RT2 PCR Research Articles

Neuroinflammatory responses and blood–brain barrier injury in chronic alcohol exposure: role of purinergic P2 × 7 Receptor signaling

Alcohol consumption leads to neuroinflammation and blood‒brain barrier (BBB) damage, resulting in neurological impairment. We previously demonstrated that ethanol-induced disruption of barrier function in human brain endothelial cells was associated with mitochondrial injury, increased ATP and extracellular vesicle (EV) release, and purinergic receptor P2 × 7R activation. Therefore, we aimed to evaluate the effect of P2 × 7R blockade on peripheral and neuro-inflammation in ethanol-exposed mice. In a chronic intermittent ethanol (CIE)-exposed mouse model, P2 × 7R was inhibited by two different methods: Brilliant Blue G (BBG) or gene knockout. We assessed blood ethanol concentration (BEC), brain microvessel gene expression by using RT2 PCR array, plasma P2 × 7R and P-gp, serum ATP, EV-ATP, number of EVs, and EV mtDNA copy numbers. An RT2 PCR array of brain microvessels revealed significant upregulation of proinflammatory genes involved in apoptosis, vasodilation, and platelet activation in CIE-exposed wild-type animals, which were decreased 15–50-fold in BBG-treated–CIE-exposed animals. Plasma P-gp levels and serum P2 × 7R shedding were significantly increased in CIE-exposed animals. Pharmacological or genetic suppression of P2 × 7R decreased receptor shedding to levels equivalent to those in control group. The increase in EV number and EV-ATP content in the CIE-exposed mice was significantly reduced by P2 × 7R inhibition. CIE mice showed augmented EV-mtDNA copy numbers which were reduced in EVs after P2 × 7R inhibition or receptor knockout. These observations suggested that P2 × 7R signaling plays a critical role in ethanol-induced brain injury. Increased extracellular ATP, EV-ATP, EV numbers, and EV-mtDNA copy numbers highlight a new mechanism of brain injury during alcohol exposure via P2 × 7R and biomarkers of such damage. In this study, for the first time, we report the in vivo involvement of P2 × 7R signaling in CIE-induced brain injury.

Alterations in the expression of homologous recombination repair (HRR) genes in breast cancer tissues considering germline BRCA1/2 mutation status

IntroductionHomologous recombination (HR) is a crucial DNA-repair mechanism, and its disruption can lead to the accumulation of mutations that initiate and promote cancer formation. The key HR genes, BRCA1 and BRCA2, are particularly significant as their germline pathogenic variants are associated with a hereditary predisposition to breast and/or ovarian cancer.Materials and methodsThe study was performed on 45 FFPE breast cancer tissues obtained from 24 and 21 patients, with and without the germline BRCA1/2 mutation, respectively. The expression of 11 genes: BRCA1, BRCA2, ATM, BARD1, FANCA, FANCB, FANCI, RAD50, RAD51D, BRIP1, and CHEK2 was assessed using Custom RT2 PCR Array (Qiagen), and results were analysed using R.ResultsCancer tissues from patients with BRCA1 or BRCA2 germline mutations displayed no significant differences in the expression of the selected HR genes compared to BRCA1 or BRCA2 wild-type cancer tissues. In BRCA1mut cancer tissues, BRCA1 expression was significantly higher than in BRCA2mut and BRCA wild-type cancer tissues.ConclusionsIn cancer tissues harbouring either BRCA1 or BRCA2 germline mutations, no significant differences in expression were observed at the mRNA level of any tested HR genes, except BRCA1. However, the significant differences observed in BRCA1 expression between germline BRCA1mut, germline BRCA2mut and BRCA1/2wt tissues may indicate a compensatory mechanism at the mRNA level to mitigate the loss of BRCA1 function in the cells.

