Normal Control Cells Research Articles

E74-like factor 4 promotes the proliferation, invasion, and migration of colorectal cancer cells via long non-coding RNA integrin subunit beta 8 antisense RNA 1-mediated histone H3 lysine 27 trimethylation modification.

Colorectal cancer (CRC) is a common malignancy in the gastrointestinal tract. The main objective of this study is to explore the potential mechanisms of E74-like factor 4 (ELF4) in CRC progression, providing a novel therapeutic target for CRC treatment. CRC cells and normal control cells were cultured. Levels of ELF4/long non-coding RNA integrin subunit beta 8 antisense RNA 1 (LncRNA ITGB8-AS1)/claudin-23 (CLDN23) were detected by real-time quantitative polymerase chain reaction or Western blot assay. ELF4 siRNA, ITGB8-AS1 pcDNA3.1, or CLDN23 siRNA were transfected into cells. Cell proliferation, migration, and invasion were evaluated. The enrichment of ELF4 on the ITGB8-AS1 promoter was detected. Dual-luciferase assay was employed to assess the binding between ELF4 and the ITGB8-AS1 promoter. RNA pull-down and RNA immunoprecipitation assays were conducted to investigate the binding between ITGB8-AS1 and enhancer of zeste homolog 2 (EZH2). The binding of EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to the CLDN23 promoter was detected. ELF4 and ITGB8-AS1 were upregulated in CRC cells, while CLDN23 was downregulated. Knockdown of ELF4 inhibited cell proliferation, invasion, and migration. Mechanistically, ELF4 transcriptionally activated ITGB8-AS1 and promoted the binding between ITGB8-AS1 and EZH2. EZH2 catalyzed H3K27me3 modification on the CLDN23 promoter, leading to decreased CLDN23 expression. Overexpression of ITGB8-AS1 or downregulation of CLDN23 could reduce the inhibitory effects of silencing ELF4 on CRC cell proliferation, migration, and invasion. ELF4 accelerates CRC progression through the ITGB8-AS1/CLDN23 axis, providing new therapeutic targets for CRC.

New synergistic combination therapy approaches with HDAC inhibitor quisinostat, cisplatin or PARP inhibitor talazoparib for urothelial carcinoma.

Urothelial carcinoma (UC) urgently requires new therapeutic options. Histone deacetylases (HDAC) are frequently dysregulated in UC and constitute interesting targets for the development of alternative therapy options. Thus, we investigated the effect of the second generation HDAC inhibitor (HDACi) quisinostat in five UC cell lines (UCC) and two normal control cell lines in comparison to romidepsin, a well characterized HDACi which was previously shown to induce cell death and cell cycle arrest. In UCC, quisinostat led to cell cycle alterations, cell death induction and DNA damage, but was well tolerated by normal cells. Combinations of quisinostat with cisplatin or the PARP inhibitor talazoparib led to decrease in cell viability and significant synergistic effect in five UCCs and platinum-resistant sublines allowing dose reduction. Further analyses in UM-UC-3 and J82 at low dose ratio revealed that the mechanisms included cell cycle disturbance, apoptosis induction and DNA damage. These combinations appeared to be well tolerated in normal cells. In conclusion, our results suggest new promising combination regimes for treatment of UC, also in the cisplatin-resistant setting.

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug-Resistant Human Breast Cancer.

The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.

Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures (RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

Trimethylamine-N-oxide, a New Risk Factor for Non-alcoholic Fatty Liver Disease Changes the Expression of miRNA-34a, and miRNA-122 in the Fatty Liver Cell Model.

