been targeted for mining mineral resources, concerns about the potential impacts of mining on vent ecosystems have gradually increased. The vent alvinocaridid shrimp is one of the dominant endemic vent organisms. We collected an experimental species, Nautilocaris saintlaurentae, from the Tonga Arc in the Southwest Pacific Ocean, and firstly determined the sequences of seven housekeeping genes (HKGs) and three stress-related genes in this study. The stability of the HKGs was evaluated and the expression levels of targeted genes were measured in different body parts, the cephalothorax, abdomen, and whole body, to provide information on which parts of nonmodel species can be used for ecotoxicological studies. Based on geNorm platform, multiple combinations of HKGs were tested. The stability of the HKG expression in each body part indicated that the cephalothorax can be used alone and also demonstrated that using at least three HKGs provides suitable interpretations for mRNA real-time quantitative PCR arrays in N. saintlaurentae. Although analysis of specific body part subdivisions was not carried out, our results suggested that anatomical characteristics should be considered when investigating biological functions at a molecular level. Finally, this study revealed upregulation of stress-responsive genes in the cephalothorax of N. saintlaurentae with environmental changes.

P5374Genetic deletion of IL-22 increased cardiac rupture after myocardial infarction in mice

Abstract Background Interleukin-22 (IL-22) is a member of the IL-10 cytokine family, which mainly targets epithelial cells and does not target immune cells. Recently, it has been reported that IL-22 play roles in tissue repair in the skin and the liver; however, role of IL-22 in the process of tissue repair after myocardial infarction (MI) is unknown. Here, we investigated the role of IL-22 in tissue repair process after MI. Methods and results First, we examined the expression of IL-22 and its receptor IL-22RA1 in the wild type (WT) mice by real-time PCR. The expression of IL-22 and IL-22RA1 in the hearts were significantly increased 3 days after MI (p<0.05). To clarify the role of IL-22 in the heart after MI, we produced MI model in the WT mice and IL-22 knockout (KO) mice. We found that the IL-22 KO mice had significantly higher mortality than the WT mice after MI (p<0.05). Approximately 80% of the IL-22 KO mice died with cardiac rupture after MI. The infarct size which was estimated by evans blue dye and triphenyltetrazolium chloride staining at 3 days after MI was comparable between the IL-22 KO mice and the WT mice. Next, we performed real time PCR and PCR array analysis for tissue fibrosis and repair genes. We found that alpha-smooth muscle actin (aSMA), NF-kB, TNF-a and MMP13 (also known as collagenase-3) were significantly increased in the infarct area of IL-22 KO mice compared to WT mice. Immunostaining showed that the myofibroblast marker aSMA positive cells in the border area after MI were markedly higher in the IL-22 KO mice compared with the WT mice (p<0.05). Approximately 70% of cardiac rupture after MI in the IL-22 KO mice were occurred in the infarct area adjacent to the border area. Furthermore, we found aSMA positive cells and MMP13 positive cells around the ruptured site of the heart. Conclusion Thus, IL-22 KO mice exhibit high mortality and increased cardiac rupture after MI. And expression of aSMA and MMP13 were highly expressed in the ruptured site after MI in the IL-22 KO mice. These results suggest that IL-22 may play an important role in the tissue repair process after MI.

Serum microRNA profiles in patients with autosomal dominant polycystic kidney disease show systematic dysregulation partially reversible by hemodialysis

