Colony-forming Activity Research Articles

OTUB2 stabilizes U2AF2 to promote the Warburg effect and tumorigenesis via the AKT/mTOR signaling pathway in non-small cell lung cancer.

Increasing evidence has confirmed that deubiquitinating enzymes play an important role in lung cancer progression. In the current study, we investigated the expression profile of deubiquitinating enzymes in non-small cell lung cancer (NSCLC) tissues and identified OTUB2 as an upregulated deubiquitinating enzyme. The role of OTUB2 in NSCLC is unknown.Methods: Quantitative, real-time PCR and Western blot were used to detect OTUB2 and U2AF2 expression in NSCLC tissues. The correlations between OTUB2 and U2AF2 expression and clinicopathologic features were then analyzed. We used In vitro Cell Counting Kit-8 (CCK-8) , colony formation , and trans-well invasion assays to investigate the function of OTUB2 and U2AF2 in tumorigenesis. The regulation of glycolysis by OTUB2 and U2AF2 was assessed by determining the extracellular acid ratio, glucose consumption, and lactate production. The mechanism of OTUB2 was explored through co-immunoprecipitation and mass spectrometry analyses. A xenograft model was also used to study the tumorigenesis role of OTUB2 In vivo.Results: OTUB2 expression was significantly upregulated in primary NSCLC tissues and greatly associated with metastasis, advanced tumor stages, poor survival, and recurrence. In NSCLC cell lines, OTUB2 promoted cell growth, colony formation, migration, and invasive activities. Mechanistic investigations showed that OTUB2 stimulated the Warburg effect and induced the activation of the serine/threonine kinase/mechanistic target of rapamycin kinase (AKT/mTOR) pathway in different NSCLC cells. More importantly, OTUB2 promoted NSCLC progression, which was largely dependent on the direct binding to and deubiquitination of U2AF2, at least in NSCLC cells. U2AF2 expression was also significantly upregulated in primary NSCLC tissues and dramatically associated with metastasis, advanced tumor stages, poor survival, and recurrence. Importantly, a positive correlation between the protein expression of OTUB2 and U2AF2 in NSCLC tissues was found. In vivo experiments indicated that OTUB2 promoted xenograft tumor growth of NSCLC cell. In addition, our results suggest that high expression of OTUB2, U2AF2 and PGK1 is significantly associated with worse prognosis in NSCLC patients.Conclusion: Taken together, the present study provides the first evidence that OTUB2 acts as a pivotal driver in NSCLC tumorigenesis by stabilizing U2AF2 and activating the AKT/mTOR pathway and the Warburg effect. It may serve as a new potential prognostic indicator and therapeutic target in NSCLC.

ASSESSMENT OF RADIOPROTECTIVE ACTION OF BASIDIOMYCOTIC MELANIN PIGMENTS ON THE HEMATOPOIETIC SYSTEM OF Balb/C MICE UNDER EXPOSURE TO IONIZING RADIATION IN SUBLETHAL DOSE.

Assessment of radioprotective action of basidiomycotic melanin pigments on hematopoietic stem and progenitor bone marrow cells of Balb/C mice in case of exposure to ionizing radiation in sublethal dose. Using original method of cultivation in gel diffusion chambers in vivo of bone marrow cells of Balb/C mice we investigated the colony-forming efficiency of hematopoietic progenitor cells of the ani- mals, which were exposed to ionizing radiation action in sublethal dose, in case of treatment with melanin pig- ments solution of basidiomycotic fungi as radioprotector. Investigation of functional activity of bone marrow progenitor cells of Balb/C mice allowed assessing their hematopoiesis state in case of ionizing radiation action, as well as in case of previous treat- ment of the animals with the solution of melanin pigments as radioprotector. It was determined that under the influence of ionizing radiation the colony-forming activity of mice bone marrow has decreased comparing to con- trol. Solution of melanin pigments was able to enhance the functional activity of bone marrow of irradiated ani- mals. Obtained results of radioprotective action of basidiomycotic melanin pigments solution on irradiated stem cells and their descendants (progenitor cells) may become the evidence for development of the protective means for human organism from the injuring action of ionizing radiation.