Non-alcoholic fatty liver disease is a multifactorial disorder with complicated pathophysiology ranging from simple steatosis to steatohepatitis and liver fibrosis. Trimethylamine-N-oxide (TMAO) production is believed to be correlated with choline deficiency. This study investigated the expression of miRNA-34a, miRNA-122, and miRNA-192 in the fatty liver cell model treated with different concentrations of TMAO. A fatty liver cell model was developed by exposing HepG2 cells to a mixture of palmitate and oleate in a ratio of 1:2 at a final concentration of 1200μM for 24h. The confirmed fatty liver cells were treated with 37.5, 75, 150, and 300μM of TMAO for 24h. RT-qPCR was used to quantify the expression of microRNAs in a cellular model. The cellular expression of all microRNAs was significantly higher in treated fatty liver cells compared to normal HepG2 cells (P < 0.05). Only 75 and 150µM of TMAO significantly increased the expression of miRNA-34a and miRNA-122 compared to both fatty and normal control cells (P < 0.05). Our results provided an experimental documentation for the potential effect of TMAO to change the expression of miR-34a and miR-22 as a mechanism for contributing to the pathogenesis of non-alcoholic fatty liver disease.

Deep Learning-Based Identification of Intraocular Pressure-Associated Genes Influencing Trabecular Meshwork Cell Morphology

Genome-wide association studies have recently uncovered many loci associated with variation in intraocular pressure (IOP). Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs) and thus, IOP regulation. Experimental study. Primary TMCs collected from human donors. Sixty-two genes at 55 loci associated with IOP variation were knocked out in primary TMC lines. All cells underwent high-throughput microscopy imaging after being stained with a 5-channel fluorescent cell staining protocol. A convolutional neural network was trained to distinguish between gene knockout and normal control cell images. The area under the receiver operator curve (AUC) metric was used to quantify morphological variation in gene knockouts to identify potential pathological perturbations. Degree of morphological variation as measured by deep learning algorithm accuracy of differentiation from normal controls. Cells where LTBP2 or BCAS3 had been perturbed demonstrated the greatest morphological variation from normal TMCs (AUC 0.851, standard deviation [SD] 0.030; and AUC 0.845, SD 0.020, respectively). Of 7 multigene loci, 5 had statistically significant differences in AUC (P<0.05) between genes, allowing for pathological gene prioritization. The mitochondrial channel most frequently showed the greatest degree of morphological variation (33.9% of cell lines). We demonstrate a robust method for functionally interrogating genome-wide association signals using high-throughput microscopy and AI. Genetic variations inducing marked morphological variation can be readily identified, allowing for the gene-based dissection of loci associated with complex traits. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Generation of three isogenic human induced pluripotent stem cell lines from normal neonate skin fibroblasts

Characterized human induced pluripotent stem cell lines are important for basic research. Here, we report the establishment of three isogenic human induced pluripotent stem cell (hiPSC) lines generated from normal neonate male skin fibroblasts. Pluripotency was induced using the integration free Sendai virus reprogramming method. The pluripotency, identity, quality, and safety of the lines were confirmed to establish characterized human induced pluripotent stem cell lines to be used as normal control cell lines in future studies.

A conserved ZFX/WNT3 axis modulates the growth and imatinib response of chronic myeloid leukemia stem/progenitor cells

BackgroundZinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear.MethodsReal-time quantitative PCR (RT–qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT–qPCR and western blotting were used to study the effect of ZFX on β-catenin signaling.ResultsWe showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/β-catenin signaling, including c-MYC and CCND1 expression.ConclusionThe present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells.

Multiproperty Polyethylenimine-Caged Platinum Nanoclusters Promote Apoptosis of Osteosarcoma Cells via Regulating the BAX-Bcl-2/Caspase-3/PARP Axis.

Osteosarcoma, a prevalent primary bone cancer in children, exhibits a poor prognosis due to the high prevalence of drug resistance. The objective of this study was to investigate the potential of fluorescent ultrafine polyethylenimine-coated caged platinum nanoclusters (PEI-Pt NCs) as an antitumor agent in osteosarcoma. The primary focus of this study involved the utilization of osteosarcoma cells (U2-OS and MG-63) and normal control cells (hBMSC) as the primary subjects of investigation. The capacity of PEI-Pt NCs to enter osteosarcoma cells was observed through the implementation of confocal microscopy. The impact of PEI-Pt NCs on migration and proliferation was assessed through the utilization of various methodologies, including the CCK8 assay, Ki-67 immunofluorescence, clone formation assay, transwell assay, and wound healing assay. Furthermore, the influence of PEI-Pt NCs on apoptosis and its underlying mechanism was explored through the implementation of flow cytometry and Western blotting techniques. The PEI-Pt NCs demonstrated the capability to enter osteosarcoma cells, including the nucleus, while also exhibiting fluorescent labeling properties. Furthermore, the PEI-Pt NCs effectively impeded the migration and proliferation of osteosarcoma cells. Additionally, the PEI-Pt NCs facilitated apoptosis by modulating the BAX-Bcl-2/Caspase 3/PARP axis. The novel nanomaterial PEI-Pt NCs possess diverse advantageous capabilities, including the ability to impede cell proliferation and migration, as well as the capacity to modulate the BAX-Bcl-2/Caspase 3/PARP axis, thereby promoting cell apoptosis. Consequently, this nanomaterial exhibits promising potential in addressing the issue of inadequate platinum-based treatment for osteosarcoma.