IntroductionThe impact of autosomal dominant polycystic kidney disease (ADPKD) on serum microRNAs (miRNA) is unknown.Material and methodsFor profiling experiment we recruited 30 patients from three equinumerous groups: controls, ADPKD and ADPKD on hemodialysis. From the last group extra samples were collected for in pre-/postdialysis analysis. Additionally, 23 healthy volunteers were used for selected biomarker verification. Real-time PCR arrays were used for quantification of 752 miRNAs. Validation of selected miRNAs was performed in total RNA extracted from the serum and the exosomal fraction in pre-/postdialysis samples.ResultsIn total, 37 significant circulating miRNAs were found to differ between ADPKD patients and controls. In validation, 3 miRNAs with the highest fold change in comparison of dialyzed vs non-dialyzed patients (miR-532-3p, miR-320b, miR-144-5p) were not significantly altered by hemodialysis and from the top down-regulated ones, miR-27a-3p was significantly lower after dialysis in both total and exosomal fractions, miR-20a-5p was down-regulated in the exosomal fraction and miR-16-5p was unaltered by hemodialysis. MiR-16-5p was selected as the best circulating biomarker of ADPKD. Circulating representatives of the miR-17 family sharing the same seed region (miR-20a-5p, miR-93-5p and miR-106a-5p) showed significantly lower expression among dialyzed vs. non-dialyzed patients and their exosomal fraction dropped after hemodialysis.ConclusionsThe serum miRNAs among ADPKD patients differ substantially depending on the stage of CKD. The exosomal fraction of miRNA was more affected by dialysis than the total one. There was a common pattern of down-regulation for circulating miR-17 family members sharing the same seed region.

Characterization of Dermal Stem Cells of Diabetic Patients.

Diabetic foot ulcers (DFUs) are lesions that involve loss of epithelium and dermis, sometimes involving deep structures, compartments, and bones. The aim of this work is to investigate the innate regenerative properties of dermal tissue around ulcers by the identification and analysis of resident dermal stem cells (DSCs). Dermal samples were taken at the edge of DFUs, and genes related to the wound healing process were analyzed by the real-time PCR array. The DSCs were isolated and analyzed by immunofluorescence, flow cytometry, and real-time PCR array to define their stemness properties. The gene expression profile of dermal tissue showed a dysregulation in growth factors, metalloproteinases, collagens, and integrins involved in the wound healing process. In the basal condition, diabetic DSCs adhered on the culture plate with spindle-shaped fibroblast-like morphology. They were positive to the mesenchymal stem cells markers CD44, CD73, CD90, and CD105, but negative for the hematopoietic markers CD14, CD34, CD45, and HLA-DR. In diabetic DSCs, the transcription of genes related to self-renewal and cell division were equivalent to that in normal DSCs. However, the expression of CCNA2, CCND2, CDK1, ALDH1A1, and ABCG2 was downregulated compared with that of normal DSCs. These genes are also related to cell cycle progression and stem cell maintenance. Further investigation will improve the understanding of the molecular mechanisms by which these genes together govern cell proliferation, revealing new strategies useful for future treatment of DFUs.

Oxidative stress and related gene expression effects of cyfluthrin in human neuroblastoma SH-SY5Y cells: Protective effect of melatonin

This study was designed to assess oxidative stress induction in human neuroblastoma SH-SY5Y cells in response to cyfluthrin exposure. Cell viability MTT assay was carried out to assess cyfluthrin cytotoxicity; IC30 and IC50 values for cyfluthrin were calculated to be 4.81 ± 0.92 μM and 19.39 ± 3.44 μM, respectively. Cyfluthrin induced a significant increase in ROS generation, lipid peroxides measured as malondialdehyde (MDA) and nitric oxide (NO) production and a significant decrease in NQO1 activity. The antioxidant activity of melatonin (MEL), Trolox, N-acetylcysteine (NAC) and Sylibin against cyfluthrin-induced oxidative stress was examined. Cyfluthrin increased significantly gene expressions of apoptosis, proinflammation and oxidative stress (Bax, Bcl-2, Casp-3, BNIP3, AKT1, p53, APAF1, NFκB1, TNFα and Nrf2) mediators. In the most genes, the mRNA levels induced by cyfluthrin were partially reduced by MEL (1 μM). Cyfluthrin effects on gene expression profiling of oxidative stress pathway by Real-Time PCR array analysis showed that of the 84 genes examined, (fold change > 1.5) changes in mRNA levels were detected in 31 genes: 13 upregulated and 18 down-regulated. A fold change >3.0 fold was observed on upregulated CYBB, DUOX1, DUOX2, AOX1, BNIP3, HSPA1A, NOS2, and NQO1 genes. The greater fold change reversion (2.5 fold) by MEL (1 μM) was observed on cyfluthrin-upregulated CYBB, AOX1, BNIP3 and NOS2 genes. These results demonstrated that oxidative stress is a key element in cyfluthrin induced neurotoxicity as well as MEL may play a role in reducing cyfluthrin-induced oxidative stress.