Investigating the paracrine and juxtacrine abilities of adipose-derived stromal cells in angiogenesis triple cell co-cultures

The pro-angiogenic abilities of adipose-derived stromal cells (ASCs) make them attractive candidates for cellular therapy, especially for ischemic disease indications. However, details regarding the underlying mechanisms remain elusive. Therefore, this study aimed to investigate paracrine and juxtacrine abilities of ASCs in angiogenesis triple cell co-cultures by detailed image analysis of the vascular-like structures. Fibroblast-endothelial cell co-cultures were established, and ASCs were added directly or indirectly through inserts. The cultures were treated with antibodies or subjected to analyses using ELISA and RT2 PCR Arrays. The model consistently generated vascular-like structures. ASCs increased the total branch lengths equally well in paracrine and juxtacrine conditions, by increasing the number of branches and average branch lengths (ABL). In contrast, addition of VEGF to the model increased the number of branches, but not the ABL. Still, ASCs increased the VEGF levels in supernatants of paracrine and juxtacrine co-cultures, and anti-VEGF treatment decreased the sprouting. ASCs themselves up-regulated collagen type V in response to paracrine signals from the co-cultures. The results suggest that ASCs initiate sprouting through secretion of several paracrine factors, among which VEGF is identified, but VEGF alone does not recapitulate the paracrine actions of ASCs. By employing neutralizing antibodies and dismantling common model outputs using image analysis, the triple cell co-culture is an attractive tool for discovery of the paracrine factors in ASCs’ secretome which act in concert with VEGF to improve angiogenesis.

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Senescence-associated secretory phenotypes in rat-derived dedifferentiated fat cells with replicative senescence.

Senescence-associated secretory phenotype (SASPs) secreted from senescent cells often cause the deleterious damages to the surrounding tissues. Although dedifferentiated fat (DFAT) cells prepared are considered a promising cell source for regenerative therapies, SASPs from DFAT cells undergoing long-term cell culture, which latently induce replicative senescence, have barely been explored. The present study was designed to investigate senescent behaviors in rat-derived DFAT cells at high passage numbers and to analyze the possible types of SASPs. Our data show that DFAT cells undergo senescence during replicative passaging, as determined by multiple senescent hallmarks including morphological changes in cell shape and nucleus. Moreover, RT2 PCR array analysis indicated that senescent DFAT cells expressed higher levels of 16 inflammatory cytokines (Ccl11, Ccl12, Ccl21, Ccl5, Csf2, Cxcl1, Cxcl12, Ifna2, IL11, IL12a, IL13, IL1a, IL1rn, IL6, Mif, and Tnf) associated with SASPs than non-senescent cells. This study implicates that rat DFAT cells undergo cellular senescence after long-term cell culture; cautious consideration should be paid to treat SASP secretion when senescent DFAT cells are used in regenerative medicine.

Diverse pathogen-associated molecular patterns affect transcription of genes in the toll-like receptor signaling pathway in goat blood

Pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan (PGN), Polyinosinic-polycytidylic acid (poly I:C), and CpG Oligodeoxynucleotides (ODN) are recognized by Toll-like receptors (TLR). This study aimed to investigate the effect of diverse PAMPs on the transcription of TLR signaling pathway genes in goat blood. Whole blood was collected from 3 female BoerXSpanish goats and treated with the following PAMPs: 10 µg/ml LPS, PGN, CpG ODN (2216), CpG ODN (2006), and 12.5 µg/ml Poly I:C. Blood-treated PBS served as a control. The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array (Qiagen) was evaluated using real-time PCR. Treatment with PBS affected the expression of 74 genes, Poly I:C affected the expression of 40 genes, t ODN 2006 affected the expression of 50 genes, ODN 2216 affected the expression of 52 genes, LPS affected the expression of 49 genes, while PGN affected the expression of 49 genes. Our results show that PAMPs modulated and increased the expression of genes in the TLR signaling pathway. These results highlight important insights into how the host responds to different pathogens and may help design adjuvants for therapeutics and vaccines that target different.