OR07-01 Aberrant Expression Of Agouti Signaling Protein (ASIP) As A Cause of Monogenic Severe Childhood Obesity

Abstract Disclosure: K. Landgraf: None. E.T. Kempf: None. R. Stein: None. M. Hanschkow: None. Y. Tseng: None. T. Schöneberg: None. E. Zeggini: None. M. Blüher: None. A. Körner: None. Here we identified and characterized a heterozygous tandem duplication at the ASIP (agouti-signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female index patient with extreme childhood obesity.From an adipose tissue sample of the patient, we observed pronounced and sustained differentiation potential of stroma-vascular fraction (SVF) cells into adipocytes in the patient compared to normal control cells. We further found reduced mitochondrial maximum respiration, spare capacity, and proton leak in the patient’s SVF cells as proxies for reduced energy expenditure. By transcriptome screen, we identified overexpression of one single gene, ASIP. We found and functionally confirmed a chromosomal rearrangement at the ASIP locus, that places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase (ITCH) promoter. The patient's phenotype of early-onset obesity, overgrowth, red hair, and hyperinsulinemia is concordant with that of mutant, so called agouti mice ubiquitously expressing the homolog nonagouti. Physiologically, ASIP antagonises the melanocortin 1 receptor (MC1R) in the skin. Due to its homology to the hypothalamic regulator of eating behaviour agouti-related protein (AGRP), we hypothesize that ectopic ASIP similarly influences eating behaviour via repressing melanocyte-stimulating hormone-mediated activation of MC4R and that the mutation might therefore represent a novel monogenic cause for severe childhood obesity. We show that the tandem duplication results in the ectopic generation of ASIP in patient-derived native stroma-vascular fraction (SVF) cells, adipocytes and blood cells as well as in induced pluripotent stem cells (iPSCs) generated from SVF cells of the patient, thus indicating ubiquitous ectopic ASIP expression. This overexpression persisted after differentiation of iPSCs into the three germ layers, mesoderm, ectoderm and endoderm, and after differentiation into hypothalamic-like neurons. Furthermore, we observed that ASIP causes a reduction of MC4R activity in vitro supporting the hypothesis that the obesity phenotype of our patient is caused by ectopic ASIP acting at the MC4R in the hypothalamus. Since the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig childhood obesity cohort of 1745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity and might be potentially treatable with melanocortin 4 receptor agonists. Presentation: Thursday, June 15, 2023

Danshenxinkun B protects human umbilical vein endothelial cells against ox-LDL-induced injury by inhibiting pyroptosis and the NF-κB/NLRP3 pathway