Microarray analysis of apoptosis gene expression in liver injury induced by chronic exposure to arsenic and high-fat diet in male mice.

Rapid growth in the incidence of liver disease is largely attributable to lifestyle and environmental contaminants, which are often overlooked as the leading causes of this problem. Thus, the possible contribution of arsenic (As) to high-fat diet (HFD)-induced liver damage was examined via microarray analysis. To perform this experiment, a total number of 40 healthy adult male NMRI mice (22-30 g) were used. To this end, these animals were randomly assigned to four groups of 10. Oxidative stress and histopathological parameters were also evaluated in the liver of the mice exposed to a minimally cytotoxic concentration of As (50 ppm) in drinking water while being fed with a HFD for 20 weeks. Subsequently, apoptosis gene expression profiling was utilized via real-time (RT) PCR array analysis. The results showed that As had increased the amount of HFD-induced liver damage and consequently amplified changes in oxidative stress factors, histopathological parameters, as well as apoptosis pathway genes. Investigating the expression profile of apoptosis pathway genes similarly revealed that caspase-8, as a main upstream contributor to the apoptosis pathway, might play an important role in the induction of apoptosis generated by As and HFD. Ultimately, this study highlighted that As in drinking water could increase sensitivity in mice to HFD-induced liver disease through strengthening apoptosis pathway.

Abstract 1797: Serum microRNAs as immune modulators and biomarkers of breast cancer recurrence

Abstract MicroRNAs (microRNAs) are short sequences of RNA (about 22 nucleotides) that are involved in the regulation of gene expression. Previous studies have suggested that a number of microRNAs are recognized as new biomarkers for cancers. The aim of this study is to identify specific microRNAs in serum, which may serve as potential diagnostic and prognostic biomarkers and therapeutic targets for breast cancer. Quantitative real-time PCR (qRT-PCR) array analyses of microRNAs in sera from four pairs of recurrent and nonrecurrent breast cancer patients were performed. Those differentially expressed microRNAs were verified in serum samples from 42 breast cancer patients. The prognostic values of the selected microRNAs were statistically analyzed, determined by the correlation between microRNA expression and tumor parameters. High serum levels of miR-134 and miR-483-5p were found to be associated with some aggressive tumor behaviors. Kaplan-Meier analysis of four upregulated microRNAs (miR-134, miR-483-5p, miR-493-3p and miR-139-3p) indicated that serum level of miR-134 can predict tumor recurrence in breast cancer patients after primary treatment. Further functional analysis showed that overexpression of miR-134 has no significant effects on tumor growth but regulates macrophage M1/M2 polarization. Overall, our data suggested that serum microRNAs may promote tumor progression and recurrence via modulating inflammation and immune response and potentially serve as blood biomarkers for prediction of recurrence in breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Lauren Chen, Mithil Soni. Serum microRNAs as immune modulators and biomarkers of breast cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1797.