PD02-08 ESR1+ LUMINAL EPITHELIAL CELLS DRIVE THE PROSTATE GROWTH AND SURVIVAL IN THE ABSENCE OF SRD5A2

You have accessJournal of UrologyCME1 Apr 2023PD02-08 ESR1+ LUMINAL EPITHELIAL CELLS DRIVE THE PROSTATE GROWTH AND SURVIVAL IN THE ABSENCE OF SRD5A2 Christina Sharkey, Xingbo Long, Ra'ad Al-Faouri, Aria F. Olumi, and Zongwei Wang Christina SharkeyChristina Sharkey More articles by this author , Xingbo LongXingbo Long More articles by this author , Ra'ad Al-FaouriRa'ad Al-Faouri More articles by this author , Aria F. OlumiAria F. Olumi More articles by this author , and Zongwei WangZongwei Wang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Steroid 5α reductase 2 (SRD5A2) is the predominant enzyme responsible for prostatic development and growth. 5α reductase inhibitors (5ARI) are the only class of benign prostate hyperplasia-related medications that reduce prostate size. However, why the patients respond variably to 5ARI therapies is still largely unknown. We previously demonstrated that 30% of adult human prostatic tissues do not express the SRD5A2 gene and protein via epigenetic modifications. We established a SRD5A2 null (Srd5a2-/-) mouse model and through single cell RNA sequencing, we identified LE2, a sub-cell population of ESR1+ luminal epithelial that is upregulated in Srd5a2-/- mice. Our goal in this study is to characterize the spatial expression of LE2 markers and its functional phenotype when SRD5A2 is absent. METHODS: Homozygous Srd5a2-/- mice and littermate control heterozygous SRD5A2+/- mice were generated and differentially expressed gene profiles of LE2 were obtained from single-cell RNA sequencing (scRNA seq). Spatial expression of LE2 markers was evaluated by RNA fluorescence in-situ hybridization (RNA-FISH). Cell proliferation was detected by Ki67 and BrdU staining. The Cell Death and ER signaling pathways were evaluated with RT2 PCR profiler arrays (84 genes per array) and downstream signaling pathways were analyzed by western blot. Prostate biopsies from the clinical trial of 5ARI for Medical Therapy of Prostatic Symptoms (MTOPS) were used for immunohistochemistry. RESULTS: The prostate weight of Srd5a2-/- mice was significantly decreased compared to Srd5a2+/- littermate control mice (p<0.001). The prostate cell proliferation was suppressed in Srd5a2-/- mice (p=0.05). The cell death signaling akt1, caspase 7, caspase 9, and ER target genes cyp1a1 and vegfa were downregulated (p<0.05). On the other hand, in the anterior lobe of prostate of Srd5a2-/- mice, the cell proliferation was higher than that in control. scRNA seq showed that ESR1 was expressed mainly in the prostate anterior lobe and the number of ESR1+ LE2 cells was significantly increased upon SRD5A2 absence. This was confirmed by RNA-FISH using the probes of LE2 cell markers ESR1 and PVT1. Co-staining of LE2 cell markers ESR1 and PKCa in prostate biopsies of MTOPS further confirmed the existence of an equivalent LE2 cell population in the human prostate with BPH. CONCLUSIONS: Our data demonstrate that LE2, the subpopulation of prostate cells may play an important role in alternative pathways for prostate growth and survival in absence of SRD5A2 and androgen deprivation. Understanding the underlying androgen-independent pathways contributing to prostate growth can help identify novel therapeutic targets for the management of prostatic diseases. Source of Funding: NIH/R01 DK124502 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e71 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christina Sharkey More articles by this author Xingbo Long More articles by this author Ra'ad Al-Faouri More articles by this author Aria F. Olumi More articles by this author Zongwei Wang More articles by this author Expand All Advertisement PDF downloadLoading ...

Targeting Features of Curaxin CBL0137 on Hematological Malignancies In Vitro and In Vivo.