To investigate the protective effect of Danshenxinkun B against oxidized low-density lipoprotein (ox-LDL)- induced human umbilical vein endothelial cell (HUVEC) injury and explore the underlying mechanism. HUVECs cultured in the presence of 10% fetal bovine serum were treated with ox-LDL (100 μg/mL), ox-LDL+0.1% dimethyl sulfoxide (DMSO), or ox-LDL+Danshenxinkun B (100 ng/mL, dissolved in DMSO) for 24 h. The changes in lactate dehydrogenase (LDH) release was detected, and qRT-PCR was used to detect the mRNA expressions of nuclear factor-κB1 (NF-κB1), nucleotide binding oligomerization domain-like receptor family pyrin domain protein 3 (NLRP3), gasdermin D (GSDMD) and interleukin- 1β (IL-1β). The protein expressions of NF-κB1, NLRP3, caspase-1, IL-1β and GSDMD-N were detected with Western blotting. Immunofluorescence assay was performed to examine the changes in GSDMD expression in the cells. Compared with the normal control cells, the cells treated with ox-LDL alone or in combination with DMSO exhibited significantly increased LDH release, mRNA expressions of NF-κB1, NLRP3, GSDMD, and IL- 1β and the protein levels of NF-κB1, NLRP3, IL- 1β, GSDMD-N and caspase-1 (P<0.01), which were all significantly lowered by treatment with Danshenxinkun B (P<0.05 or 0.01). Danshenxinkun B treatment significantly inhibited GSDMD expression on the cell membrane and restricted its entry into the cell nucleus. Danshenxinkun B alleviates ox-LDL-induced HUVEC injury possibly by suppressing pyroptosis mediated by NLRP3 inflammatory bodies and inhibiting the NF-κB/NLRP3 signaling pathway.

An Aptamer That Rapidly Internalizes into Cancer Cells Utilizes the Transferrin Receptor Pathway

Strategies to direct drugs specifically to cancer cells have been increasingly explored, and significant progress has been made toward such targeted therapy. For example, drugs have been conjugated into tumor-targeting antibodies to enable delivery directly to tumor cells. Aptamers are an attractive class of molecules for this type of drug targeting as they are high-affinity/high-specificity ligands, relatively small in size, GMP manufacturable at a large-scale, amenable to chemical conjugation, and not immunogenic. Previous work from our group revealed that an aptamer selected to internalize into human prostate cancer cells, called E3, can also target a broad range of human cancers but not normal control cells. Moreover, this E3 aptamer can deliver highly cytotoxic drugs to cancer cells as Aptamer-highly Toxic Drug Conjugates (ApTDCs) and inhibit tumor growth in vivo. Here, we evaluate its targeting mechanism and report that E3 selectively internalizes into cancer cells utilizing a pathway that involves transferrin receptor 1 (TfR 1). E3 binds to recombinant human TfR 1 with high affinity and competes with transferrin (Tf) for binding to TfR1. In addition, knockdown or knockin of human TfR1 results in a decrease or increase in E3 cell binding. Here, we reported a molecular model of E3 binding to the transferrin receptor that summarizes our findings.

Transcriptome sequencing identifies prognostic genes involved in gastric adenocarcinoma.

Gastric adenocarcinoma (GAC) is one of the world's most lethal malignant tumors. It has been established that the occurrence and progression of GAC are linked to molecular changes. However, the pathogenesis mechanism of GAC remains unclear. In this study, we sequenced 6 pairs of GAC tumor tissues and adjacent normal tissues and collected GAC gene expression profile data from the TCGA database. Analysis of this data revealed 465 differentially expressed genes (DEGs), of which 246 were upregulated and 219 were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that DEGs were observably enriched in ECM-receptor interaction, protein digestion and absorption, and gastric acid secretion pathways. Six key genes (MATN3, COL1A1, COL5A2, P4HA3, SERPINE1 and VCAN) associated with poor GAC prognosis were screened from the protein‒protein interaction (PPI) network by survival analysis, and P4HA3 and MATN3 have rarely been reported to be associated with GAC. We further analyzed the function of P4HA3 in the GAC cell line SGC-7901 by RT‒qPCR, MTT, flow cytometry, colony formation, wound healing, Transwell and western blot assays. We found that P4HA3 was upregulated in the SGC-7901 cell line versus normal control cells. The outcomes of the loss-of-function assay illustrated that P4HA3 significantly enhanced the ability of GAC cells to proliferate and migrate. This study provides a new basis for the selection of prognostic markers and therapeutic targets for GAC.

Cinnamomum zeylanicum Blume Essential Oil Inhibits Metastatic Melanoma Cell Proliferation by Triggering an Incomplete Tumour Cell Stress Response.