Inhibition of hsa_circ_0001313 (circCCDC66) induction enhances the radio-sensitivity of colon cancer cells via tumor suppressor miR-338-3p: Effects of cicr_0001313 on colon cancer radio-sensitivity

Circular RNA_0001313 (circ_0001313), also known as circCCDC66, is a novel circRNA that recently found to be upregulated in colon cancer tissues and promote colon cancer progression. However, the role of circ_0001313 in regulating radio-sensitivity of colon cancer and its molecular mechanism remain undetermined. Here we found circ_0001313 was significantly upregulated and miR-338-3p was downregulated in radio-resistant colon cancer tissues compared to radio-sensitive tissues. Radiation treatment in colon cells triggered a remarkable upregulation of circ_0001313 and a downregulation of miR-338-3p. Knockdown of circ_0001313 reduced cell viability, colony formation rate and increased caspase-3 activity in colon cancer cells under irradiation. Moreover, circ_0001313 act as a sponge for miR-338-3p in colon cancer cells. Furthermore, miR-338-3p could reverse the effects of circ_0001313 knockdown on cell viability, colony formation, and caspase-3 activity. These findings revealed that knockdown of circ_0001313 could induce radio-sensitivity of colon cancer cells by negatively regulating miR-338-3p.

Defining a Characteristic Gene Expression Set Responsible for Cancer Stem Cell-Like Features in a Sub-Population of Ewing Sarcoma Cells CADO-ES1.

One of the still open questions in Ewing sarcoma, a rare bone tumor with weak therapeutic options, is to identify the tumor-driving cell (sub) population and to understand the specifics in the biological network of these cells. This basic scientific insight might foster the development of more specific therapeutic target patterns. The experimental approach is based on a side population (SP) of Ewing cells, based on the model cell line CADO-ES1. The SP is established by flow cytometry and defined by the idea that tumor stem-like cells can be identified by the time-course in clearing a given artificial dye. The SP was characterized by a higher colony forming activity, by a higher differentiation potential, by higher resistance to cytotoxic drugs, and by morphology. Several SP and non-SP cell fractions and bone marrow-derived mesenchymal stem cell reference were analyzed by short read sequencing of the full transcriptome. The double-differential analysis leads to an altered expression structure of SP cells centered around the AP-1 and APC/c complex. The SP cells share only a limited proportion of the full mesenchymal stem cell stemness set of genes. This is in line with the expectation that tumor stem-like cells share only a limited subset of stemness features which are relevant for tumor survival.

Activated Leukocyte Cell Adhesion Molecule (ALCAM) Regulate Myeloma Cancer Stem Cell Mediated Myelomagenesis and Chemoresistance