The anticancer activity of Curaxin CBL0137, a DNA-binding small molecule with chromatin remodulating effect, has been demonstrated in different cancers. Herein, a comparative evaluation of CBL0137 activity was performed in respect to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia and multiple myeloma (MM) cultured in vitro. MTT assay showed AML and MM higher sensitivity to CBL0137's cytostatic effect comparatively to other hematological malignancy cells. Flow cytometry cell cycle analysis revealed an increase in subG1 and G2/M populations after CBL0137 cell treatment, but the prevalent type of arrest varied. Apoptosis activation by CBL0137 measured by Annexin-V/PI dual staining was more active in AML and MM cells. RT2 PCR array showed that changes caused by CBL0137 in signaling pathways involved in cancer pathogenesis were more intensive in AML and MM cells. On the murine model of AML WEHI-3, CBL0137 showed significant anticancer effects in vivo, which were evaluated by corresponding changes in spleen and liver. Thus, more pronounced anticancer effects of CBL0137 in vitro were observed in respect to AML and MM. Experiments in vivo also indicated the perspective of CBL0137 use for AML treatment. This in accordance with the frontline treatment approach in AML using epigenetic drugs.

TAK-242 treatment and its effect on mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice

Intervertebral disc (IVD) degeneration is accompanied by mechanical and gene expression changes to IVDs. SPARC-null mice display accelerated IVD degeneration, and treatment with (toll-like receptor 4 (TLR4) inhibitor) TAK-242 decreases proinflammatory cytokines and pain. This study examined if chronic TAK-242 treatment impacts mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice. Male and female SPARC-null and WT mice aged 7-9 months were given intraperitoneal injections with TAK-242 or an equivalent saline vehicle for 8 weeks (3x/per week, M-W-F). L2-L5 spinal segments were tested in cyclic axial tension andcompression. Gene expression analysis (RT-qPCR) was performed on male IVD tissues using Qiagen RT2 PCR arrays. SPARC-null mice had decreased NZ length (p = 0.001) and increased NZ stiffness (p < 0.001) compared to WT mice. NZ length was not impacted by TAK-242 treatment (p = 0.967) despite increased hysteresis energy (p = 0.024). Tensile stiffness was greater in SPARC-null mice (p = 0.018), and compressive (p < 0.001) stiffness was reduced from TAK-242 treatment in WT but not SPARC-null mice (p = 0.391). Gene expression analysis found upregulation of 13 ECM and 5 inflammatory genes in SPARC-null mice, and downregulation of 2 inflammatory genes after TAK-242 treatment. TAK-242 had limited impacts on SPARC-null mechanical properties and did not attenuate NZ mechanical changes associated with IVD degeneration. Expression analysis revealed an increase in ECM and inflammatory gene expression in SPARCnull mice with a reduction in inflammatory expression due to TAK-242 treatment. This study provides insight into the role of TLR4 in SPARC-null mediated IVD degeneration.

Differential Expression of Cardiac Fat Long Non‐Coding RNAs in Coronary Artery Disease

Coronary artery disease (CAD) is characterized by the buildup of atherosclerotic plaques within the coronary arteries, leading to serious complications. CAD affects 7.2% and 4.2% of males and females, respectively. Epicardial adipose tissue (EAT) interacts with the heart and vasculature, but its mechanistic role in CAD is not completely understood. A more comprehensive view of non‐coding RNA (ncRNA) expression in EAT is needed to better characterize its role in CAD. To conduct this characterization, in an IRB‐approved investigation, we isolated the microRNA (miRNA), long non‐coding RNA (lncRNA), and messenger RNA (mRNA) from the EAT and subcutaneous adipose tissue (SAT) of male and female CAD patients undergoing coronary artery bypass graft as well as from non‐CAD patients undergoing aortic valve replacement. Differential expression of these RNAs between tissues, disease status, and sex was determined using next generation sequencing (mRNA) and microarrays (miRNA and lncRNA). Functional and pathological associations of the differentially expressed RNAs were determined using Ingenuity Pathway Analysis, GO, KEGG, and TAM 2.0. DIANA‐LncBase was used to identify experimentally verified miRNA‐lncRNA interactions. NGS showed differential expression of mRNAs associated with cardiovascular function in EAT but not in SAT. miRNA microarray showed that ncRNA expression within the EAT of CAD patients is distinct from its expression in non‐CAD patients. 14 miRNAs were differentially expressed in EAT from patients with CAD compared to those without CAD. Similarly, lncRNA RT2 PCR array showed a downregulation of three lncRNAs: IPW, TINCR, and linc00853, between the EAT of CAD compared to non‐CAD male patients but not in the EAT of female patients. DIANA‐LncBase showed high confidence pairings of IPW with three miRNAs that were upregulated in the EAT of patients with CAD: hsa‐miR‐24‐3p, hsa‐miR‐26b‐5p, and hsa‐29c‐3p. This suggests that IPW may serve as a ‘sponge’ for these three miRNAs in the EAT of patients without CAD, but its downregulation in CAD reduces that interaction. TAM 2.0 showed these three miRNAs to be associated with functional pathways such as vascular inflammation, apoptosis, adipogenesis, etc. Overall, these findings both reveal potential ncRNA biomarkers for CAD and elucidates possible pathological roles of EAT in CAD mediated by ncRNAs.