Given the known pro-oxidant status of tumour cells, the development of anti-proliferative strategies focuses on products with both anti- and pro-oxidant properties that can enhance antitumour drug cytotoxicity. We used a C. zeylanicum essential oil (CINN-EO) and assessed its effect on a human metastatic melanoma cell line (M14). Human PBMCs and MDMs from healthy donors were used as normal control cells. CINN-EO induced cell growth inhibition, cell cycle perturbation, ROS and Fe(II) increases, and mitochondrial membrane depolarization. To assess whether CINN-EO could affect the stress response, we analysed iron metabolism and stress response gene expression. CINN-EO increased HMOX1, FTH1, SLC7A11, DGKK, and GSR expression but repressed OXR1, SOD3, Tf, and TfR1 expression. HMOX1, Fe(II), and ROS increases are associated with ferroptosis, which can be reversed by SnPPIX, an HMOX1 inhibitor. Indeed, our data demonstrated that SnPPIX significantly attenuated the inhibition of cell proliferation, suggesting that the inhibition of cell proliferation induced by CINN-EO could be related to ferroptosis. Concurrent treatment with CINN-EO enhanced the anti-melanoma effect of two conventional antineoplastic drugs: the mitochondria-targeting tamoxifen and the anti-BRAF dabrafenib. We demonstrate that CINN-EO-mediated induction of an incomplete stress response specifically in cancer cells affects the proliferation of melanoma cells and can enhance drug cytotoxicity.

Regulation of cellular contractile force, shape and migration of fibroblasts by oncogenes and Histone deacetylase 6.

The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasis.

Metabolomic characterization of congenital microtia: a possible analysis for early diagnosis.

Although metabolic abnormalities have been deemed one of the essential risk factors for growth and development, the relationship between metabolic abnormalities and microtia is still unclear. In this study, we aimed to establish a cell model of microtia and the changes of serum metabolites in patients with microtia. After constructing a cell model of microtia with low expression of BMP5, we performed integrative metabolomics analysis. For the altered metabolites, the content of glycerophosphocholine (PC), triacylglycerol (TG), and choline in the serum of 28 patients (15 patients with microtia and 13 controls) with microtia was verified by enzyme-linked immunosorbent assay (ELISA). Detailed metabolomic evaluation showed distinct clusters of metabolites between BMP5-low expressing cells and normal control (NC) cells. The cell model of microtia had significantly higher levels of TG, PC, glycerophosphoethanolamine (PE), sphingomyelin, sulfatide, glycerophosphoglycerol, diacylglycerol, and glycosphingolipid. The main abnormal metabolites were mainly concentrated in the glycerophospholipid metabolism pathway, and PC and choline were closely related. In the serum of patients with microtia, the contents of PC, TG, and choline were significantly increased. The individual serum samples confirmed the different metabolites between patients with microtia and controls. In particular, we showed that a newly developed metabolic biomarker panel has a high sensitivity and specificity for separating patients with microtia from controls.

Dual Targeting of HDM2 and PPM1D As a Novel Therapeutic Approach for Myelofibrosis