IntroductionMultiple myeloma (MM) is an incurable plasma cell cancer. People introduced the concept of cancer stem cells (CSCs) in MM, known as MM-CSCs, to characterize a small population of malignant cells with distinguishable activities of self-renewal and differentiation into regular MM cells. In this study, we showed that activated leukocyte cell adhesion molecule (ALCAM) interacted with epidermal growth factor receptor (EGFR). ALCAM-EGFR axis regulated MM-CSC mediated myelomagenesis and chemoresistance.MethodsMM-CSCs in MM cell lines and primary MM cells were identified by Hoechst staining or ALDEFLUOR dye, followed by flow cytometry analyses. MM-CSC clonalization activity was examined by soft agar colony formation assay. The protein-protein interaction was identified by co-immunoprecipitation, proximity ligation assay, and proteins' structure simulation. MM-CSC mediated myelomagenesis in vivo was examined in human MM xenograft mouse model and murine adoptive MM model. MM-CSC mediated chemoresistance in vivo was examined using 5T MM model. The clinical significance of ALCAM expression in MM was analyzed using patients' gene expression profiles.ResultsBoth MM cell lines and primary MM cells expressed ALCAM. When cultured with bone marrow stromal cells (BMSCs) or in BMSC-conditioned medium, ALCAM knocked down (AL-KD) MM cells exhibited increased colony formation activity. Hoechst staining showed that under such culture conditions, AL-KD MM cells had increased stemness side population (SP), which was MM-CSC. Depletion of MM-CSC reduced MM colony formation activity. In human MM xenograft model, subcutaneous co-injection of AL-KD MM cells and BMSCs had large amount of MM-CSC and fast tumorigenesis. In murine adoptive MM model, which had tumor derived from viral transformed plasma cells with mandatory expression of MYC, KRAS genes and a shRNA sequence, ALCAM shRNA expression resulted significant more rapid tumor development than non-target shRNA.Mechanistic studies showed that ALCAM and EGFR co-immunoprecipitated in MM cells. Proximity ligation assay showed interaction of ALCAM and EGFR in primary MM cells. Immunofluorescent staining showed that ALCAM colocalized with unphosphorylated EGFR in MM. Epidermal growth factor (EGF), the canonical ligand of EGFR, stimulated EGFR phosphorylation and abolished ALCAM-EGFR colocalization. Protein structure simulation suggested that EGF and ALCAM mutual exclusively bound to the same region of EGFR. EGF addition stimulated MM-CSC in cultured MM. Inhibitors of EGFR, ERK, or hedgehog pathway repressed MM-CSC. EGFR activation led to hedgehog pathway activation via RAS/MEK/ERK cascade, while ALCAM inhibited such pro-MM-CSC cell signaling by binding to EGFR.Chemotherapeutics, such as bortezomib and melphalan, induced MM apoptosis, but also stimulated MM-CSCs, which were resistant to chemotherapeutics. Although CTR-KD and AL-KD MM had similar death rate after the drug treatment, AL-KD MM cells had more MM-CSC generated. MM-CSCs derived in AL-KD MM cells had more active cell proliferation even in the presence of drugs. Further study showed that chemotherapy stimulated EGFR expression and repressed ALCAM expression, therefore switched the balance of ALCAM-EGFR axis to pro-MM-CSC direction. EGFR inhibitors, such as gefitinib, did not have anti-MM activity, but repressed MM-CSCs. In 5T MM model, Gefitinib/melphalan combination therapy resulted prolonged remission period, and significant better mouse survival.We identified MM-CSC in patients BM. ALCAM expression correlated with MM overall survival (OS): ALCAMhigh patients had better OS. In ALCAM-EGFR axis, only ALCAM expression, but not EGFR expression, correlated with MM OS. ALCAM expression varied in different patients, while EGFR expression was more even. Therefore we believe in ALCAM-EGFR axis, it was ALCAM, but not the EGFR, that played the regulatory role. Furthermore, pathway enrichment analysis showed that MM-CSC, identified by Hoechst staining, and MM minimum residual disease (MRD) shared similarities in signaling pathways, as well as pattern of ALCAM expressions.Conclusion and DiscussionALCAM-EGFR axis regulates MM-CSC mediated myelomagenesis and chemoresistance. EGFR inhibitors repress chemoresistant MM-CSC. Combination therapy with conventional chemotherapeutics and an EGFR inhibitor might be promising in MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Modeling Fanconi Anemia Using Human Induced Pluripotent Stem Cells By Reversible Complementation

Modeling of Fanconi anemia (FA) using human induced pluripotent stem cells (iPSCs) has been hindered by the requirement for an intact FA DNA repair pathway for effective reprogramming and maintenance of pluripotency in patient-derived iPSCs. Temporary complementation of FA pathway defects could permit effective reprogramming, with removal of complementation upon directed differentiation to hematopoietic stem and progenitor cells (HSPCs) to model the hematopoietic phenotypes of FA. In this study, we used three FA patient-derived iPSC lines engineered with doxycycline inducible complementation of disease-causing FANCA mutations. We maintained complementation with doxycycline exposure in the pluripotent state and during morphogen directed differentiation of hemogenic endothelium (HE) within embryoid bodies, which possesses the capacity to differentiate to HSPCs. Upon initiation of the endothelial-to-hematopoietic transition (EHT) in purified iPSC-derived HE, doxycycline was either removed or maintained in culture to either inactivate or sustain FANCA expression. Morphologic EHT and emergence of CD34+CD45+ immunophenotypic human HSPCs were not affected by the status of FANCA expression, which allowed for the isolation of otherwise isogenic FANCA-expressing and FANCA-deficient HSPCs for disease modeling. FANCA-deficient HSPCs were unable to form FANCD2/γH2AX foci relative to FANCA-expressing HSPCs in response to genotoxic stress, confirming impairment of the function of the FA pathway. We found that uncomplimented, FANCA-deficient HSPCs showed impaired cell cycle progression, increased apoptosis, and markedly decreased colony forming activity in methylcellulose compared to complemented, FANCA-expressing HSPCs. Unexpectedly, we also found that FANCA-deficient HSPCs showed accelerated erythroid differentiation compared to cells with an intact FA pathway. RNA sequencing analysis showed enrichment of signatures of normal HSPCs in complemented HSPCs, while uncomplimented HSPCs showed signatures of more mature hematopoietic cells and cells undergoing erythroid differentiation. Together, these findings demonstrate the utility of reversible complementation in modeling FA with patient-derived iPSCs, and provide an inexhaustible source of otherwise isongenic complimented and uncomplimented human FA HSPCs for use in the investigation of new therapies. DisclosuresNo relevant conflicts of interest to declare.