Peritoneal Modulators of Endometriosis-Associated Ovarian Cancer.

Ovarian cancer is the 4th largest cause of cancer death in women. Approximately 10–15% of women of childbearing age suffer from endometriosis. Endometriosis is defined by the growth and presence of endometrial tissue (lesions) outside of the uterus. The women with endometriosis also have an increased presence of peritoneal fluid (PF) that comprises of inflammatory cells, growth factors, cytokines/chemokines, etc. Epidemiological studies have shown that >3% of women with endometriosis develop ovarian cancer (low-grade serous or endometrioid types). Our hypothesis is that the PF from women with endometriosis induces transformative changes in the ovarian cells, leading to ovarian cancer development. PF from women with and without endometriosis was collected after IRB approval and patient consent. IOSE (human normal ovarian epithelial cells) and TOV-21G cells (human ovarian clear cell carcinoma cell line) were treated with various volumes of PF (no endometriosis or endometriosis) for 48 or 96 h and proliferation measured. Expression levels of epigenetic regulators and FoxP3, an inflammatory tumor suppressor, were determined. A Human Cancer Inflammation and Immunity Crosstalk RT2 Profiler PCR array was used to measure changes in cancer related genes in treated cells. Results showed increased growth of TOV-21G cells treated with PF from women with endometriosis versus without endometriosis and compared to IOSE cells. Endo PF treatment induced EZH2, H3K27me3, and FoxP3. The RT2 PCR array of TOV-21G cells treated with endo PF showed upregulation of various inflammatory genes (TLRs, Myd88, etc.). These studies indicate that PF from women with endometriosis can both proliferate and transform ovarian cells and hence this microenvironment plays a major mechanistic role in the progression of endometriosis to ovarian cancer.

Transcriptomic Profiling of In Vitro Tumor-Stromal Cell Paracrine Crosstalk Identifies Involvement of the Integrin Signaling Pathway in the Pathogenesis of Mesenteric Fibrosis in Human Small Intestinal Neuroendocrine Neoplasms.

AimAnalysis of the pathophysiology of mesenteric fibrosis (MF) in small intestinal neuroendocrine tumors (SI-NETs) in an in vitro paracrine model and in human SI-NET tissue samples.MethodsAn indirect co-culture model of SI-NET cells KRJ-I and P-STS with stromal cells HEK293 was designed to evaluate the paracrine effects on cell metabolic activity, gene expression by RT2 PCR Profilers to analyse cancer and fibrosis related genes, and RNA sequencing. The integrin signaling pathway, a specific Ingenuity enriched pathway, was further explored in a cohort of human SI-NET tissues by performing protein analysis and immunohistochemistry.ResultsRT Profiler array analysis demonstrated several genes to be significantly up- or down-regulated in a cell specific manner as a result of the paracrine effect. This was further confirmed by employing RNA sequencing revealing multiple signaling pathways involved in carcinogenesis and fibrogenesis that were significantly affected in these cell lines. A significant upregulation in the expression of various integrin pathway – related genes was identified in the mesenteric mass of fibrotic SI-NET as confirmed by RT-qPCR and immunohistochemistry. Protein analysis demonstrated downstream activation of the MAPK and mTOR pathways in some patients with fibrotic SI-NETs.ConclusionThis study has provided the first comprehensive analysis of the crosstalk of SI-NET cells with stromal cells. A novel pathway – the integrin pathway – was identified and further validated and confirmed in a cohort of human SI-NET tissue featured by a dual role in fibrogenesis/carcinogenesis within the neoplastic fibrotic microenvironment.