Dysregulation of TP53 in myeloproliferative neoplasms (MPNs) has been hypothesized to be a key driver of disease progression (Kubesova et al, Leukemia. 2018;32 (2):450-61). Targeting of double minute protein 2 (HDM2), a negative regulator of TP53, results in activation of the TP53 pathway which induces cell death in hematopoietic stem and progenitor cells (HSPCs). Nutlins, antagonists of HDM2, are currently being evaluated in multiple MPN clinical trials with promising results to date. Protein Phosphatase Mg2+/Mn2+ dependent 1D protein (PPM1D) is another key negative regulator of the TP53 pathway that dephosphorylates TP53 and stabilizes HDM2 (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). We and others have previously reported the presence of PPM1D mutations in MPNs that result in overexpression of the protein (Grinfeld et al, N Engl J Med. 2018 Oct 11;379 (15):1416-30). We have also reported overexpression of PPM1D in the mononuclear cells (MNC) of a subset of MPN patients not harboring PPM1D mutations. We hypothesize that inhibition of PPM1D in myelofibrosis (MF) would target the MF HSPC and sensitize MF HSPCs to HDM2 inhibitors resulting in further depletion of MF HSPCs. To further characterize PPM1D expression in the MF HSPC compartment, we utilized quantitative real time-PCR, to assess PPM1D transcript levels in CD34+ cells from 28 MF patients and compared these transcript levels to levels in CD34+ normal control cells (n=6). Forty-three percent (12/28) of the MF samples had overexpression of PPM1D (>1.5 fold increase) and the fold increase ranged from 1.8-59. To identify whether PPM1D is a viable therapeutic target for MF HSPCs, we utilized hematopoietic colony assays to test the activity of GSK2830371, an allosteric inhibitor of PPM1D (PPM1Di) alone and in combination with a low dose HDM2 inhibitor (HDM2i). Eight samples from distinct MF patients were tested under these conditions. There was not a significant reduction in the number of JAK2V617F+ colonies upon treatment with single agent HDM2i (50 nM) (p>0.05) or PPM1Di (1uM) (p>0.05). Combination of PPM1Di (1 uM) + HDM2i (50 nM) resulted in decreased JAK2V617F+ colony formation in all 8 samples (p<0.005) (Fig. 1). The reduction in JAK2V617F+ colony number was significant compared to either single agent treatment with HDM2i (p<0.001) or PPM1Di (p<0.05) (Fig. 1). Four samples had persistence of or an increase in JAK2 wild-type (WT) colony number with combination treatment. The MF samples utilized were from a heterogenous population of patients that all had WT TP53 and PPM1D but included patients with high risk myeloid mutations (in ASXL1, NRAS), complex cytogenetics and prior exposure to standard MF directed therapies. Additionally, to validate the on-target effect of PPM1Di, we showed that treatment of leukemia cell lines with PPM1Di led to increased protein levels of phosphorylated-p53 and p21 and resulted in decreased viability and increased apoptosis. To determine the effect of PPM1Di +/- HDM2i on distinct subclones we employed a multi-omics approach that allows for single cell mutation and epitope analysis of a clonally complex patient sample. We analyzed MNCs from a JAK2V617F, NRASG12D, TET2T1884A, ASXL1W960* MF patient treated in suspension culture with PPM1Di (1uM) alone, low dose HDM2i (50 nM) alone and PPM1Di (1uM) + low dose HDM2i (50 nM) in combination. Compared to vehicle alone or either drug alone, a marked effect was seen with the combination approach whereby the percent of WT cells increased by nearly 5 fold (4% to 19%) and 4/6 mutated clones were decreased in size, demonstrated by the fishplot (Fig. 2). These results show that the combination of the HDM2i + PPM1Di depletes multiple MF clones, while allowing for the emergence of more WT cells. Furthermore these studies demonstrate the utility of single cell genotyping to evaluate the effects of novel therapeutic strategies on MF clonal architecture. These studies indicate that PPM1Di sensitizes MF HSPCs to an HDM2i and is a promising strategy to treat MF patients. To this purpose, we are currently developing a novel clinical grade PPM1D targeting agent. Dual targeting of PPM1D and HDM2 has the potential to further deplete multiple MF HSPC clones while allowing the persistence of WT cells. Additional combinatorial mutation + epitope studies are underway to determine degree of MF HSPC subpopulation depletion utilizing this treatment strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Suppressive effect of YY1-mediated RGS22 regulation on the proliferation, migration and invasion of pancreatic ductal adenocarcinoma.

Regulator of G-protein signaling 22 (RGS22) is specifically expressed in the testis and in tumors of epithelial origin, but the expression and role of RGS22 in pancreatic cancer are unclear. In this study, 52 pairs of pancreatic ductal adenocarcinoma (PDAC) and adjacent non-neoplastic tissue samples with the corresponding clinical data were used to examine the expression of RGS22 and its relationship with PDAC prognosis. The findings showed that the expression of RGS22 was higher in the PDAC tissues than in the adjacent non-tumorous tissues and its expression was associated with the degree of blood vessel invasion. The in vitro experiments with PDAC cell lines and a normal control cell line showed that the proliferation, invasion, and metastasis of PDAC cells were suppressed by RGS22 overexpression and enhanced by RGS22 knockdown. The in vivo effect of RGS22 on PDAC xenografts was studied using subcutaneous implantation of tumor cells in BALB/cA-nu mice, and the results corroborated the in vitro findings. Analysis of the regulators of RGS22 showed that it was positively regulated by the transcription factor Yin Yang-1 (YY1). Thus, YY1-mediated RGS22 regulation suppressed the proliferation, migration, and invasion of PDAC.