Regulation of plasticity and biological features of endothelial progenitor cells by MSC-derived SDF-1

Bone marrow (BM) is a source of mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs). MSCs provide a specific niche in the BM and biological features of EPCs may be changed with this niche. Stromal cell-derived factor 1 (SDF-1) secreted from primary BM-MSCs and biological features of this niche on EPC development are still yet to be understood. The aim of this study was to evaluate the role of SDF-1 produced by MSCs on EPC development. We applied the CRISPR/Cas9 system for the knock-out of the SDF-1 gene in BM-derived MSCs. BM-derived EPCs were then cocultured with MSCsSDF-1−/− or MSCsSDF-1+/+ to identify the role of MSC-derived SDF-1α on proliferation, migration and angiogenic activity of EPCs. Next, pre-expanded EPCs were harvested and co-transplanted with MSCsSDF-1−/− or MSCsSDF-1+/+ into sublethally irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed that proliferation, colony formation, migration and angiogenic activity of EPCs was significantly increased after coculture with MSCsSDF-1+/+. We also found that co-transplantation of EPCs with MSCsSDF-1+/+, in contrast to MSCsSDF-1−/−, into irradiated mice resulted in marrow repopulation and hematologic recovery, leading to improved survival of transplanted mice. In conclusions, MSC-derived SDF-1 niche plays an important role in the development of EPCs and this niche is essential for bone marrow repopulation by these cells and can enhance the efficiency of EPC therapy for ischemic diseases.

The comparative study of the effects of Fe2 O3 and TiO2 micro- and nanoparticles on oxidative states of lung and bone marrow tissues and colony stimulating factor secretion.

Nowadays, increased use of nanomaterials in industry and biomedicine poses potential risks to human health and the environment. Studying their possible toxicological effects is therefore of great significance. The present investigation was designed to examine the status of oxidative stress induced by nanoparticles (NPs) of ferric oxide (Fe2 O 3 ) and titanium oxide (TiO 2 ) with their micro-sized counterpart on mouse lung and bone marrow-derived normal tissue cells. We assessed the induction of oxidative stress by measuring its indicators such as antioxidant scavenging activity of superoxide dismutase and catalase as well as malondialdehyde concentration. Moreover, colony formation of bone marrow cells was assayed following induction with colony stimulating factor (CSF) from lung cells. NPs had a more potent stimulatory effect on the oxidative stress status than their micron-sized counterparts. In addition, the highest level of oxidative stress derived from TiO 2 NPs was observed in both tissue types. Cotreatment with NPs and the antioxidant α-tocopherol reduced antioxidant activities and membrane lipid peroxidation (LPO) in the lung cells, but increased CSF-induced colony formation activity of bone marrow cells, suggesting that oxidative stress may be the cause of the cytotoxic effects of NPs. It is concluded that free radicals generated following exposure to NPs resulted in significant oxidative stress in mouse cells, indicated by increased LPO and antioxidant enzyme activity and decreased colony formation.