The profiling, identification, quantification and analysis of differentially expressed genes (DEGs) in response to drug treatment in lung cancer

The profiling and identification of genes that are differentially expressed is frequently used to underpin the underlying molecular mechanisms of biological conditions and provides a molecular foothold on biological questions of interest. However, this can be a daunting task since there is a cross talk and overlap of some of the components of the signalling pathways. The deregulation of the cell cycle signalling pathway is a hallmark of cancer, including lung cancer. Proper regulation of the cell cycle results in cellular homeostasis between cell proliferation and cell death. The comprehension of the cell cycle regulation in drug metabolism studies is of significance. This study aimed at elucidating the regulation of cell cycle genes’ in response to LPV/r in lung cells. Thus, this study describes methodology for revealing molecular mechanisms employed by LPV/r to induce stress on genomic DNA. This approach is based on the interrogation of a panel of 84 genes related to the cell cycle pathway, and how the differentially expressed genes’ expression pattern corroborates loss in nuclear integrity (phenotypic observation). MAD2L2, AURKB and CASP3 gene expressions were further confirmed by RT-qPCR. Furthermore, the use of in-silico bioinformatics tools integrates the molecular profiles and phenotypic changes. This approach revealed the activation of the DNA damage response (DDR) pathway in response to LPV/r treatment. The proposed methodology will aid in the comprehension of drug metabolism at genotypic and phenotypic levels.•Gene profiling often reveals the underlying molecular mechanisms.•RT2 PCR gene arrays have integrated patented quality controls and allow reliable gene expression analysis.•In-silico bioinformatics analysis help reveal pathways affected, that often correspond to phenotypic changes/features.

The dual protease inhibitor lopinavir/ritonavir (LPV/r) exerts genotoxic stress on lung cells

The Sub-Saharan countries, particularly South Africa has the largest number of people living with HIV, accompanied by the largest antiretroviral treatment (ART) programme in the world. The Highly Active Antiretroviral Treatment (HAART) is the most effective regimen against HIV/AIDS and has improved the lifespan and quality of life of HIV positive patients. HAART has also led to a decrease in the incidence of AIDS defining cancers (ADCs) while there is an increased incidence of the non-AIDS Defining Cancers (NADCs), such as lung cancer in the HAART era. The association between lung tumourigenesis and the use of HAART components such as the dual protease inhibitor (PI) lopinavir/ritonavir (LPV/r) is poorly understood. Using cell and molecular biological approaches, this study aimed at elucidating the effects of LPV/r on the regulation of the cell cycle related genes in normal (MRC-5) and adenocarcinoma (A549) lung cells. Initially, the nuclear integrity of these cells in response to LPV/r was determined using DAPI staining. The effect of LPV/r on cell cycle genes was evaluated through the use of a RT2 PCR gene array of 84 genes related to the cell cycle signaling pathway. The PCR array data was validated by Real-Time Quantification PCR (RT-qPCR). Ingenuity Pathway Analysis (IPA) bio-informatics tool was employed to disclose the molecular mechanism/s observed at cellular and gene expression levels. Loss of nuclear integrity and the upregulation of the p53 DNA damage response (DDR) pathway was revealed by DAPI staining, differential gene expression and IPA core analysis. Furthermore, MAD2L2 and AURKB which also play a role in the DDR pathway were shown to be differentially expressed. The activation of the CASP3 gene in response to LPV/r in A549 cells was also observed. The findings of this study suggest genotoxic properties of LPV/r in healthy normal lung fibroblasts cells and anti-tumour properties in the A549 cells.

Pharmacology and Molecular Mechanisms of Clinically Relevant Estrogen Estetrol and Estrogen Mimic BMI-135 for the Treatment of Endocrine-Resistant Breast Cancer.