Abstract 2207: The dual role of BI 2536, a small-molecule inhibitor that targets PLK1, in induction of apoptosis and attenuation of autophagy in neuroblastoma cells

Abstract Neuroblastoma(NB) is the most common extra-cranial solid tumor in childhood with the overall survival less than 40%. Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase expressed during mitosis and overexpressed in multiple cancers, including neuroblastoma. We found the PLK1 expression related to the outcome of NB patients. BI2536, a small molecule inhibitor against PLK1, significantly reduced cell viability in a panal of NB cell lines, with IC50 less than 100 nM. PLK1 inhibition by BI 2536 treatment induced cell cycle arrest at G2/M phase and cell apoptosis in NB cells. Realtime PCR array revealed the PLK1-regulated genes, such as BIRC7, TNFSF10, LGALS1 and DAD1 et al. Moreover, autophagy activity was investigated in the NB cells treated with BI 2536. BI 2536 treatment in NB cells increased LC3-II puncta formation and LC3-II expression. Formation of autophagosome induced by BI 2536 was observed by TEM. However, BI2536 abrogated the autophagic flux in NB cells by reducing SQSTM1/p62 expression and AMPKαT172 phosphorylation. These results provide new clues for the molecular mechanism of cell death induced by BI 2536 and suggest that BI 2536 may act as new candidate drug for neuroblastoma. Citation Format: Zhiheng Li. The dual role of BI 2536, a small-molecule inhibitor that targets PLK1, in induction of apoptosis and attenuation of autophagy in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2207.

NLRP3 inhibition improves heart function in GPER knockout mice

The molecular mechanisms of postmenopausal heart diseases in women may involve the loss of estrogen-deactivation of its membrane receptor, G-protein coupled estrogen receptor (GPER), and subsequent activation of the cardiac NLRP3 inflammasome, a component of the innate immune system. To study the potential effects of cardiac GPER on NLRP3-mediated inflammatory pathways, we characterized changes in innate immunity gene transcripts in hearts from 6-month-old cardiomyocyte-specific GPER knockout (KO) mice and their GPER-intact wild type (WT) littermates using RT2 Profiler™ real-time PCR array. GPER deletion in cardiomyocytes decreased %fractional shortening (%FS) and myocardial relaxation (e′), and increased the early mitral inflow filling velocity-to-early mitral annular descent velocity ratio (E/e′), determined by echocardiography, and increased the mRNA levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), determined by real-time PCR. Of the 84 inflammasome-related genes tested, 9 genes were upregulated, including NLRP3 and IL-18, while 1 gene, IL-12a, was downregulated in GPER KO when compared to WT. The importance of NLRP3 upregulation in GPER KO-induced heart failure was further confirmed by an in vivo study showing that, compared to vehicle-treated KO mice, 8 weeks of treatment with a NLRP3 inhibitor, MCC950 (10 mg/kg, i.p., 3 times per week), significantly limited hypertrophic remodeling, defined by reductions in heart weight/body weight, and improved systolic and diastolic functional indices, including increases in %FS and e′, and decreases E/e′ (P < 0.05). Both ANF and BNP mRNA levels were also significantly reduced by chronic MCC950 treatment. The findings from this study point toward a new understanding for the increased occurrence of heart diseases in women following loss or absence of estrogenic protection and GPER activation that involves cardiac NLRP3 inflammatory pathways.

Epigenetically Down-Regulated Acetyltransferase PCAF Increases the Resistance of Colorectal Cancer to 5-Fluorouracil