Establishment of a transgenic mouse to model ETV7 expressing human tumors

The ETS transcription factor ETV7 has been characterized as a hematopoietic oncoprotein, which requires cooperating mutations for its leukemogenic activity. Although the ETV7 gene is highly conserved among vertebrates, part of the rodents, including Mus musculus, deleted the Etv7 gene locus. Many human hematopoietic malignancies upregulate ETV7 expression but contrary to ETV7’s role in oncogenesis, its physiological role in normal tissues is unknown. To determine the physiological function of ETV7 in vivo and determine its role in tumorigenesis in a mouse model, we have generated an ETV7 transgenic mouse that carries a single copy of human BAC DNA containing the ETV7 gene locus and its regulatory sequences. ETV7 heterozygous (ETV7Tg+/WT) mice were fertile, normal in size and born at a normal Mendelian frequency. They had a normal blood count, did not display any gross physical or behavioral abnormalities, and were not tumor-prone. The ETV7 expression pattern in hematopoietic cells of ETV7Tg+/WT mice is very similar to that in human hematopoietic cells. To examine the oncogenic potential of ETV7 in vivo, we crossed ETV7Tg+/WT mice with tumor-prone mouse models. ETV7 greatly accelerated loss of Pten (phosphatase and tensin homolog)-evoked leukemogenesis in PtenΔ/ΔETV7Tg+/WT mice after deletion of the conditional Pten allele. Consistent with this observation, ETV7 expression enhanced the colony-forming and self-renewal activities of primary myeloid Pten−/− cells. In this study we established a transgenic mouse in which we can more accurately model ETV7-associated human tumorigenesis in vivo.

Blocking Interleukin-6 and Interleukin-8 Signaling Inhibits Cell Viability, Colony-forming Activity, and Cell Migration in Human Triple-negative Breast Cancer and Pancreatic Cancer Cells.

Interleukin-6 (IL-6) and interleukin-8 (IL-8) play important roles in the progression of triple-negative breast cancer (TNBC) and pancreatic ductal adenocarcinoma (PDAC). This is the first experiment to combine small molecules targeting these two signaling pathways to treat TNBC and PDAC cells. Cell viability, colony formation and cell migration assays were conducted when TNBC or PDAC cells were treated with bazedoxifene (targeting IL-6) or reparixin/SCH527123 (targeting IL-8) or their combination. The combined treatment had a more potent inhibition of cell viability, colony formation and cell migration than monotherapy in TNBC and PDAC cells. The results also showed that the combination of bazedoxifene with SCH527123 seemed to be more effective than that with reparixin in inhibiting cell viability and colony formation of TNBC. Novel drug combinations of bazedoxifene and reparixin, as well as bazedoxifene and SCH527123 may provide more effective treatments for TNBC and PDAC.

High glucose condition limited the angiogenic/cardiogenic capacity of murine cardiac progenitor cells in in vitro and in vivo milieu.

Murine c-kit+ cardiac cells were isolated and enriched by magnetic activated cell sorting technique. c-kit+ cells viability and colony-forming activity were evaluated by MTT and clonogenic assay. c-kit+ cells were exposed to endothelial, pericyte, and cardiomyocyte induction media containing 30mM glucose for 7days. We monitored the level of endothelial (VE-cadherin, CD31, and vWF), pericyte (NG2 , α-SMA, and PDGFR-β), and cardiomyocyte markers (cTnT) using flow cytometry, real-time Polymerase Chain Reaction (PCR), and Enzyme-Linked Immunosorbent Assay (ELISA) analyses. Ultrastructural changes were studied by transmission electron microscopy (TEM) in cells treated with 5-Azacytidine and 30mM glucose. Matrigel plug assay was performed to determine the angio/cardiogenic property of c-kit+ cells in a diabetic mouse model. Glucose of 30mM decreased c-kit+ cells viability and clonogenicity (P<0.05). The transdifferentiation capacity of c-kit+ cells into the endothelial lineage, pericytes, and cardiomyocytes were reduced through the inhibition of related genes (P<0.05). TEM analysis revealed cardiomyocyte differentiation rate in c-kit+ cells coincided with an increased intracellular lipid accumulation and reduced number of mitochondria. Similar to in vitro condition, the angiogenic capacity of c-kit+ cells was aborted in vivo indicated by reduced NG2 , α-SMA, CD31, and vWF levels. High glucose condition reduces the angio/cardiogenic capacity of cardiac c-kit+ cells in vitro and in vivo. SIGNIFICANCE OF THE STUDY: High glucose condition seen in diabetes mellitus could affect the regenerative potential of cardiac tissue. The current experiment showed that the exposure of murine cardiac progenitor cells (CD117+ cells) to condition containing 30mM glucose could decrease the differentiation properties into endothelial cells, pericytes, and mature cardiomyocytes in vitro and in vivo. Our finding confirmed that the angiogenic/cardiogenic potential cardiac progenitor cells decrease under treatment with high glucose content as seen in the diabetic condition.

Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation.

Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.

Abstract 5006: Kit-dependent tissue resident macrophage progenitors drive cancer progression

Abstract Macrophages play a key role in promoting tumor growth and resistance to therapy. Here we show that tissue-resident as well as bone marrow-derived macrophages play critical roles in promoting tumor growth. Tissue-resident macrophages were recently shown to originate in the yolk sac or fetal liver during embryogenesis; these cells self-maintain in post-natal tissues independent of hematopoietic stem cells. Tissue-resident macrophages and bone marrow-derived macrophages rapidly accumulate in tumors where they play independent roles in promoting tumor growth. Tissue-resident macrophages are CD11b+Gr1-F4/80hiCX3CR1hiCCR2-Ki67+ cells that accumulate in tumors independently of trafficking receptors. In contrast, bone marrow-derived CD11b+Gr1+F4/80loCX3CR1loCCR2+ macrophages accumulate in tumors in an integrin α4β1/αLβ2 and CCR2 or CXCR4-dependent manner. Gene expression studies show that tumor-associated tissue-resident macrophages are highly proliferative, immune-suppressive myeloid cells that are less proangiogenic than bone marrow-derived macrophages. Our studies show that tumor cells induce the expansion of tissue-resident macrophage progenitor cells by secreting stem cell factor and mCSF. Here we identify a Kit/KitL-dependent tissue-resident macrophage progenitor that is abundant in tumors but not in normal tissues. Notably, tumor growth and colony-forming activity are significantly inhibited in mice treated with SCF and Kit inhibitors. Tumors adoptively transferred with tissue-resident macrophage progenitor cells exhibited a significant growth advantage over control tumors. Furthermore, Kit inhibitors synergize with other immune therapy regimens to suppress tumor growth. Our studies show that tissue-resident macrophage progenitors promote aggressive tumor growth that can be targeted by Kit/SCF inhibition. Citation Format: Paulina Pathria, Hideyuki Takahashi, Megan Kaneda, Judith A. Varner. Kit-dependent tissue resident macrophage progenitors drive cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5006.

Endothelial progenitor cells from aged subjects display decreased expression of sirtuin 1, angiogenic functions, and increased senescence.

Studies have demonstrated that aging is associated with a substantial decline in numbers and angiogenic activity of endothelial progenitor cells (EPCs). In view of senescence being an important regulator of age-related cell survival and function, in the current study, we correlated EPCs numbers and functions with their senescence status and mechanisms in young and elderly subjects. Healthy young subjects (n = 30, below 60 y) and old subjects (n = 30, equal to or above 60 y) participated in the study. Subjects had no significant disease or risk factors of disease and aging was the only risk factor in the aged subjects. Enumeration of CD34-vegfr2 dual positive EPCs was performed. The ex vivo culture of EPCs was done to study colony formation, migration, and senescence-associated beta-galactosidase activity. The expression of cell cycle and senescence regulatory proteins including, p53, p21, and sirtuin 1 (SIRT1), a deacetylase protein was studied in cultured EPCs by RT-PCR and immunofluorescence staining. In vivo proliferation, ex vivo colonies, migration, and secretory ability of EPCs was significantly higher in young subjects than that in elderly subjects. EPCs in old subjects showed enhanced senescence and decreased expression of SIRT1 in comparison to that observed in young subjects. An inhibition of SIRT1 in EPCs of young subjects led to significant increase in senescence and reduction of cell differentiation. The study suggests that EPCs have decreased proliferation and functions in aged subjects due to increased senescence which may be attributable to decreased expression of SIRT1.