Exosomal microRNA-222-3p increases UVB sensitivity of lens epithelium cells by suppressing MGMT.

Age-related cataract (ARC) is a leading cause of blindness worldwide with multiple pathogenic factors. Oxidative damage of lens epithelium cells (LECs) is one of the well-accepted pathogenesis of ARC which can be regulated by DNA repair genes (DRGs). The present research aimed to clarify the regulatory mechanism of exosomal microRNAs (miRNAs) on DRGs in LECs. The LECs oxidative damage model was established by UVB-irradiation on SRA01/04 (human lens epithelium cell line). Exosomes from UVB-irradiated cells (UVB-exo) and exosomes from normal control cells (NC-exo) were collected from the culture medium. To explore the functions of LECs exosomes, SRA01/04 were incubated with UVB-exo/NC-exo. Then, we detected SRA01/04 proliferation, viability and apoptosis respectively using 5'-ethynyl-2'-deoxyuridine(EdU), cell-counting kit-8(CCK-8) and TdT-mediated dUTP Nick-End Labeling(TUNEL) assay. Next, the miRNA expression profiles of UVB-exo and NC-exo were identified by miRNA microarrays. RNA expression in exosomes, cells, and clinical samples was verified by qRT-PCR. The location and expression of MGMT and CD63 proteins were detected by immunofluorescence and western blot. The 3'UTR regulation of miR-222-3p to MGMT was verified by luciferase analyses. MGMT down-regulated while miR-222-3p up-regulated in LECs sub-central anterior capsule from ARC lenses. MGMT and miR-222-3p expressions in central and peripheral LECs from anterior lens capsules were differential. UVB-exo can transport the up-regulated miR-222-3p from oxidative-damaged LECs to normal LECs, which could suppress MGMT expression and increase UVB sensitivity of LECs. Findings on exosomal miRNA functions provided novel insights into pathogenesis of ARC. Exosomal miR-222-3p can be a potential target for prevention and cure of ARC.

Study of DNA ploidy in newly diagnosed multiple myeloma by flow cytometry and its correlation with disease prognosis and outcome

Objectives: Multiparameter flow cytometry is increasingly being used in determining DNA content in several hematological malignancies where abnormal DNA ploidy is a useful prognostic marker. This study was done to analyze the DNA ploidy of plasma cells in multiple myeloma (MM) by flow cytometry and explore its role as a prognostic factor. Material and Methods: Propidium-iodide staining technique and gating based on light scatter properties were used. DNA index (DI) of the tumor sample was calculated as the ratio of the DNA content of the G0-G1 population of the myeloma cells with the normal control cells (lymphocytes) present in the same sample. Based on DI, ploidy was categorized as diploidy, hypodiploidy, and hyperdiploidy and the results were correlated with the clinical outcome. Results: Among 32 patients, none had hypodiploid DNA content, 8 (25%) patients had hyperdiploid DNA, and 24 (75%) patients had diploid DNA. There was no significant association between DI and international staging system staging (P = 0.68), Eastern Cooperative Oncology Group Performance Status (P = 0.59), and post-induction remission status (P = 0.10). The median overall survival (OS) in the study patients was 20 (CI 11.4–28.8) months and the median progression-free survival was not reached. There was no difference in the OS among patients with diploid MM and hyperdiploid MM (P = 0.84). Conclusion: Although hyperdiploid MM has been reported to have a better prognosis than diploid MM, we did not find any significant difference possibly due to the small sample size. Nevertheless, flow cytometry is a useful tool in DNA ploidy analysis and its role as a prognostic factor in various hematologic malignancies including MM can be further explored.