Long-term estrogen deprivation (LTED) with tamoxifen (TAM) or aromatase inhibitors leads to endocrine-resistance, whereby physiologic levels of estrogen kill breast cancer (BC). Estrogen therapy is effective in treating patients with advanced BC after resistance to TAM and aromatase inhibitors develops. This therapeutic effect is attributed to estrogen-induced apoptosis via the estrogen receptor (ER). Estrogen therapy can have unpleasant gynecologic and nongynecologic adverse events. Here, we study estetrol (E4) and a model Selective Human ER Partial Agonist (ShERPA) BMI-135. Estetrol and ShERPA TTC-352 are being evaluated in clinical trials. These agents are proposed as safer estrogenic candidates compared with 17β-estradiol (E2) for the treatment of endocrine-resistant BC. Cell viability assays, real-time polymerase chain reaction, luciferase reporter assays, chromatin immunoprecipitation, docking and molecular dynamics simulations, human unfolded protein response (UPR) RT2 PCR profiler arrays, live cell microscopic imaging and analysis, and annexin V staining assays were conducted. Our work was done in eight biologically different human BC cell lines and one human endometrial cancer cell line, and results were compared with full agonists estrone, E2, and estriol, a benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. Our study shows the pharmacology of E4 and BMI-135 as less-potent full-estrogen agonists as well as their molecular mechanisms of tumor regression in LTED BC through triggering a rapid UPR and apoptosis. Our work concludes that the use of a full agonist to treat BC is potentially superior to a partial agonist given BPTPE’s delayed induction of UPR and apoptosis, with a higher probability of tumor clonal evolution and resistance.SIGNIFICANCE STATEMENTGiven the unpleasant gynecologic and nongynecologic adverse effects of estrogen treatment, the development of safer estrogens for endocrine-resistant breast cancer (BC) treatment and hormone replacement therapy remains a priority. The naturally occurring estrogen estetrol and Selective Human Estrogen-Receptor Partial Agonists are being evaluated in endocrine-resistant BC clinical trials. This work provides a comprehensive evaluation of their pharmacology in numerous endocrine-resistant BC models and an endometrial cancer model and their molecular mechanisms of tumor regression through the unfolded protein response and apoptosis.

Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8+ T cells

BackgroundThe mitogen-activated protein kinases (MAPKs) are important for T cell survival and their effector function. Mixed lineage kinase 3 (MLK3) (MAP3K11) is an upstream regulator of MAP kinases and emerging...

10H-3,6-Diazaphenothiazines triggered the mitochondrial-dependent and cell death receptor-dependent apoptosis pathways and further increased the chemosensitivity of MCF-7 breast cancer cells via inhibition of AKT1 pathways.

Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H-3,6-diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF-7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC50 = 0.895 µM) toward MCF-7 cells. Further, cell cycle analysis illustrated that the S-phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF-10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT2 PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria-dependent and cell death receptor-dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF-7 cells, thus suggesting its potential as a chemotherapeutic drug.

The role of cancer stem cells in immunotherapy for bladder cancer: An in vitro study