Only 10%–20% of colorectal cancer (CRC) patients observe effective responses to 5-fluorouracil (5-FU) based chemo-treatment. We used real-time PCR array and Western blot analysis to examine the expression alteration of acetyltransferases and deacetylases in 5-FU resistant CRC cell lines as compared to their respective parental CRC cell lines. Unlike other acetyltransferases and deacetylases, we found that the expression of acetyltransferase P300/CBP-associated factor (PCAF) is consistently decreased in three 5-FU resistant CRC cell lines. Similarly, knockdown of PCAF in HCT116 CRC parental cell line also increases the resistance to 5-FU and attenuates 5-FU-induced apoptosis. Mechanistically, we demonstrated that increased binding of trimethylated histone H3K27 in the promoter region of PCAF attenuated its transcription in 5-FU resistant HCT116/5-FU cells. Decreased PCAF impairs the acetylation of p53 and attenuates the p53-dependent transcription of p21, which results in the increased cyclin D1 and phosphorylation of Retinoblastoma 1. Conversely, overexpression of PCAF in CRC cell lines increases p21 and their susceptibility to 5-FU in vitro and in vivo. However, knockdown of p21 abolishes the beneficial effects of PCAF overexpression on increasing the sensitivity of HCT116/5-FU cells to 5-FU. Also, the reduced intensity of PCAF immunostaining was observed in the precancerous lesion, and microarray data from the public database further demonstrated the association between PCAF down-regulation and poor survival outcome. Our data suggest that PCAF-mediated p53 acetylation is an essential regulatory mechanism for increasing the susceptibility of CRC to 5-FU.

Leptin enhances cytokine/chemokine production by normal lung fibroblasts by binding to leptin receptor

BackgroundObesity is a known risk and exacerbation factor for bronchial asthma. Leptin is an adipokine secreted by adipocytes and enhances energy consumption. Earlier studies have shown that leptin also activates inflammatory cells and structural cells, including airway epithelial cells, thereby exacerbating inflammation. However, little is known about leptin's effect on normal human lung fibroblasts (NHLFs), which are deeply involved in airway remodeling in asthma. This study aimed to elucidate the direct effect of leptin on NHLFs. MethodsNHLFs were co-cultured with leptin, and production of cytokines/chemokines was analyzed with real-time PCR and cytometric bead arrays (CBA). Expression of alpha smooth muscle actin (α-SMA) in the lysate of NHLFs stimulated with leptin was assessed by western blotting. Expression of leptin receptor (Ob-R) was analyzed by real-time PCR and flow cytometry. NHLFs were transfected with Ob-R small interference ribonucleic acid (siRNA) by electroporation and used for experiments. ResultsLeptin enhanced production of CCL11/Eotaxin, monocyte chemoattractant protein-1 (CCL2/MCP-1), CXCL8/IL-8, interferon gamma-induced protein 10 (CXCL10/IP-10) and IL-6 by NHLFs at both the protein and messenger ribonucleic acid (mRNA) levels. Leptin also slightly, but significantly, elevated expression of α-SMA. We found robust Ob-R expression on cell surfaces, and transfection with Ob-R siRNA suppressed the enhanced production of CCL11/Eotaxin, CXCL10/IP-10 and IL-6 by leptin, although not completely. ConclusionsThese findings indicate that leptin may contribute to worsening of asthma in obese patients by enhancing production of inflammatory mediators by binding to Ob-R and accelerating myofibroblast differentiation.

Establishment and Characterization of 5-Fluorouracil-Resistant Human Colorectal Cancer Stem-Like Cells: Tumor Dynamics under Selection Pressure.

5-Fluorouracil (5-FU) remains the gold standard of first-line treatment for colorectal cancer (CRC). Although it may initially debulk the tumor mass, relapses frequently occur, indicating the existence of cancer cells that are therapy-resistant and are capable of refueling tumor growth. To identify mechanisms of drug resistance, CRC stem-like cells were subjected to long-term 5-FU selection using either intermittent treatment regimen with the IC50 drug dose or continuous treatment regimen with escalating drug doses. Parental cancer cells were cultivated in parallel. Real-time PCR arrays and bioinformatic tools were used to investigate gene expression changes. We found the first method selected for cancer cells with more aggressive features. We therefore transplanted these cancer cells or parental cells in mice, and again, found that not only did the 5-FU-selected cancer cells generate more aggressive tumors with respect to their parental counterpart, but they also showed a different gene expression pattern as compared to what we had observed in vitro, with ID1 the top upregulated gene. We propose ID1 as a stemness marker pervasively expressed in secondary lesions emerging after completion of chemotherapy.