Novel antibacterial mouthguard material manufactured using silver-nanoparticle-embedded ethylene-vinyl acetate copolymer masterbatch.

The purpose of the present study was to develop an antibacterial mouthguard (MG) material using a masterbatch of silvernanoparticle-embedded ethylene-vinyl acetate (EVA) copolymers. In order to verify that the testing material was clinically applicable as an antibacterial MG material, we conducted an antibacterial test, a shock absorption test, and analysis of in vitro silver release. The colony-forming activity of Streptococcus sobrinus, Porphyromonas gingivalis, and Escherichia coli were significantly inhibited on the testing materials compared with the commercial EVA sheet (p<0.05). The shock absorption capability of the testing material was not significantly different from that of the commercial EVA sheet. Cumulative silver release (in pure water) from the testing materials were infinitesimal after soaking for 20 days, which implied that there could be no harm in wearing the MG during exercise. These results showed that this testing material could be clinically applicable as an antibacterial MG material.

Antifungal, antibiofilm, and antiproliferative activities of Guapira graciliflora Mart.

The aim of this study was to evaluate in vitro the antifungal, antibiofilm and antiproliferative activities of the extract from the leaves of Guapira graciliflora Mart. The phytochemical characterization of the extract was performed using electrospray ionization mass spectrometry (ESI-MS). The antimicrobial activity of the extract and its fractions was evaluated using the broth microdilution method against species of Candida. The inhibition of C. albicans biofilm was evaluated based on the number of colony-forming units (CFU) and metabolic activity (MTT). The antiproliferative activity of the extract and its fraction was evaluated in the presence of human tumor and non-tumor cells, and the cytotoxicity of the extract was determined on the RAW 264.7 macrophage line - both using the sulforhodamine B method. The phytochemical characterization indicated the presence of the flavonoids rutin and kaempferol. The extract and the methanol fraction exhibited moderate antifungal activity against C. albicans, C. krusei, and C. glabrata, and strong activity against C. dubliniensis. In the biofilms at 24 and 48 hours, the concentration of 12500 µg/mL of the extract was the most effective at reducing the number of CFU s/mL (44.4% and 42.9%, respectively) and the metabolic activity of C. albicans cells (34.6% and 52%, respectively). The extract and its fractions had no antiproliferative effect on the tumor lines tested, with mean activity (log GI50) equal to or greater than 1.71 µg/mL. Macrophage cell viability remained higher than 80% for concentrations of the extract of up to 62.5 µg/mL. G. graciliflora has flavonoids in its chemical composition and demonstrates potential antifungal and antibiofilm activity, with no evidence of a significant change in the viability of human tumor and non-tumor cell lines.

Upregulation of miR‐374a promotes tumor metastasis and progression by downregulating LACTB and predicts unfavorable prognosis in breast cancer

Breast cancer (BRCA) is the second leading cause of cancer‐related death among female worldwide. Recent studies have revealed that LACTB was frequently repressed and functioned as a bona fide new tumor suppressor in a series of cancers, including BRCA. However, the molecular mechanisms underlying LACTB dysregulation in BRCA have not been reported. In the present study, we find that LACTB is repressed in BRCA and associated with poor prognosis by BRCA tissue microarray (TMA) analysis. Moreover, we confirm that LACTB is a direct target of miR‐374a, which is significantly overexpressed and associated with malignancies in BRCA. Mechanistically, applying loss‐of‐function and gain‐of‐function approaches in a series of in vitro and in vivo experiments show that miR‐374a knockdown suppresses the cell proliferative and colony formation activity, as well as migration and invasion capacity, but LACTB silencing in these cells reverses this change. Furthermore, we find that miR‐374a silencing markedly reduces the tumor growth in xenograft mouse models. In summary, our findings suggest the miR‐374a/LACTB axis plays a critical role in the tumorigenicity and progression of BRCA. miR‐374a/LACTB axis may be a potential target in the development of therapeutic strategies for BRCA patients.

Tympanic Membrane Derived Stem Cell-Like Cultures for Tissue Regeneration.