ObjectiveBladder cancer is characterized by frequent recurrence and progression. CD44+ cancer stem cells (CSCs) might be one of the main reasons for recurrence. Although Bacillus Calmette Guerin (BCG) has become a gold standard immunotherapy, after treatment recurrence frequently occur. Based on this knowledge, the aim of this study was to evaluate the changes in cytokine and chemokine expressions in bladder cancer and CSCs cultures in vitro with BCG only and in combination with IL2 and lymphocyte (MNCs) applications. Material and methodsIn this study, 3 cell lines of human bladder cancer cells with different characteristics (T24, 5637, and JMSU-1) and CD44+ bladder CSCs isolated by magnetic bead isolation (Miltenyl Magtech) were used. Bladder cancer cell lines and bladder CSCs in complete medium were cultured under humidified conditions of 37°C temperature in 5% CO2. BCG only and its combination with IL2 and MNCs were applied to bladder cancer cell lines and bladder CSCs for 24, 48, and 72 hours. Annexin V-PI was used to detect the percentages of apoptotic and necrotic cells in treatment groups and control groups. After treatments, total RNAs were isolated and converted to cDNA for each group and controls. Quantitative fold changes in terms of gene expression were measured by RT2–PCR array and fold changes for expression levels of genes were compared among groups. Eighty-four genes were analyzed in standard array of chemokines and cytokines (Biorad). ResultsBCG treatment with 7.32 µg/ml dose alone and in combination with IL2 (1000 IU/ml) and MNCs (1000 cells/ml) were found to be most effective on bladder cancer cells. When BCG and its combinations were applied to CSCs of the 3 cell lines, BCG treatment showed cytotoxic effect on CSCs as well as cancer cells. CSCs of 3 cell lines over expressed CXCL5, CCL8, CNTF, and CSF2 compared with cancer cells. Cancer cells over expressed IL6, TNSFF11, FASLG, and CXCL9 compared with CSCs. In all 3 cell lines, BCG application increased expression of CXCL5 and LTB and also decreased CCL20 and IL6. When BCG was combined with IL2 and MNCs, CXCL10, CXCL5, and IFNG were increased and CXCL12, IL6, and TNSF11 were decreased. BCG treatment of CSCs caused increases in ADIPOQ, CXCL10, and XCL1 and a decrease in CCL8. When IL2 and MNCs were combined with BCG, the expression of many cytokines and chemokines decreased. ConclusionBCG treatment changes the expression of many cytokines and chemokines in bladder cancer. The expression differs in 3 different cell lines and their CSCs. Immune modulation of each case differs from each other. The effectivity of BCG-based immunotherapy in bladder cancer on CSCs might decrease in combination with IL2. Our results indicate that recurrence after BCG treatment for bladder cancer may not occur mainly based on the CSCs hypothesis considering bladder cancer occurs at different loci of surface epithelium.

Hydroxylapatite-collagen hybrid scaffold induces human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro and bone regrowth in patients.

Tissue engineering‐based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite‐collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3‐year follow‐up. Among DEGs, osteogenesis‐related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in “in vitro” experiments with hASCs.

PSVI-3 The effect of oligodeoxynucleotides on Toll-like receptor pathway genes in goat blood

Abstract The objective of this study was to investigate the effect of two classes of oligodeoxynucleotides (ODNs) on the expression of genes involved in Toll-like receptor signaling in goat blood. Toll-like receptors play a critical role in initiating innate immune responses. Select synthetic oligodeoxynucleotides containing methylated CpG motifs (CpG ODN) have immunologic effects similar to those seen with bacterial DNA. These short synthetic single-stranded DNA molecules are being explored as adjuvants to enhance animal health. Blood samples were collected from the jugular vein of BoerXSpanish goats (n = 3) into tubes containing an anticoagulant. Blood was treated with the 10 µg/ml CpG ODN (2216, 2006) class A and B, or 200µl of PBS which served as control. Cells were incubated at 37°C with 5% CO2 and 85% humidity for 30 minutes. Total RNA was isolated using Trizol and then converted to cDNA using RETROscript kit (Qiagen). The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array was evaluated using real-time PCR. Fold change in gene expression was calculated using the 2−ΔΔCt method. The housekeeping genes GAPDH, ACTB, HPRT1, TBP, and YWHAZ was used to normalize the data. Of 84 genes tested, 74 genes were expressed in the control group. Following treatment with ODN 2006, 50 genes were expressed, 29 were up-regulated while 17 genes were down-regulated. The genes HSPD1, IFNG, MAPK8, and PTGS2 were induced by ODN 2006 treatment. Following treatment with ODN 2216, 52 genes were expressed. Treatment with ODN 2216, upregulated 26 genes and down-regulated the expression of 19 genes. The genes CLEC4E, HSPD1, IFNG, IRF1, MAPK8, PTGS2, and UBE2V1 were induced by ODN 2216. Thus specific patterns of TLR signaling may be involved in response to the immunostimulatory effect of CpG ODN in goats with implicates for their use as adjuvants to enhance animal health.