Epidermal cells with stem cell-like characteristics have been identified in the tympanic membrane (TM) and localized specifically to the umbo and annulus regions. While they have been proposed to play a role in the regeneration of both acute and chronic TM perforations, evidence for the mechanism and regulation of their contribution is not yet described. Indeed, the behavior of these putative stem cells is largely unknown, in part due to a lack of refined methods for efficient cell isolation. In this study, we compared different explant techniques using normal and perforated rat TM tissues and investigated their ex vivo characteristics. TM after perforation in vivo showed increased staining for epidermal stem cell markers integrin β1 and cytokeratin (CK) 19, and for proliferation Ki-67, indicating activation of the proliferative centers. A mixed population of fibroblasts and epithelial cells were isolated from explant cultures. Excised TM umbo implanted on a culture well insert was the most effective technique. Explants made from perforated TM produced cells before those from unperforated TM. More importantly, the implanted TM umbo organoid was capable of producing cells in a continuous manner, allowing subsequent harvest using trypsin. Primary rat TM epithelial cell cultures positive for pancytokeratin had colony forming activity and could be enriched for CK 19-positive cells that were capable of culture expansion by proliferation and cell migration when subject to a wound assay. Taken together, trauma to the TM activated the proliferative centers and prompted early cell production from TM umbo organoid cultures, which produced TM stem cell-like cultures that proved suitable for tissue engineering of the TM.

CAMP-CRP acts as a key regulator for the viable but non-culturable state in Escherichia coli.

A variety of bacteria, including Escherichia coli, are known to enter the viable but non-culturable (VBNC) state under various stress conditions. During this state, cells lose colony-forming activities on conventional agar plates while retaining signs of viability. Diverse environmental stresses including starvation induce the VBNC state. However, little is known about the genetic mechanism inducing this state. Here, we aimed to reveal the genetic determinants of the VBNC state of E. coli. We hypothesized that the VBNC state is a process wherein specific gene products important for colony formation are depleted during the extended period of stress conditions. If so, higher expression of these genes would maintain colony-forming activities, thereby restraining cells from entering the VBNC state. From an E. coli plasmid-encoded ORF library, we identified genes that were responsible for maintaining high colony-forming activities after exposure to starvation condition. Among these, cpdA encoding cAMP phosphodiesterase exhibited higher performance in the maintenance of colony-forming activities. As cpdA overexpression decreases intracellular cAMP, cAMP or its complex with cAMP-receptor protein (CRP) may negatively regulate colony-forming activities under stress conditions. We confirmed this using deletion mutants lacking adenylate cyclase or CRP. These mutants fully maintained colony-forming activities even after a long period of starvation, while wild-type cells lost most of this activity. Thus, we concluded that the lack of cAMP-CRP effectively retains high colony-forming activities, indicating that cAMP-CRP acts as a positive regulator necessary for the induction of the VBNC state in E. coli.

Effects of LPA1 and LPA6 on the regulation of colony formation activity in colon cancer cells treated with anticancer drugs

Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits a variety of cellular responses via the activation of G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA1) to LPA6). The aim of our study was to investigate effects of LPA receptors on soft agar colony formation in colon cancer cells treated with anticancer drugs. DLD1 cells were treated with fluorouracil (5-FU) or cisplatin (CDDP) for at least six months (DLD-5FU and DLD-CDDP cells, respectively). LPAR1 gene expression was markedly elevated in DLD-5FU cells. In contrast, DLD-CDDP cells showed the high expression of LPAR6 gene. In colony formation assay, DLD-5FU cells formed markedly large-sized colonies, while no colony formation was observed in DLD1 and DLD-CDDP cells. The large-sized colonies formed in DLD-5FU cells were suppressed by LPA1 knockdown. In contrast, LPA6 knockdown increased the size of colonies. In addition, DLD-5FU cells were further treated with CDDP for three months (DLD-C-F cells). DLD-CDDP cells were also treated with 5-FU (DLD-F-C cells). DLD-C-F cells formed large-sized colonies, but not DLD-F-C cells, correlating with LPAR1 and LPAR6 gene expression levels. These results suggest that LPA1 and LPA6 may regulate the colony formation activity in DLD1 cells treated with anticancer drugs.